Towards Corneal Gene Therapy in the Aniridia Mouse Model Pax6^Sey/+

Abstract

Purpose : Aniridia is a rare autosomal dominant disorder caused by mutations in PAX6 (Paired Box 6). Although named for lack of iris, aniridia is a panocular disorder, with vision loss attributable to three main causes: 1) hypomorphic fovea, 2) progressive corneal clouding, and 3) progressive glaucoma. Current treatments may delay vision loss, but do not prevent the blindness experienced by most people with aniridia. Gene therapy is a promising treatment strategy that may delay, or even prevent, vision loss. Here we focus on gene therapy for the corneal pathology.

Methods : EmGFP (emerald GFP), PAX6, 3xFLAG/PAX6, and PAX6/3xFLAG mRNAs were injected into Xenopus laevis embryos, which were fixed and examined 14 days later. Additionally, EmGFP and 3xFLAG/PAX6 constructs, driven by smCBA (ubiquitous) or a human MiniPromoter (targeted to the cornea) were cloned into a custom rAAV genome, and packaged into rAAV9 at the University of Pennsylvania Vector Core. Viruses were introduced into wild-type and Pax6^Sey/+ mice using several different injections routes. Mouse eyes were longitudinally scored for corneal opacification and then harvested by enucleation, and fixed in 4% paraformaldehyde. Cryosections were stained with antibodies against EmGFP, PAX6, and FLAG tag for immunofluorescent analysis.

Results : Since human and mouse PAX6 are indistinguishable by immune detection, we required a tagged version of PAX6 that did not jeopardize PAX6 in vivo function. Thus, we tested PAX6 fused to either amino- or carboxyl-terminal 3xFLAG for the ability to generate ectopic eye structures in X. laevis tadpoles. PAX6 and 3xFLAG/PAX6 mRNA drove similar ectopic eye formation, suggesting that this amino-terminal 3xFLAG tag did not disrupt PAX6 function. Delivery of rAAV to the cornea was then explored using topical, intravitreal, intravenous, and subconjunctival routes with ssAAV9 smCBA-EmGFP-WPRE and ssAAV9 Ple253-EmGFP-WPRE. The MiniPromoter Ple253 was derived from the human PITX3 gene and had previously shown, using a different methodology, to express in the corneal stroma (de Leeuw et al., 2016). In this study, stromal expression was achieved with both intravenous and subconjunctival routes.

Conclusions : These results have laid the foundation for rAAV9 delivery of in vivo functional 3xFLAG/PAX6, with either a ubiquitous or cell-type restricted promoter, by either intravenous or subconjunctival routes, to treat corneal pathology in Pax6^Sey/+ mice.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.