Activation of Gene Expression from Silenced Adenovector Genomes

Abstract

Abstract: : Purpose:Transgene expression driven by the human cytomegalovirus (hCMV) promoter from adenovectors following intravitreal administration into the eye is characterized by an initial high–level burst of expression that drops quickly and is followed by eventual loss of transgene expression. The decrease in expression could arise from elimination of vector genomes or by loss of promoter activity. In the current studies we investigate the cause for the diminution of activity, evaluate quantitatively vector genomes and expression levels, describe a means in which to follow transgene expression in the eye in the same animal over time, and discover means in which silenced adenovector genomes can be reactivated to express transgenes. Methods:In these studies, replication–deficient adenovectors deleted of E1, E3 and E4 that express nothing, GFP, or luciferase from the hCMV promoter were delivered to the eyes of mice by direct intravitreal injection. The amount of vector genome was quantitated by qPCR, the amount of luciferase transgene expression quantitated by luminesence, and the level of GFP transgene expression monitored by fluorescence. Results:From these analyses the loss of transgene expression over time from adenovectors can be attributed to loss of promoter activity. Adenovector genomes from which there was no detectable transgene expression still retained the ability to express their protein product. This was shown by the ability to completely restore expression from a silenced hCMV promoter by multiple means. The administration of non–expressing adenovector particles to the vitreous activated expression from the silenced adenovector genome to levels equal to or greater than that originally observed on day 1. These experiments also showed that quiescent adenovector genomes retain the ability to be reactivated for transgene expression even after being silenced for more than a year. A screen of potential molecules capable of activating the hCMV promoter found retinoic acid (RA) to be a potent activator of expression and systemic administration of RA was found to activate expression from silenced adenovector genomes to day 1 levels. Furthermore, we found that RA could activate expression from silenced adenovector genomes even after 2 months following initial administration into the eye, which further demonstrates that adenovector genomes remain expression competent. Conclusions:These data suggest that adenovectors may provide a means to deliver proteins to the eye requiring repeated expression for the treatment of ocular disease and expands the utility of adenovectors in ocular gene delivery.

Keywords: gene transfer/gene therapy • adenovirus • gene/expression