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Aniridia is a rare congenital disorder in which there is a variable degree of hypoplasia or the absence of iris tissue associated with multiple other ocular changes, some present from birth and some arising progressively over time. Most cases are associated with dominantly inherited mutations or deletions of the PAX6 gene. This article will review the clinical manifestations, the molecular basis including genotype-phenotype correlations, diagnostic approaches and management of aniridia.

Aniridia is a congenital and progressive panocular condition with poor visual prognosis that is associated with brain, olfactory, and pancreatic abnormalities. Development of aniridia is linked with nonsense mutations that result in paired box 6 (PAX6) haploinsufficiency. Here, we used a mouse model of aniridia to test the hypothesis that manipulation of Pax6 dosage through a mutation-independent nonsense mutation suppression strategy would limit progressive, postnatal damage in the eye. We focused on the nonsense suppression drugs 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid (ataluren) and gentamicin. Remarkably, we demonstrated that nonsense suppression not only inhibited disease progression but also stably reversed corneal, lens, and retinal malformation defects and restored electrical and behavioral responses of the retina. The most successful results were achieved through topical application of the drug formulation START (0.9% sodium chloride, 1% Tween 80, 1% powdered ataluren, 1% carboxymethylcellulose), which was designed to enhance particle dispersion and to increase suspension viscosity. These observations suggest that the eye retains marked developmental plasticity into the postnatal period and remains sensitive to molecular remodeling. Furthermore, these data indicate that other neurological developmental anomalies associated with dosage-sensitive genetic mutations may be reversible through nonsense suppression therapeutics.

Aniridia is a rare congenital condition of abnormal eye development arising principally from heterozygous mutation of the PAX6 gene. Among the multiple complications arising in the eye, aniridia-associated keratopathy (AAK) is a severe vision-impairing condition of the cornea associated with a progressive limbal stem cell deficiency that lacks suitable treatment options. Current mouse models of aniridia do not accurately represent the onset and progression dynamics of human AAK, hindering therapy development. Here, we performed deep phenotyping of a haploinsufficient Pax6+/- small-eye (Sey) mouse model on the 129S1/SvImJ background, which exhibits key features of mild presentation at birth and progressive AAK with aging, mimicking human disease. The model exhibits a slowly progressing AAK phenotype and provides insights into the disease, including disturbed basal epithelial cell organization, function, and marker expression; persistent postnatal lymphangiogenesis; disrupted corneal innervation patterns; and persisting yet altered limbal stem cell marker expression with age. The model recapitulates many of the known features of human disease, enabling investigation of underlying disease mechanisms and, importantly, access to a well-defined temporal window for evaluating future therapeutics.

Patients with large iris defects not only suffer from functional disadvantages but also from aesthetic limitations. The aim of this study was to evaluate the aesthetic outcome of iris reconstruction using an artificial iris (AI). In this study, 82 eyes of 79 consecutive patients with mostly traumatic partial or total aniridia that underwent iris reconstruction surgery using a custom-made silicone AI (HumanOptics, Erlangen, Germany). Pre- and postoperative photographs of 66 patients were analysed subjectively and objectively. Subjective evaluation was based questionnaires. Objective evaluation included measurement of pupil centration and iris colour analysis. Averaged hues from iris areas were transferred to numerical values using the LAB-colour-system. Single parameters and overall difference value (ΔE) were compared between AI and remaining iris (RI), as well as AI and fellow eye iris (FI). Patients, eye doctors and laymen rated the overall aesthetic outcome with 8.9 ±1.4, 7.7 ±1.1 and 7.3 ±1.1 out of 10 points, respectively. Mean AI decentration was 0.35 ±0.24 mm. Better pupil centration correlated with a higher overall score for aesthetic outcome (p<0.05). The AI was on average 4.65 ±10 points brighter than RI and FI. Aniridia treatment using a custom-made artificial iris prosthesis offers a good aesthetic outcome. Pupil centration was a key factor that correlated with the amount of aesthetic satisfaction. The AI was on average slightly brighter than the RI and FI.

Haploinsufficiency of the PAX6 gene is the primary pathogenic mechanism underlying classical congenital aniridia. Notably, at least 50% of patients with this condition develop glaucoma. Prostaglandin analogues, such as travoprost, are widely used to lower intraocular pressure in this patient population. At the same time, limbal epithelial cells (LECs) are believed to play a key role in the development and progression of aniridia-associated keratopathy (AAK). Therefore, the aim of this study was to investigate the effects of travoprost on cell viability, proliferation, migration, and the expression of key genes and proteins in both normal and PAX6-knockdown LECs. Primary human LECs were isolated from seven corneal donors. To simulate the haploinsufficient state characteristic of congenital aniridia, a PAX6-knockdown model, using siRNA was employed. LECs were treated with 0.039-40 μg/mL travoprost concentration for 20 minutes. Cell viability was assessed using the XTT assay, while cell proliferation was evaluated by the BrdU assay. Based on XTT results, 0.156 and 0.313 μg/mL travoprost were selected for further measurements in both LECs and PAX6-knockdown LECs. A scratch assay was conducted to measure cell migration. The expression levels of PAX6, FOSL2, MAPKs, inflammatory markers, caspase-3, and MMP9 were analyzed at both the gene and protein levels using qRT-PCR, Western blot, and ELISA. Travoprost significantly reduced LEC viability at 0.156 μg/mL (p = 0.028), while only higher concentrations (20 and 40 μg/mL) inhibited significantly LEC proliferation (p ≤ 0.004). PAX6-knockdown LECs exhibited reduced migration compared to control cells (p ≤ 0.046); however, treatment with 0.313 μg/mL travoprost significantly enhanced their migration (p = 0.047), accompanied by upregulation of JNK1/2 protein (p = 0.039) and MMP9 mRNA and protein levels (p = 0.021, p = 0.027). PAX6 knockdown led to suppression of inflammation-related genes (p ≤ 0.031) and travoprost did not exacerbate inflammatory responses (p ≥ 0.155). Additionally, 0.313 μg/mL travoprost significantly increased caspase-3 protein levels in PAX6-knockdown LECs (p = 0.044). Travoprost, at specific concentrations, can reduce the viability and proliferation of limbal epithelial cells. At 0.313 μg/mL, it significantly upregulates JNK1/2 and MMP9 expression, thereby enhancing the migratory capacity of PAX6-knockdown LECs. These findings may offer valuable insights for the selection of antiglaucomatous medications in patients with congenital aniridia.

Progressive aniridia associated keratopathy is worsening visual acuity of congenital aniridia subjects lifelong. Restoration of PAX6 expression in PAX6 haploinsufficient limbal epithelial cells could be one therapeutic option. In a previous study using aniridia-like CRISPR/Cas9 genome-edited corneal epithelial cells, the antipsychotic drugs duloxetine and ritanserin increased PAX6 mRNA and protein expression. Our purpose was to investigate the effect of duloxetine and ritanserin on cultured primary limbal epithelial cells (pLECs) without and with PAX6 knockdown. pLECs were isolated from 11 aniridia patients and corneoscleral rims of 8 healthy human donors and were treated with 5 µM duloxetine or ritanserin for 24 hours. In addition, pLECs were transfected with small interfering RNA (siRNA) (PAX6 knockdown) in the siRNA-based aniridia cell model and were also treated by 5 µM duloxetine or ritanserin for 24 hours. Gene and protein expression were analyzed using qPCR and Western blot. In both primary aniridia limbal epithelial cells and the siRNA-based aniridia cell model, the expression of PAX6 at the transcriptional or translational level did not show significant changes through duloxetine or ritanserin treatment (p > 0.5). The target genes of PAX6 such as KRT3, KRT12, DSG1 , ALDH1A1, ADH7 , FABP5 , ABCG2 also did not change significantly (p ≥ 0.2). Our study shows that primary cultures of limbal epithelial cells from both aniridia patients and healthy donors were unresponsive to drug treatment. Therefore, our data suggest that different aniridia cell models or cell culture conditions exhibit varying responses to duloxetine and ritanserin. The use of in vivo models could further enhance our understanding of duloxetine and ritanserin treatment in aniridia-associated keratopathy.

To study structural changes in naïve and surgically treated corneas of aniridia patients with advanced aniridia-related keratopathy (ARK). Two naïve corneal buttons from patients with advanced ARK submitted to penetrating keratoplasty for the first time, one corneal button from an ARK patient that had undergone a keratolimbal allograft (KLAL), two corneal buttons from ARK patients who had previously undergone centered or decentered transplantation and were now retransplanted and two adult healthy donor control corneas were processed for immunohistochemistry. Antibodies against extracellular matrix components in the stroma and in the epithelial basement membrane (collagen I and IV, collagen receptor α11 integrin and laminin α3 chain), markers of fibrosis, wound healing and vascularization (fibronectin, tenascin-C, vimentin, α-SMA and caveolin-1), cell division (Ki-67) and macrophages (CD68) were used. Naïve ARK, KLAL ARK corneas and transplanted corneal buttons presented similar histopathological changes with irregular epithelium and disruption or absence of epithelial basal membrane. There was a loss of the orderly pattern of collagen lamellae and absence of collagen I in all ARK corneas. Vascularization was revealed by the presence of caveolin-1 and collagen IV in the pannus of all ARK aniridia corneas. The changes observed in decentered and centered transplants were analogous. Given the similar pathological features of all cases, conditions inherent to the host seem to play an important role on the pathophysiology of the ARK in the long run.

In congenital aniridia, not only limbal epithelial cells but also limbal stromal cells may contribute to the development of aniridia associated keratopathy (AAK). Secondary glaucoma affects 50-75% of patients with congenital aniridia, and prostaglandin analogs are commonly used for conservative treatment. This study aimed to explore the effect of travoprost on corneal limbal stromal cells from healthy (LSCs) and congenital aniridia subjects (AN-LSCs), in vitro. Cells were extracted from aniridia (AN-LSCs) (n=7) and healthy donors (LSCs) (n=7). In culture, the cells were treated with travoprost at concentrations ranging from 0.039-40 μg/mL for 20 minutes. Cell viability, proliferation and migration were determined to assess the effect of travoprost on AN-LSCs and LSCs. Analysis of inflammation-, retinoic acid signaling-, and apoptosis-related genes and proteins was performed using qPCR, Western blot, and ELISA. One-way ANOVA was used to analyze cell viability and proliferation. The Mann-Whitney test was applied to compare between-group differences, while the Friedman test was used to assess within-group differences. Both in LSCs and AN-LSCs, travoprost treatment at 0.078 μg/mL and higher concentrations significantly reduced cell viability (p≤0.033; p<0.001) and proliferation decreased both in LSCs and AN-LSCs at 40 μg/mL travoprost concentration (p=0.006; p=0.002). At 6 and 12 hours, 0.313 μg/mL travoprost significantly increased the migration rate of AN-LSCs (p=0.021; p=0.021). AN-LSCs displayed lower PAX6 and JNK (MAPK8) mRNA (p<0.001) but higher MMP-3, MMP-9, ADH7, FABP5 and VEGFA mRNA levels (p≤0.037) than LSCs. PTGFR and JNK mRNA levels, MMP9 and ADH7 protein levels increased significantly in AN-LSCs after 0.313 μg/mL travoprost treatment (p≤0.039), while NF-κB and ADH7 protein levels decreased significantly in LSCs using 0.313 μg/mL travoprost (p=0.039; p<0.001). Travoprost may affect viability, proliferation, and migration of both LSCs and AN-LSCs, with AN-LSCs exhibiting greater sensitivity than LSCs. Additionally, travoprost may regulate MMP-9 expression in AN-LSCs via the JNK signaling pathway. Furthermore, in AN-LSCs, travoprost treatment does not lead to a decrease in NF-κB and ADH7 protein levels.

Aniridia is a panocular disorder that severely affects vision in early life. Most cases are caused by dominantly inherited mutations or deletions of the PAX6 gene, which encodes a transcription factor that is essential for the development of the eye and the central nervous system. In this issue of the JCI, Gregory-Evans and colleagues demonstrate that early postnatal topical administration of an ataluren-based formulation reverses congenital malformations in the postnatal mouse eye, providing evidence that manipulation of PAX6 after birth may lead to corrective tissue remodeling. These findings offer hope that ataluren administration could be a therapeutic paradigm applicable to some major congenital eye defects.