Summary Mutations in the photoreceptor transcription factor gene cone-rod homeobox (CRX) lead to distinct retinopathy phenotypes, including early-onset vision impairment in dominant Leber congenital amaurosis (LCA). Using induced pluripotent stem cells (iPSCs) from a patient with CRX -I138fs48 mutation, we established an in vitro model of CRX -LCA in retinal organoids that showed defective photoreceptor maturation by histology and gene profiling, with diminished expression of visual opsins. Adeno-associated virus (AAV)-mediated CRX gene augmentation therapy partially restored photoreceptor phenotype and expression of phototransduction-related genes as determined by single-cell RNA-sequencing. Retinal organoids derived from iPSCs of a second dominant CRX -LCA patient carrying K88N mutation revealed the loss of opsin expression as a common phenotype, which was alleviated by AAV-mediated augmentation of CRX. Our studies provide a proof-of-concept for developing gene therapy of dominant CRX -LCA and other CRX retinopathies.

Usher syndrome (USH) is the most common form of hereditary deaf-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course, and severity, with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retinae uncovered harmonin_a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Müller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones, and outer segments of rods as well as at adhesive junctions between MGCs and photoreceptor cells (PRCs) in the outer limiting membrane (OLM). Our data provide evidence for interaction of harmonin with OLM molecules in PRCs and MGCs and with rhodopsin in PRCs. Subcellular expression and colocalization of harmonin correlate with the clinical phenotype observed in USH1C patients. We also demonstrate that primary cilia defects in USH1C patient-derived fibroblasts could be reverted by the delivery of harmonin_a1 transcript isoform. Our studies thus provide novel insights into PRC cell biology, USH1C pathophysiology, and development of gene therapy treatment(s).

Human Usher syndrome (USH) is a complex genetic disorder that comprises three clinical subtypes USH1, USH2, and USH3, and the most common form of hereditary combined deaf-blindness. Since rodent USH1 models do not reflect the ocular phenotype observed in human patients to date only little is known about the pathophysiology of USH1 in the human eye. The USH1C gene is heavily alternatively spliced and encodes for numerous harmonin isoforms that function as scaffold proteins and organizer of the USH interactome. RNA-seq analysis of USH1C revealed harmonin a1 as the most abundant transcript of USH1C in the human retina. Bulk mRNA-seq and Western blots confirmed abundant expression of harmonin in Muller glia cells (MGCs) and retinal neurons. We located harmonin in the terminal endfeet and apical microvilli of MGCs and in photoreceptor cells (PRCs), particularly in cone synapses and outer segments of rods as well as at adhesive junctions between both, MGCs and PRCs in the outer limiting membrane (OLM). We evidenced the interaction of harmonin with OLM molecules and rhodopsin in PRCs. We correlate the identified harmonin subcellular expression and colocalization with the clinical phenotype observed in USH1C patients. Furthermore, we demonstrate a phenotype in primary cilia in patient-derived fibroblasts which we were able to revert by gene addition of harmonin a1. Our data provide novel insights for retinal cell biology, USH1C pathophysiology and subsequent gene therapy development.