SOX2 Activation Using CRISPR/dCas9 Promotes Wound Healing in Corneal Endothelial Cells

Abstract

Abstract There are no effective treatments for corneal endothelial diseases, except for corneal transplantation, as human corneal endothelial cells (hCECs) do not regenerate. The regeneration of hCECs could be induced through regulation of the expression of specific genes. In this study, we investigated whether the overexpression of sex-determining region Y-box 2 (SOX2) can regenerate hCECs in vivo and in vitro. SOX2 was activated using the clustered regularly interspaced short palindromic repeats (CRISPR)/deactivated CRISPR-associated protein 9 (dCas9) activation system. Genes were transfected into the corneal endothelium of Sprague-Dawley rats. Central corneal thickness and opacity were measured, and alizarin red S staining was performed. Corneal opacity and central corneal thickness were reduced in the SOX2 group compared with the control group. The density of CECs was higher in the SOX2 group compared with the control group. Additionally, hCECs were cultured and analyzed after overexpressing SOX2. Cell viability, proliferation rate, and the number of cells in S-phase were increased after SOX2 overexpression (p < .05). Cyclin-dependent kinase 1 and cyclin D1 were found to be overexpressed (p < .05). WNT signaling was repressed, and the AKT pathway was activated by SOX2 overexpression. Mitochondrial oxidative stress and energy production were increased by SOX2 overexpression (p < .05). In conclusion, SOX2 activation promotes wound healing and regeneration in CECs. SOX2 activation using the CRISPR/dCas9 system may thus be useful for the treatment of hCEC diseases.