Granular corneal dystrophy (GCD) is an autosomal dominant hereditary disease in which multiple discrete and irregularly shaped granular opacities are deposited in the corneal stroma. GCD is caused by a point mutation in the transforming growth factor-β-induced (TGFBI) gene, located on chromosome 5q31. Here, we report the first successful application of CRISPR-Cas9-mediated genome editing for the correction of a TGFBI mutation in GCD patient-derived primary corneal keratocytes via homology-directed repair (HDR). To correct genetic defects in GCD patient cells, we designed a disease-specific guide RNA (gRNA) targeting the R124H mutation of TGFBI, which causes GCD type 2 (GCD2). An R124H mutation in primary human corneal keratocytes derived from a GCD2 patient was corrected by delivering a CRISPR plasmid expressing Cas9/gRNA and a single-stranded oligodeoxynucleotide HDR donor template in vitro. The gene correction efficiency was 20.6% in heterozygous cells and 41.3% in homozygous cells. No off-target effects were detected. These results reveal a new therapeutic strategy for GCD2; this method may also be applicable to other heredity corneal diseases.

PurposeWe describe a sporadic case of unilateral pigmented paravenous retinochoroidal atrophy (PPRCA) with retinitis pigmentosa (RP) in the contralateral eye.Observationsa 24-year-old female aware of the narrowing of visual field was examined at our hospital. Funduscopic examination revealed left eye showing retinochroidal atrophy along the retinal veins with pigment accumulation while right eye showing peripheral diffuse retinal pigmented epithelium atrophy with bone spicule pigmentation. Fundus autofluorescence, electroretinogram, visual field test and optic coherent tomography were also performed and obtained results were compatible with funduscopic observation.Conclusions and importanceSimultaneous manifestation of PPRCA and RP observed in this case is rare and supports a shared genetic basis between the two diseases. Further genetic investigations are needed to elucidate the etiology and to properly manage PPRCA.

This study investigated a rabbit model of autologous simple oral mucosal epithelium transplantation (SOMET) for limbal stem cell deficiency (LSCD). LSCD was created in the SOMET group and the Control group. In the SOMET group, oral mucosa harvested from the buccal region was treated with dispase, cut into small pieces, and placed on the exposed corneal stroma without using graft sutures, amniotic membrane, and/or glue. A soft contact lens was positioned and tarsorrhaphy was performed in both groups. Postoperative corneal neovascularization and fluorescein staining scores were evaluated by slit lamp microscopy in both groups. At 2 weeks postoperatively, eyes were excised and subjected to immunohistochemical staining for CK3, CK13, CK15, and p63. In the SOMET group, transplantation of oral mucosa led to complete recovery of LSCD, as indicated by low neovascularization scores, low fluorescein staining scores, and detection of stratified K3/K13-positive cells on the stroma at 2 weeks after surgery. In contrast, corneal epithelial defects persisted in the Control group at 2 weeks. SOMET achieved re-epithelialization of the corneal surface in this rabbit LSCD model. It is a simple technique that does not require culture and could be a promising option for ocular surface reconstruction in bilateral LSCD.

Mutations in transforming growth factor-beta-induced (TGFBI) gene cause clinically distinct types of corneal dystrophies. To delineate the mechanisms driving these dystrophies, we focused on the R124C mutation in TGFBI that causes lattice corneal dystrophy type1 (LCD1) and generated novel transgenic mice harbouring a single amino acid substitution of arginine 124 with cysteine in TGFBI via ssODNmediated base-pair substitution using CRISPR/Cas9 technology. Eighty percent of homozygous and 9.1% of heterozygous TGFBI-R124C mice developed a corneal opacity at 40 weeks of age. Hematoxylin and eosin and Masson trichrome staining showed eosinophilic deposits in subepithelial corneal stroma that stained negative for Congo-red. Although amyloid deposition was not observed in TGFBI-R124C mice, irregular amorphous deposits were clearly observed via transmission electron microscopy near the basement membrane. Interestingly, we found that the corneal deposition of TGFBI protein (TGFBIp) was significantly increased in homozygous TGFBI-R124C mice, suggesting a pathogenic role for the mutant protein accumulation. Furthermore, as observed in the LCD1 patients, corneal epithelial wound healing was significantly delayed in TGFBI-R124C mice. In conclusion, our novel mouse model of TGFBI-R124C corneal dystrophy reproduces features of the human disease. This mouse model will help delineate the pathogenic mechanisms of human corneal dystrophy. Corneal dystrophy is a hereditary disease that causes corneal opacity; 22 types of corneal dystrophy are currently classified according to the International Committee for Classification of Corneal Dystrophies (IC3D)0 1. Among these, transforming growth factor-beta-induced (TGFBI) corneal dystrophies occur most frequently in East Asian populations 2. TGFBI corneal dystrophies are caused by a point mutation, or insertion or deletion of some frames, in TGFBI, which is located on chromosome 5q31 1,2. Various mutations in TGFBI cause corneal opacities with different phenotypes, such as granular corneal dystrophy (GCD), lattice corneal dystrophy (LCD), Reis-Bücklers corneal dystrophy (RBCD), and Thiel-Behnke corneal dystrophy (TBCD) 3. There are genotype-phenotype correlations in TGFBI corneal dystrophy; for example, the R124H mutation causes GCD type2 (GCD2), and the R124C mutation causes LCD type1 (LCD1) 2. Even though we know that the corneal opacities are caused by accumulated TGFBI protein (TGFBIp) 4 , the mechanism or pathophysiology is not yet clearly understood 5. Animal models help elucidate the pathophysiology of corneal dystrophies. Previously, Bustamante et al. used a lentiviral vector to generate knock-in mice overexpressing the human R555W mutation, which causes GCD type1 (GCD1) 6. However, R555W mutant mice did not show any corneal phenotype according to this report 6. Tgfbi-knockout mice also failed to show corneal abnormalities even after systemic depletion of Tgfbi expression 7. Recently, Yamazoe et al. established an R124H mutant transgenic mouse model showing corneal opacities 8. Ho...

The purpose of this study was to investigate the association between fundus autofluorescence (FAF) and visual field (VF) sensitivities in eyes with retinitis pigmentosa (RP). We also investigated the model we developed to predict VF sensitivity using the FAF ring and its prediction accuracy. METHODS. The training dataset consisted of 51 eyes of 28 patients, and the testing dataset consisted of 42 eyes of 25 patients with RP. VF and FAF measurements were conducted using the Humphrey Field Analyzer (HFA) 10-2 test and Optos. The HFA 10-2 test was divided into three sectors according to the association with the FAF (IN, ON, and OUT). Moreover, concentric curves were drawn at 1-degree intervals outside the FAF ring and OUT was divided into six sectors (from OUT1 to OUT6 toward the periphery). Finally, the total deviation (TD) value was predicted using age and visual acuity (VA) in the whole field, and each of the eight sectors was compared. RESULTS. The TD value decreased significantly from IN, ON, and then toward OUT6. The absolute prediction error with the FAF ring (average, 7.6 dB) was significantly smaller than that without the FAF ring (average, 8.7 dB). The absolute prediction error with the FAF ring was significantly smaller in the central areas (IN, 4.4 dB and ON, 5.3 dB) than those in the peripheral areas (OUT1-6, 6.8-9.1 dB). CONCLUSIONS. VF sensitivity decreases in association with the FAF ring. We developed a model to predict 10-2 VF sensitivity values using the FAF ring, which enabled us to predict 10-2 TD values.