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Jack W Hickmott, Beatrice M Tam, Andrea J. Korecki, Siu Ling Lam, Orson L Moritz, Elizabeth M Simpson; Towards an rAAV PAX6-Gene Therapy for Aniridia. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1178.
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© 2017 Association for Research in Vision and Ophthalmology.
Aniridia is an autosomal dominant panocular disorder caused by mutations in PAX6 (Paired Box 6). People with aniridia are born with low vision which often progresses towards legal blindness in young adulthood, leaving a window for therapeutic intervention. Current treatments can delay vision loss, but do not prevent the blindness experienced by many people with aniridia. Gene therapy is a promising treatment platform that may delay, or even prevent, such vision loss. Here we tested rAAV (recombinant adeno-associated viruses) with specially designed PAX6-MiniPs (MiniPromoters; ≤4 kb promoters designed from human regulatory sequences) driving a 3xFLAG/PAX6 protein, in a mouse model of aniridia.
EmGFP (emerald GFP), PAX6, or 3xFLAG/PAX6 mRNA was injected into Xenopus laevis embryos, which were fixed and examined 14 days later. Additionally, EmGFP and 3xFLAG/PAX6 constructs, driven by PAX6 MiniPs, were cloned into a custom “plug-and-play” rAAV genome, and packaged into rAAV at the University of Pennsylvania vector core. Viruses were injected into wild-type and Sey (Pax6 mutant) mice. Eyes were longitudinally scored for corneal opacification and then harvested by enucleation, fixed in 4% paraformaldehyde, and weighed. Cryosections were stained with antibodies against EmGFP, Pax6, and FLAG tag for immunofluorescent analysis.
Injection of PAX6 and 3xFLAG/PAX6 mRNA into X. laevis embryos both drove ectopic eye formation in the resulting tadpoles, suggesting that the FLAG tag did not disrupt PAX6 function. In mice, wild-type eyes (21 ± 0.32 mg) were found to be significantly heavier (p < 0.001) than Sey eyes (16.06 ± 0.31 mg). Additionally, opacities were observed only in the Sey corneas. Using PAX6 MiniPs (Ple254 and Ple255) to drive expression in rAAV, EmGFP was detected in combinations of ganglion, amacrine, and horizontal cells, which all endogenously express Pax6. Ple254, which drove EmGFP expression in ganglion and amacrine cells, also drove 3xFLAG/PAX6 expression in the ganglion cell and the inner nuclear layers of wild-type and Sey retinas two months after injection.
rAAV is a platform that in combination with PAX6 MiniPs has the capacity to transduce the mouse retina and drive PAX6 expression in cells that endogenously express Pax6. Delivery and expression of a functional 3xFLAG/PAX6 construct in mouse eyes lays the groundwork for preclinical trials of a PAX6-gene therapy for aniridia in mice.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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