Retroviral vector-mediated gene transfer into keratocytes in vitro and in vivo

Berthold Seitz, Luciane Moreira, Edwin Baktanian, Daniel Sanchez, Brad Gray, Erlinda M. Gordon, W. French Anderson, Peter J. McDonnell

Research output: Contribution to journalArticlepeer-review

Abstract

PURPOSE: To determine the potential of somatic gene transfer as a technique for modulating corneal wound healing after superficial keratectomy. METHODS: The transduction of human and rabbit keratocytes with β- galactosidase and herpes simplex virus thymidine kinase genes was performed. In vitro, human and rabbit keratocytes were transduced with retrovital vectors bearing β-galactosidase or HStk (herpes simplex virus thymidine kinase) genes. In vivo, rabbit keratocytes were transduced by topical application of vector supernatant after a superficial keratectomy. In vitro and in vivo, expression of the β-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 determining the degree of confluency of cells in 6-well plates after 10 days of incubation. Corneal haze in rabbits was measured after transduction with Hstk and subsequent treatment with topical ganciclovir. RESULTS: In vitro, both human and rabbit keratocytes were transduced successfully with both β-galactosidase and HStk genes. Transduction efficiency was greater with human (22%) than with rabbit (16%) cells, and both HStk-transduced cell lines showed dose-dependent ganciclovir cytotoxicity and a significant bystander effect. In vivo, expression of β- galactosidase within vimentin-positive corneal stromal cells confirmed transduction of keratocytes in the rabbit after superficial stromal keratectomy with an efficiency of 25% to 40%. Postoperative application of topical ganciclovir reduced corneal stromal haze in rabbits. CONCLUSIONS: The ability to genetically transduce stromal keratocytes provides a new strategy for understanding the important cellular and molecular events that influence corneal wound healing, thus offering a potential approach to decrease or prevent corneal haze and scarring after superficial keratectomy.

Original languageEnglish (US)
Pages (from-to)630-639
Number of pages10
JournalAmerican journal of ophthalmology
Volume126
Issue number5
DOIs
StatePublished - Nov 1998

ASJC Scopus subject areas

  • Ophthalmology

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