RETROVIRAL VECTOR-MEDIATED GENE-TRANSFER INTO KERATOCYTES - IN-VITRO EFFECTS OF POLYBRENE AND PROTAMINE SULFATE

Background: To determine the potential of somatic gene transfer as a n ovel technique for modulating corneal wound healing on a cellular leve l, the successful transduction of human keratocytes should be ascertai ned in vitro. In addition, the ability of different polycations to inc rease the transduction efficiency and their antiproliferative and cyto toxic effects should be assessed. Methods: To test transduction effici ency (X-Gal staining), cultured human keratocytes were incubated for 2 h with a retroviral vector bearing the beta-galactosidase gene, with and without the addition of polybrene or protamine sulfate. To test th e antiproliferative and cytotoxic effects, cultured human keratocytes were incubated with various concentrations of polybrene and protamine sulfate (0.08 to 800 mu g/ml) for 2, 24 and 72 h, and evaluations were performed by means of an XTT-based colorimetric assay and phase-contr ast microscopy. Results: Human keratocytes in vitro were transduced su ccessfully with the beta-galactosidase gene (3.5 +/- 1.0%). Transducti on efficiency was significantly (P less than or equal to 0.01) improve d by addition of a polycation (from 12.3+/-1.7% to 18.6+/-2.3%), but t here was no significant difference between the effects of polybrene an d those of protamine sulfate. Both drugs induced a highly significant dose-dependent inhibition of proliferation (P < 0.001). ID50 ranged fr om 11 to 22 mu g/ml with polybrene and from 15 to 244 mu g/ml with pro tamine sulfate. Only with doses of 80 and 800 mu g/ml did protamine su lfate produce less antiproliferative effects than polybrene (P less th an or equal to 0.04). The lowest concentrations induced no morphologic al signs of cytotoxicity, whereas these signs were mild at 8 mu g/ml a nd moderate to severe at the highest concentrations. Conclusions: Both polybrene and protamine sulfate can significantly improve the in vitr o efficiency of successful retroviral vector-mediated gene transfer in to keratocytes. Mild cytotoxic and moderate antiproliferative effects are to be expected in cultured keratocytes with a standard transductio n procedure (8 mu g/ml for 2 h).