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 novel technique for modulating corneal wound healing on a cellular level, the successful transduction of human keratocytes should be ascertained in vitro. In addition, the ability of different polycations to increase the transduction efficiency and their antiproliferative and cytotoxic effects should be assessed.

METHODS

To test transduction efficiency (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 the antiproliferative and cytotoxic effects, cultured human keratocytes were incubated with various concentrations of polybrene and protamine sulfate (0.08 to 800 micrograms/ml) for 2, 24 and 72 h, and evaluations were performed by means of an XTT-based colorimetric assay and phase-contrast microscopy.

RESULTS

Human keratocytes in vitro were transduced successfully with the beta-galactosidase gene (3.5 +/- 1.0%). Transduction efficiency was significantly (P < or = 0.01) improved by addition of a polycation (from 12.3 +/- 1.7% to 18.6 +/- 2.3%), but there was no significant difference between the effects of polybrene and those of protamine sulfate. Both drugs induced a highly significant dose-dependent inhibition of proliferation (P < 0.001). ID50 ranged from 11 to 22 micrograms/ml with polybrene and from 15 to 244 micrograms/ml with protamine sulfate. Only with doses of 80 and 800 micrograms/ml did protamine sulfate produce less antiproliferative effects than polybrene (P < or = 0.04). The lowest concentrations induced no morphological signs of cytotoxicity, whereas these signs were mild at 8 micrograms/ml and moderate to severe at the highest concentrations.

CONCLUSIONS

Both polybrene and protamine sulfate can significantly improve the in vitro efficiency of successful retroviral vector-mediated gene transfer into keratocytes. Mild cytotoxic and moderate antiproliferative effects are to be expected in cultured keratocytes with a standard transduction procedure (8 micrograms/ml for 2 h).