A New in Vitro Model of GDLD by Knocking Out and Its Paralogous Gene in Human Corneal Epithelial Cells.

PURPOSE

Gelatinous drop-like corneal dystrophy (GDLD) is a rare autosomal recessive corneal dystrophy that causes severe vision loss. Because of its poor prognosis, there is a demand for novel treatments for GDLD. Here, we establish a new in vitro disease model of GDLD based on immortalized human corneal epithelial (HCE-T) cells.

METHODS

By using transcription activator-like effector nuclease plasmids, tumor-associated calcium signal transducer 2 () and its paralogous gene, epithelial cell adhesion molecule (), were knocked out in HCE-T cells. Fluorescence-activated cell sorting was performed to obtain cells in which both and were knocked out (DKO cells). In DKO cells, the expression levels and subcellular localizations of claudin (CLDN) 1, 4, and 7, and ZO-1 were investigated, along with epithelial barrier function. By using DKO cells, the feasibility of gene therapy for GDLD was also investigated.

RESULTS

DKO cells exhibited decreased expression and aberrant subcellular localization of CLDN1 and CLDN7 proteins, as well as decreased epithelial barrier function. Transduction of the gene into DKO cells nearly normalized expression levels and subcellular localization of CLDN1 and CLDN7 proteins, while significantly increasing epithelial barrier function.

CONCLUSIONS

We established an in vitro disease model of GDLD by knocking out and its paralogous gene, in HCE-T cells. This cell line accurately reflected pathological aspects of GDLD.

TRANSLATIONAL RELEVANCE

We expect that the cell line will be useful to elucidate the pathogenesis of GDLD and develop novel treatments for GDLD.