Efficient and sustained transgene expression in human corneal cells mediated by a lentiviral vector

The development of vectors and techniques able to transfer potentially ther apeutic genetic information to corneal tissues efficiently may have broad c linical applications. Although a variety of vectors have been tested for th eir ability to transduce corneal tissue, these systems have been ineffectiv e at transducing all cell types or have been associated with a relatively s hort duration of transgene expression. Towards the implementation of effici ent, long-term transgene expression in all corneal cell types, we have stud ied the ability of a recombinant lentiviral vector containing the enhanced green fluorescent protein (eGFP), to mediate gene transfer into human corne al tissue in vitro and in situ. Human primary keratocytes, cultured in vitr o, were efficiently transduced by a lentiviral Vector as determined by fluo rescent-activated cell sorting (FAGS) and by fluorescent microscopy. Transd uction efficiency was found to be dependent upon multiplicity of infection (MOI); 92% of keratocytes were transduced at an MOI of 1000. The proportion of eGFP-positive cells remained unchanged throughout continuous culture fo r 60 days, indicating stable expression and a lack of selective pressure fo r or against transduced cells Human corneal tissue, obtained at the time of penetrating keratoplasty, demonstrated efficient in situ transduction with this vector. Endothelial cells, epithelial cells and stromal keratocytes a t the exposed cut edge of the corneal tissue in situ demonstrated eGFP expr ession. Underlying stromal cells not in direct contact with vector-containi ng media, were not transduced, implying that virus-cell contact is required far transduction. Transduced corneal tissues expressed eGFP in situ for th e life of the corneal button in extended organ culture (60 days). These res ults imply that lentiviral vectors may prove to be useful tools, able to tr ansduce corneal tissue efficiently, and that transgene expression is tempor ally stable.