Dynamics of gene transfer to retinal pigment epithelium.
OBJECTIVE
To examine the nature and dynamics of gene transfer to human retinal pigment epithelium (RPE) using an adenoviral vector and adjuvants that may enhance the uptake of recombinant adenoviruses.


METHODS
Human RPE cultures (HRPE7) were transfected in vitro with varying concentrations (4, 20, 40, 120, and 200 pfu/microliter) and for varying periods (1, 2, 4, 16, 24, 48, and 72 hours) with a replication-deficient adenovirus (Ad.RSV. beta gal) containing the bacterial beta-galactosidase transgene (beta gal). The expression of beta gal was monitored by counting after X gal staining. The transgene expression profiles were compared to those of human F2000 fibroblasts under the same conditions. The adjuvant effect of sodium hyaluronate (HA) on the expression of beta gal was tested in F2000 and early and late passage human RPE cells for differing concentrations of HA, viral titers, and incubation times. Immunofluorescent 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 motility (RHAMM).


RESULTS
The number of HRPE7 and F2000 cells expressing the adenoviral transgene increased consistently with increasing incubation time and viral titer. There was a higher uptake of Ad.RSV. beta gal in HRPE7 cells compared to the F2000 fibroblasts under the same conditions. There was an increase of 28.1% and 41.4% in the number of RPE7 cells expressing adenoviral transgene and 16.2% and 15.8% F2000 fibroblast cells expressing the adenoviral transgene in the presence of 0.001% and 0.005% HA, respectively. Significant adjuvant effects on transgene expression also were shown in HRPE51 cells. It appears that the effects 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 on F2000 cells was similar. The RHAMM receptors in HRPE7 cells, however, were shown preferentially over the nucleus.


CONCLUSIONS
On the basis of these results, the authors propose that adenovirus transgene expression increases with increasing incubation time and viral titer in cell culture. The rate of increase of expression differs between human RPE cells and the F2000 fibroblast cells, which may offer a targeting opportunity. The authors propose that the use of HA can offer both an adjuvant effect and a targeting advantage in terms of transferring adenoviral transgenes to human RPE in culture.
da Cruz
L
L
Molecular Biology Group, Lions Eye Institute, Perth, Australia.
Rakoczy
P
P
Perricaudet
M
M
Constable
I J
IJ
D000260:Adenoviruses, Human / Q000235:genetics*; D002648:Child; D003673:Defective Viruses; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000821:virology; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D006820:Hyaluronic Acid / Q000494:pharmacology / Q000502:physiology; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000201:enzymology / Q000821:virology; D008875:Middle Aged; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000821:virology; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D018960:Hyaluronan Receptors; D018799:Intercellular Adhesion Molecule-1; D006820:Hyaluronic Acid; D001616:beta-Galactosidase