Inhibition of rabbit keratocyte and human fetal lens epithelial cell proliferation by retrovirus-mediated transfer of antisense cyclin G1 and antisense MAT1 constructs

The aim of this study is to evaluate the potential of gene transfer of cell cycle control genes as treatment of corneal haze or secondary cataract for mation. The guiding hypothesis is that strategic modulation of the cyclin G 1 or MAT1 gene by retrovirus-mediated gene transfer will inhibit proliferat ion of rabbit keratocytes (RabK) and fetal human lens epithelial (FHLEpi) c ells in vitro. RabK and FHLEpi cell cultures were transduced in triplicate with retroviral vectors bearing either a nuclear-targeted beta-galactosidas e, an antisense cyclin G1 (aG1), an antisense MAT1 (aMAT1) construct, or th e neo(r) gene, The presence of beta-galactosidase activity in the transduce d cultures was detected by immunohistochemical X-Gal staining, while cyclin G1 and MAT1 protein expression levels were evaluated by Western analysis. Proliferation of RabKs and FHLEpi cells was analyzed by counting the number of cells in the aG1 and aMAT1 vector-transdued cultures over 5 days. The m ean transduction efficiency was 34.4% (SD 1.41) for RabKs and 19.7% (SD 1.8 3) for FHLEpi cells, Downregulation of cyclin G1 and MAT1 protein expressio n was noted 24 hr after transduction of RabK cultures with the respective v ectors, Cytostatic effects of the aG1 and aMAT1 vectors in both RabKs and F BLEpi cells were most pronounced on the fifth day (RabKs, p < 0.0007; FHEpi cells, p < 0.001). An increased incidence of apoptosis was identified in b oth aG1 and MAT1-transduced FHLEpi cells. Taken together, these data sugges t the potential utility of developing aG1 and aMAT1 retroviral vectors in g ene therapy protocols for corneal haze and secondary cataract formation.