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

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. Overall design: Genome editing of aniridia-related keratopathy and rescue. Gene expression comparison between normal and genome-edited PAX6 mutation containing T-limba cells

导致单倍体剂量不足（haploinsufficiency）的杂合PAX6基因突变，是先天性无虹膜症的主要致病原因。该病属于罕见且呈进行性发展的全眼疾病，以视力下降为核心临床特征。
高达90%的患者会并发无虹膜相关角膜病（aniridia-related keratopathy, ARK），其发病由多种因素共同介导，包括角膜缘上皮干细胞（limbal epithelial stem-cell, LSC）缺乏、愈合反应受损以及分化异常。该病通常始发于患者生命最初十年，可引发复发性角膜糜烂、上皮下纤维化与角膜混浊。
遗憾的是，目前针对此类患者尚无有效的治疗方案，也缺乏该病理状态的体外研究模型。本研究借助CRISPR/Cas9技术，在LSCs的PAX6基因中引入了ARK患者体内检出的杂合无义突变，最终获得9个在单一等位基因上携带p.E109X突变的克隆，且未检测到脱靶突变。
与亲本野生型LSCs相比，杂合突变型LSCs的PAX6表达水平显著降低，细胞增殖、迁移与脱落过程均出现明显减缓。值得注意的是，向培养基中添加融合了细胞穿透肽的重组PAX6蛋白，可激活内源性PAX6基因并修复突变型LSCs的表型缺陷，这提示此类重组PAX6蛋白的给药策略有望成为先天性无虹膜症的潜在治疗手段。
更广泛而言，本研究结果证实，通过CRISPR/Cas9基因组编辑技术在LSCs中引入疾病相关突变，可构建具有病理相关性的眼部疾病细胞模型，这将极大推动新型治疗策略的筛选工作。
总体实验设计：无虹膜相关角膜病的基因组编辑与表型挽救。对正常细胞与经基因组编辑引入PAX6突变的T-角膜缘细胞进行基因表达比较。