Postnatal manipulation of Pax6 dosage reverses congenital tissue malformation defects

Abstract

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.

Figure 1. Postnatal treatment of Pax6 mice with ataluren.

(A) Effect of systemic ataluren treatment on mice with the Pax6^Sey+/– phenotype. The black arrowhead indicates the lenticular stalk; the black arrow indicates the cornea; and the asterisk indicates the ciliary margin. WT, Pax6^+/+; Mt, Pax6^Sey/+; L, lens; r, retina. Original magnification, ×5. (B) Histological comparison of 1% ataluren suspension and the START formulation instilled topically in Pax6^Sey+/– eyes. Original magnification, ×5. (C) PAX6 protein measurements in the retinas and corneal epithelia from Pax6^+/+ (WT), Pax6^Sey/+ (Sey), and Pax6^Sey–1Neu (Neu) mice. Black bars depict wild-type mice; white bars depict untreated mice; checkered bars depict mice after START therapy. *P < 0.001, n = 6. (D) Box-and-whisker plots comparing maximum spatial frequency threshold of topical ataluren (At.) in H₂0 and the START formulation. Box-and-whisker plots were prepared showing the 5% and 95% quantiles (whiskers), 25% and 75% quartiles (box), and the median marked by a horizontal line.

Figure 2. Retina and corneal histology in Pax6^Sey+/– mice.

(A) Representative images at P14 showing that the cornea of the ataluren-treated Pax6^Sey+/– mice is thicker than wild-type (Pax6^+/+) cornea (140 μm versus 65 μm, n = 6). Scale bar: 100 μm. (B) Higher magnification of corneal tissue at P14 and P60. The stroma (green bar) is thicker and the epithelium (black bar) is abnormally thin with systemic (SYS) treatment. Arrows indicate basal cells. Scale bar: 10 μm. (C) Topical (TOP) treatment with the START formulation at P60 in Pax6^Sey+/– eyes. Untreated Pax6^Sey+/– control (CON) corneal epithelium remains thin at P60. Scale bar: 10 μm. (D) Epithelial stratification of the cornea. (E) Retinal sections from wild-type, systemically treated Pax6^Sey+/– mice and untreated mice, showing the photoreceptor inner segments (IS) and outer segments (OS) are shorter in the treated mice (n = 6). The nuclei in the outer nuclear layer (ONL) are more densely packed in the treated mice compared with those in the wild-type mice. Scale bar: 50 μm. All the retinal layers in the untreated mice are thinner than normal. INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

Figure 3. Electroretinographic analyses in response to light stimulation.

(A) Restoration of light sensitivity in Pax6^Sey+/– eyes measured at P60. Trace 1 shows wild-type mice with systemic ataluren. Trace 2 shows untreated Pax6^Sey+/– mouse responses. Trace 3 shows Pax6^Sey+/– mice with systemic ataluren. Trace 4 shows Pax6^Sey+/– mice with topical 1% ataluren suspension. Trace 5 shows Pax6^Sey+/– mice with topical START formulation. a, a-wave maximum from baseline; b, b-wave maximum from baseline. (B) Quantification of scotopic and photopic b-wave responses (n = 6). Significance determined by 1-way ANOVA with Tukey post-hoc tests *P < 0.05; **P < 0.01.