Purpose. To determine and compare the efficacy of virally-mediated gene transfer into rat RPE cells in vitro and in vivo using an amplicon HSV-1 and a recombinant adenoviral vector carrying lacZ. Methods. Primary rat RPE cultures were infected with HSVlac (0.2, 2, & 20 i.p./cell) and AdRSVntlacZ (1, 10, & 100 i.p./cell) for 2h at 37°C. X-gal histochemistry & β-galalactosidase ELISA were performed at 2 & 7d post-transfection. In vivo, 2-4 μl of AdRSVntlacZ (105, 106, 107 i.p./μl) was injected inio the subretinal space of P23 Sprague Dawley rats using the trans-scleral approach of Lazar & del Cerro (J. Neurosci. Meth.'92,43:157). The animals were sacrificed at 3 & 10d post-injection, and the eye cups were fixed as flatmounts in 0.5% glutaraldehyde for X-gal histochemistry. Results. Infection with 0.2, 2, & 20 i.p./cell HSVlac resulted in 1.5%, 9.5%, & 39% X-gal+ RPE cells at 2d. However, cytotoxicity was observed at the highest titer with a 46% decrease in total cell pop. AdRSVntlacZ infection resulted in 0.15%, 0.8%, & 9.2% X-gal+ cells at 2d and 0.3%, 2.1%, & 27% at 7d without cytotoxic effect. This increase in transgene expression was also detected using ELISA which demonstrated a 2-fold increase in β-galactosidase. In vivo, only 106 & 107 i.p./cell AdRSVntlacZ showed significant transfection efficiency with a maximum of 227 and 1200 X-gal+ RPE cells at 10d, respectively. Conclusions. High transfection efficiency in RPE cells was obtained using high titers of HSV-1 and adenoviral vectors. Cytotoxicity was observed with HSV-1 but not with adenovirus. Maximum expression of AdRSVntlacZ transgene product appeared to increase after 2d post-transfection. In vivo, AdRSVntlacZ infected a significant number of RPE cells which continued to express the transgene product for up to 10d. The results of this study are currently being applied to genetically modify RPE cells with a growth factor transgene with the aim of rescuing photoreceptors from degeneration.
|Original language||English (US)|
|Journal||Investigative Ophthalmology and Visual Science|
|State||Published - Feb 15 1996|
ASJC Scopus subject areas
- Sensory Systems
- Cellular and Molecular Neuroscience