Modulation of Contact Inhibition by ZO-1/ZONAB Gene Transfer—A New Strategy to Increase the Endothelial Cell Density of Corneal Grafts

Purpose Endothelial cell density (ECD) is the principal factor determining the success of corneal transplants. Here we explored a strategy to increase corneal ECD in human explants via modulation of the ZO-1/ZONAB pathway. In multiple cell types, ZO-1 maintains G1 cell cycle arrest via cytoplasmic sequestration of the mitosis-inducing transcription factor ZONAB. In this study, we assessed the effects of lentiviral vector-mediated downregulation of ZO-1 or overexpression of ZONAB upon ECD and the integrity of the endothelial monolayer. Methods HIV-based lentiviral vectors were used to deliver either constitutively expressed ZONAB (LNT-ZONAB), or a small hairpin RNA targeting ZO-1 (LNT-shZO1). Human corneal specimens were bisected and each half was exposed to either treatment or control vector. After 1 week in ex vivo culture, effects were assessed by quantitative RT-PCR, immunohistochemistry, and ECD assessment. Results LNT-shZO1 achieved an ∼45% knockdown of ZO-1 mRNA in corneal endothelial cells cultured ex vivo, reduced ZO-1 staining, and did not affect morphologic endothelial monolayer integrity. The proliferative effect of LNT-shZO1 correlated with control ECD but not with donor age. Within a low-ECD cohort an ∼30% increase in ECD was observed. LNT-ZONAB achieved a >200-fold overexpression of ZONAB mRNA, which led to an ∼25% increase in ECD. Conclusions ZO-1 downregulation or ZONAB upregulation increases corneal ECD via interference with contact inhibition and cell cycle control. With further development, such approaches might provide a means for improving ECD in donor corneas before transplantation.