Methods to Transduce Primary Cultured Human Limbal Epithelial Cells

Abstract

Abstract: : Purpose: To develop targeted gene therapy vectors to specifically modify limbal stem cells to treat ocular surface diseases. Limbal stem cells play a pivotal role in maintaining ocular surface function and repairing damage to the corneal epithelium. Recent work suggests that putative stem cells in the basal epithelium of the limbus express p63, ABCG2, α9 integrin, ß1 integrin, and the epidermal growth factor receptor (EGFR). Methods: To determine the feasibility of targeting gene therapy vectors to these putative stem cells, we have tested the ability of untargeted and targeted adenoviral (Ad) vectors to genetically modify human limbal cells in vitro. Human corneal epithelial cells expanded by limbal explant culture or limbal single cell suspension culture were exposed to Ad5 vectors expressing the green fluorescent protein (GFP). EGF conjugated to Ad using bifunctional polyethylene glycol (PEG) molecules to specifically target EGFR positive cells was also evaluated. The efficiency was compared with liposome transfection on these cells. Results: Unmodified Ad5 vectors transduced approximately 50% of K3 keratin–positive primary cultured limbal epithelial cells, while Ad5 conjugated to EGF transduced 7% of these epithelial cells. In comparison, liposomes transfected 21% of these cells. Conclusions: Adenovirus is the most efficient mean to transduce cultured human limbal epithelial cells. Additional work will be required to determine if PEG or biotin–mediated targeting of Ad vectors will enhance transduction of limbal stem cells in culture and in vivo. CR: N. Support: NIH Grants, EY014553 (DQL) and EY11915 (SCP), an unrestricted Grant from Research to Prevent Blindness, the Oshman Foundation and the William Stamps Farish Fund.

Keywords: cornea: epithelium • gene transfer/gene therapy • adenovirus