USE OF A RETROVIRAL VECTOR WITH AN INTERNAL OPSIN PROMOTER TO DIRECT GENE-EXPRESSION TO RETINAL PHOTORECEPTOR CELLS

Purpose. Viral-mediated gene transfer to retina, as well as to other t issues, is evolving rapidly. We have evaluated the potential of a retr oviral vector with an internal opsin promoter fragment to direct gene expression to retinal photoreceptor cells. Methods. Two recombinant re troviral vectors were prepared; in each vector, a 1.4 kb fragment of t he mouse opsin promoter was placed downstream from the neo(R) gene in the Moloney murine leukemia virus-based vector GlNa. The opsin promote r fragment was linked either to the cDNA for mouse rod photoreceptor p hosphodiesterase (PDE) beta-subunit or to the bacterial lacZ reporter gene. These vectors were tested for their ability to direct gene expre ssion after transduction of 3T3 and Y79 cells, or of dissociated retin al cell cultures or retinal explants from neonatal mice. Results. As e xpected, PDE beta-subunit and beta-galactosidase mRNAs were expressed only at low levels in 3T3 fibroblasts and Y79 retinoblastoma cells. No rthern blot analysis indicated that expression was derived from the vi ral long terminal repeat (LTR) promoter. Infection of primary retinal cell cultures or explants from neonatal mice with BAG retrovirus, in w hich beta-galactosidase is driven by the viral LTR, resulted in expres sion in many cell types, while the opsin-lacZ vector mediated the expr ession of the lacZ reporter gene specifically in photoreceptor cells. Conclusions. The internal opsin promoter fragment appears capable of s electively directing gene expression to photoreceptor cells after retr oviral-mediated gene transfer. These findings serve as a basis for fut ure studies using the opsin promoter-beta PDE retroviral vector to res cue photoreceptor cells in the rd mutant mouse, in which the beta-PDE gene is mutated resulting in degeneration of photoreceptor cells durin g the early postnatal period.