RETROVIRAL VECTOR-MEDIATED GENE-TRANSFER INTO KERATOCYTES IN-VITRO AND IN-VIVO

PURPOSE: To determine the potential of somatic gene transfer as a tech nique for modulating corneal wound healing after superficial keratecto my, METHODS: The transduction of human and rabbit keratocytes with bet a-galactosidase and herpes simplex virus thymidine kinase genes was pe rformed. In vitro, human and rabbit keratocytes were transduced with r etroviral vectors bearing beta-galactosidase or HStk (herpes simplex v irus thymidine kinase) genes. In vivo, rabbit keratocytes were transdu ced by topical application of vector supernatant after a superficial k eratectomy. In vitro and in vivo, expression of the beta-galactosidase gene was examined with histochemical staining. In vitro, ganciclovir cytotoxicity in HStk gene-transduced keratocytes and bystander effect in co cultures of HStk(+) and HStk(-) keratocytes were measured by det ermining the degree of confluency of cells in 6-well plates after 10 d ays of incubation, Corneal haze in rabbits was measured after transduc tion with Hstk and subsequent treatment with topical ganciclovir, RESU LTS: In vitro, both human and rabbit keratocytes were transduced succe ssfully with both beta-galactosidase and HStk genes. Transduction effi ciency was greater with human (22%) than with rabbit (16%) cells, and both HStk-transduced cell lines showed dose-dependent ganciclovir cyto toxicity and a significant bystander effect, In vivo, expression of be ta-galactosidase within vimentin-positive corneal stromal cells confir med transduction of keratocytes in the rabbit after superficial stroma l keratectomy with an efficiency of 25% to 40%. Postoperative applicat ion of topical ganciclovir reduced corneal stromal haze in rabbits. CO NCLUSIONS: The ability to genetically transduce stromal keratocytes pr ovides a new strategy for understanding the important cellular and mol ecular events that influence corneal wound healing, thus offering a po tential approach to decrease or prevent corneal haze and scarring afte r superficial keratectomy, (Am J Ophthalmol 1998;126: 630-639, (C) 199 8 by Elsevier Science Inc, All rights reserved.).