TY - JOUR
T1 - Retroviral vector-mediated gene transfer into keratocytes in vitro and in vivo
AU - Seitz, Berthold
AU - Moreira, Luciane
AU - Baktanian, Edwin
AU - Sanchez, Daniel
AU - Gray, Brad
AU - Gordon, Erlinda M.
AU - Anderson, W. French
AU - McDonnell, Peter J.
N1 - Funding Information:
This study was supported in part by a grant from Research to Prevent Blindness, Inc, New York, New York, to the Department of Ophthalmology; core grant EY03040 from the National Institutes of Health, Bethesda, Maryland; and Deutsche Forschungsgemeinschaft DFG-Az Se 667/5-1 (Dr Seitz).
PY - 1998/11
Y1 - 1998/11
N2 - 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.
AB - 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.
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U2 - 10.1016/S0002-9394(98)00205-0
DO - 10.1016/S0002-9394(98)00205-0
M3 - Article
C2 - 9822226
AN - SCOPUS:0032212812
SN - 0002-9394
VL - 126
SP - 630
EP - 639
JO - American journal of ophthalmology
JF - American journal of ophthalmology
IS - 5
ER -