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Développement et validation d’un modèle cellulaire de kératopathie associée à l’aniridie

Abstract : Aniridia is a rare panocular disease mainly due to PAX6 heterozygous mutations. PAX6 is the master gene of the eye development and it controls also the corneal homeostasis maintenance. Aniridia is characterized by an iris hypo/aplasia, retina and lens defects. All the aniridia patients will also develop an aniridia-related keratopathy (ARK) leading to a progressive corneal opacification. ARK is due to a limbal stem cell deficiency and alterations of corneal epithelium and stroma functions. Unfortunately, there is currently no efficient treatment to relief the patients and no cellular model for this pathology. To remedy these lacks, CRISPR/Cas9 system was used to insert a nonsense mutation into PAX6 gene of immortalized limbal epithelial cells. The mutated cells produce less PAX6 than the wild-type and PAX6 targets gene expression was modulated. They also display a marked slow-down proliferation, clonogenicity, migration and an enhanced adhesion. Moreover, we have shown that addition of recombinant PAX6 protein fused to a cell penetrating peptide to the culture medium was able to activate the endogenous PAX6 expression and to rescue some phenotypic defects of the mutated cells. Therefore, it validates the PAX6 haploinsufficiency model and suggests that PAX6 could be involved in all the rescued functions. The mutated cells can now be used to screen potential therapeutic tools for ARK and for other defects due to low levels of PAX6.
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Lauriane Roux. Développement et validation d’un modèle cellulaire de kératopathie associée à l’aniridie. Médecine humaine et pathologie. Université Sorbonne Paris Cité, 2018. Français. ⟨NNT : 2018USPCC276⟩. ⟨tel-02886220⟩

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