DYNAMICS OF GENE-TRANSFER TO RETINAL-PIGMENT EPITHELIUM

Purpose. To examine the nature and dynamics of gene transfer to human retinal pigment epithelium (RPE) using an adenoviral vector and adjuva nts that may enhance the uptake of recombinant adenoviruses. Methods. Human RPE cultures (HRPE7) were transfected in vitro with varying conc entrations (4, 20, 40, 120, and 200 pfu/mu l) and for varying periods (1, 2, 4, 16, 24, 48, and 72 hours) with a replication-deficient adeno virus (Ad.RSV.beta gal) containing the bacterial beta-galactosidase tr ansgene (beta gal). The expression of beta gal was monitored by counti ng after X gal staining. The transgene expression profiles were compar ed to those of human F2000 fibroblasts under the same conditions. The adjuvant effect of sodium hyaluronate (HA) on the expression of beta g al was tested in F2000 and early and late passage human RPE cells for differing concentrations of HA, viral titers, and incubation times. Im munofluorescent cytochemistry was carried on HRPE7 and F2000 cells for the HA receptors, homing receptor CD44 (CD44), intercellular adhesion molecule 1 (ICAM-1), and the receptor for hyaluronan mediated motilit y (RHAMM). Results. The number of HRPE7 and F2000 cells expressing the adenoviral transgene increased consistently with increasing incubatio n time and viral titer. There was a higher uptake of Ad.RSV.beta gal i n HRPE7 cells compared to the F2000 fibroblasts under the same conditi ons. There was an increase of 28.1% and 41.4% in the number of RPE7 ce lls expressing adenoviral transgene and 16.2% and 15.8% F2000 fibrobla st cells expressing the adenoviral transgene in the presence of 0.001% and 0.005% HA, respectively. Significant adjuvant effects on transgen e expression also were shown in HRPE51 cells. It appears that the effe cts of increasing viral titer, length of incubation, and the presence of HA on transgene expression are at least additive. The appearance of CD44 and ICAM receptors on RPE7 and F2000 cells and RHAMM receptors o n F2000 cells was similar. The RHAMM receptors in HRPE7 cells, however , were shown preferentially over the nucleus. Conclusions. On the basi s of these results, the authors propose that adenovirus transgene expr ession increases with increasing incubation time and viral titer in ce ll culture, The rate of increase of expression differs between human R PE cells and the F2000 fibroblast cells, which may offer a targeting o pportunity. The authors propose that the use of HA can offer both an a djuvant effect and a targeting advantage in terms of transferring aden oviral transgenes to human RPE in culture.