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| Status |
Public on Oct 22, 2019 |
| Title |
Modeling of aniridia-related keratopathy by CRISPR/Cas9 genome editing of human limbal epithelial cells and rescue by recombinant PAX6 protein |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
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.
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| Overall design |
Genome editing of aniridia-related keratopathy and rescue. Gene expression comparison between normal and genome-edited PAX6 mutation containing T-limba cells
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| Contributor(s) |
Roux LN, Petit I, Domart R, Concordet J, Qu J, Zhou H, Joliot A, Ferrigno O, Aberdam D |
| Citation(s) |
29808941 |
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| Submission date |
Oct 21, 2019 |
| Last update date |
Jun 14, 2022 |
| Contact name |
Jo Huiqing Zhou |
| E-mail(s) |
jo.zhou@radboudumc.nl
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| Organization name |
Radboud University
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| Street address |
Geert Grooteplein 26/28
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| City |
Nijmegen |
| ZIP/Postal code |
6525GA |
| Country |
Netherlands |
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| Platforms (1) |
| GPL16791 |
Illumina HiSeq 2500 (Homo sapiens) |
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| Samples (4)
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| Relations |
| BioProject |
PRJNA578775 |
| SRA |
SRP226540 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE139221_CountData.txt.gz |
366.3 Kb |
(ftp)(http) |
TXT |
| GSE139221_DEG_TableX.xlsx |
45.2 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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