ENCAPSIDATED ADENOVIRUS MINI-CHROMOSOME-MEDIATED DELIVERY OF GENES TOTHE RETINA - APPLICATION TO THE RESCUE OF PHOTORECEPTOR DEGENERATION

First (Delta E1/E3) and second (Delta E1+Delta E2/E3/E4) generation ad enovirus (Ad) vectors have been shown previously to be of limited use in the treatment of human genetic diseases due to the induction of a h ost cytotoxic T-cell mediated immune response against virally expresse d genes. In addition, a limited cloning capacity of similar to 8 kb do es not cater for the incorporation of large upstream sequences essenti al for regulated tissue-specific expression or inclusion of multiple g ene-expression cassettes. In this study we have exploited our recently developed Ad-based vector, the encapsidated adenovirus mini-chromosom e (EAM) from which all of the viral genes have been deleted. EAMs cont ain only the inverted terminal repeats required for replication and fi ve cis-acting Ad encapsidation signals necessary for packaging. We hav e shown previously that EAMs can efficiently transduce a variety of ce ll types in vitro. In this study we demonstrate that EAMs can transduc e and rescue cells from the neurosensory retina in vivo. EAM-mediated delivery of the beta subunit of cyclic GMP phosphodiesterase (PDE) cDN A to mice affected with retinal degeneration (rd) allows prolonged tra nsgene expression and rescue of rod photoreceptor cells. RT-PCR analys is from the injected retina indicates that transgene products are pres ent for at least 18 weeks post-injection. Both the alpha and beta subu nits of PDE could be detected up to 90 days postnatal in EAM-injected rd retina by western analysis. A maximal PDE activity of 150 nm/min/mg was detected at 33 days postnatal. Examination of outer nuclear thick ness showed significant differences up to 12 weeks post-injection. The se results demonstrate an improved level of rescue over first-generati on adenoviral vectors and suggest the possibility of successful EAM-me diatecl treatment of some retinal diseases in humans.