Postnatal Induction of PAX6 to Alleviate Aniridia Manifestations

Abstract

Purpose : Aniridia is a congenital panocular condition caused by a mutation in one copy of the PAX6 gene. Although the ocular problems begin in utero and are present at birth, we hypothesized that early postnatal stimulation of the normal copy of PAX6 may prevent or reduce further progression of the disease.

Methods : By a chemical screen, we identified compound X (patent pending, to be disclosed at presentation) and evaluated its effect on PAX6 expression by RT-PCR and western blot in human corneal epithelial cells (HCEC). In vivo, we treated a mouse model of aniridia (Pax6^Sey-Neu/+) with oral or topical compound X or vehicle, from postnatal day (P)5 to P30. Wild type (WT) littermates were used as normal reference. OCT and slit lamp images were used to compare the corneal epithelial/stromal thickness, clarity and scarring. H&E and immunostaining with PAX6 and Cytokeratin (CK) 12 were used to compare the morphology, protein expression and differentiation. ERG was conducted to assess retinal function.

Results : Compound X increased PAX6 mRNA and protein expressions in HCEC (2.7±0.2 & 3.0±0.3 folds). P30 PAX6 staining confirmed increase in PAX6 protein expression in corneas with both oral (2.3±0.2 folds) and topical (2.5±0.1 folds) methods (P<0.001). Corneal scarring was less in both oral (2.1±0.4% vs. 11.2±4.0%, P<0.001) and topical (2.3±0.6% vs. 10.6±2.5%, P<0.001) groups compared to vehicle controls. Corneal epithelial thickness at P30 was significantly greater than control in both oral (24.7±4.1µm vs. 15.2±1.5µm, P<0.001) and topical (25.5±4.7µm vs. 14.0±1.5µm, P<0.001) groups (WT: 50.5±1.1µm). Corneal stromal thickness at P30 was significantly less than control in both oral (80.2±25.8µm vs. 160.3±11.8µm, P<0.001) and topical (106.9±31.7µm vs. 173.6±18.1µm, P<0.001) groups (WT: 75.8±3.6µm). Both treatment groups showed a more similar histological pattern to normal. P30 CK12 immunostaining and P90 corneal whole mount staining showed better corneal epithelial differentiation in both treatment groups compared to controls (WT CK12 intensity: 100%; oral: 64±3% vs. 31±6%, P<0.01; topical: 69±32% vs. 34±13%, P<0.05). P90 ERG showed increased a and b waves in topical group compared to control (P<0.05).

Conclusions : Early postnatal induction of PAX6 can alleviate several manifestations of aniridia. This approach can potentially be used to partially treat and/or prevent aniridia, particularly the corneal disease which is known to be progressive.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.