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Modeling of aniridia-related keratopathy by CRISPR/Cas9 genome editing of human limbal epithelial cells and rescue by recombinant PAX6 protein

ABSTRACT: Heterozygous PAX6 gene mutations leading to haploinsufficiency are the main cause of congenital aniridia, a rare and progressive panocular disease characterized by reduced visual acuity. Up to 90% of patients suffer from aniridia-related keratopathy (ARK), caused by a combination of factors including limbal epithelial stem-cell (LSC) deficiency, impaired healing response and abnormal differentiation. It usually begins in the first decade of life, resulting in recurrent corneal erosions, sub-epithelial fibrosis and corneal opacification. Unfortunately, there are currently no efficient treatments available for these patients and no in vitro model for this pathology. We used CRISPR/Cas9 technology to introduce into the PAX6 gene of LSCs a heterozygous nonsense mutation found in ARK patients. Nine clones carrying a p.E109X mutation on one allele were obtained with no off-target mutations. Compared to the parental WT-LSCs, heterozygous mutant LSCs displayed reduced expression of PAX6 and marked slow-down of cell proliferation, migration and detachment. Remarkably, addition to the culture medium of recombinant PAX6 protein fused to a cell penetrating peptide was able to activate the endogenous PAX6 gene and to rescue phenotypic defects of mutant LSCs, suggesting that administration of such recombinant PAX6 protein could be a promising therapeutic approach of congenital aniridia. More generally, our results demonstrate that introduction of disease mutations into LSCs by CRISPR/Cas9 genome editing allows creating relevant cellular models of ocular disease that should greatly facilitate screening of novel therapeutic approaches.

ORGANISM(S): Homo sapiens

PROVIDER: GSE139221 | GEO | 2019/10/22

REPOSITORIES: GEO

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Project description:PAX6-related Aniridia is a sight-threatening disease due to progression of secondary glaucoma and aniridia associated keratopathy (AAK). Changes or loss of limbal epithelial progenitors causes the epithelial surfaces defects. We analyzed how PAX6 contribute to this with a two-step approach. 1) mRNA Sequencing of limbal epithelial cells isolated from controls and aniridia patients. 2) confirming the bioinformatical and literature-based result list on a siRNA based primary aniridia cell model (PAX6- knockdown). With this approach genes which are directly influenced by PAX6 should be distinguishable from genes secondary affected by AAK disease state. Therefore epithelial cells were isolated from the limbus region of two patients with aniridia and cultured in KSFM medium. Normal cells were obtained from limbus region of cadaveric control patients. For the siRNA based anridia cell model cells were transfected with lipofectamine and 5 nM siRNA against PAX6 or scrambled control. All cells were lysed to obtain DNA, RNA and protein. Reduction of PAX6 protein was controlled by Western Blot. Aniridia and Control Poly-A enriched RNA-librarys were subjected to Next Generation Sequencing. The differential analysis was a combination of quantification with RSEM and differential tests with edgeR. Gene lists were filtered by comparing to NCBI GEO Datasets, annotation with DAVID and manually annotation using literature search. For the resulting filtered gene list qPCR primer were ordered and postulated changes were verified with qPCR on siRNA based aniridia- cell model (Namely: TP63, ABCG2, ADH7, ALDH1A1, PITX1, DKK1, DSG1, K12, K3, K13, SPINK6, SPINK7, CTSV, SPERPINB1). We could identify genes which might be regulated by PAX6. We could show that SPINK7 mRNA coding for a protease inhibitor is downregulated in patients as well as in our primary aniridia cell model. ALDH1A1 and AHD7 mRNAs were reduced in limbal epithelial cells of aniridia patients (AN-LEC). Both transcripts were downregulated upon PAX6 knockdown in our cell model.Our siRNA-based aniridia cell model is a tool to proof identified PAX6 affected genes which might drive AAK pathogenesis. We could demonstrate, that this is the fact for transcripts coding for retinol converting enzymes. This provides evidence that PAX6 might drive corneal epithelial differentiation process by direct or indirect controlling retinoic acid signaling processes.

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Modeling of aniridia-related keratopathy by CRISPR/Cas9 genome editing of human limbal epithelial cells and rescue by recombinant PAX6 protein

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