Encapsidated adenovirus mini-chromosome-mediated delivery of genes to the retina: application to the rescue of photoreceptor degeneration

First (DeltaE1/E3) and second (DeltaE1+DeltaE2/E3/E4) generation adenovirus(Ad) vectors have been shown previously to be of limited use in thetreatment of human genetic diseases due to the induction of a hostcytotoxic T-cell mediated immune response against virally expressed genes.In addition, a limited cloning capacity of approximately 8 kb does notcater for the incorporation of large upstream sequences essential forregulated tissue-specific expression or inclusion of multiplegene-expression cassettes. In this study we have exploited our recentlydeveloped Ad-based vector, the encapsidated adenovirus mini-chromosome(EAM) from which all of the viral genes have been deleted. EAMs containonly the inverted terminal repeats required for replication and five cis-acting Ad encapsidation signals necessary for packaging. We have shownpreviously that EAMs can efficiently transduce a variety of cell types invitro. In this study we demonstrate that EAMs can transduce and rescuecells from the neurosensory retina in vivo. EAM-mediated delivery of thebeta subunit of cyclic GMP phosphodiesterase (PDE) cDNA to mice affectedwith retinal degeneration (rd) allows prolonged transgene expression andrescue of rod photoreceptor cells. RT-PCR analysis from the injected retinaindicates that transgene products are present for at least 18 weekspost-injection. Both the alpha and beta subunits of PDE could be detectedup to 90 days postnatal in EAM-injected rd retina by western analysis. Amaximal PDE activity of 150 nm/min/mg was detected at 33 days postnatal.Examination of outer nuclear thickness showed significant differences up to12 weeks post-injection. These results demonstrate an improved level ofrescue over first-generation adenoviral vectors and suggest the possibilityof successful EAM- mediated treatment of some retinal diseases in humans.