TACSTD2 Gene Transfer into the Epithelium Using Phosphate Groups Containing Hydrogel as the Device

Abstract

Purpose: : Gelatinous drop-like corneal degeneration (GDLD) is a refractory disease which generates sub-epithelial deposition of amyloid. While the TACSTD2 gene has been identified as the causative gene for the disease, due to the resistance to corneal transplant or similar treatments exhibited, some novel therapeutic methods are hoped for. In this study, we have investigated the transfer of the TACSTD2 gene into the corneal epithelial cells as encapsulated in a contact lens (CL) made of hydrogel which possesses phosphate groups as the carrier.

Methods: : CL with phosphate side-chain groups was synthesized and allowed to form a calcium-mediated complex with DNA (DNA-CL). Plasmid DNA was used for the DNA as obtained through implanting pLenti6.3_V5-TOPO vector with the TACSTD2 gene (TACSTD2-V5). DNA-CL was used to carry out gene transfer in vitro into SV40 immortalized human corneal epithelial cells (HCE-T). Then, Japanese white rabbits were wore with DNA-CL and observed for DNA expression in the corneal epithelial cell.

Results: : HCE-T revealed cytoplasm fluorescence in immunostaining while western blotting using anti-V5 immune body after immunoprecipiation recognized protein expression. Expression of the TACSTD2-V5 was also observed in the epithelial cells of the corneas that were fitted with DNA-CL.

Conclusions: : It has been suggested that the use of phosphate groups containing hydrogel as a gene transfer device could be applied to non-invasive novel therapeutic method for GDLD.

Keywords: gene transfer/gene therapy • degenerations/dystrophies • contact lens