Lisa Wei

Periocular injection of an adenoviral vector encoding pigment epithelium-derived factor inhibits choroidal neovascularization

Gene transfer provides an exciting new approach for the treatment of retinal and choroidal diseases. Two areas of concern are the potential for vector-related toxicity and uncertainties associated with prolonged transgene expression. One way to address these concerns for transfer of genes encoding secreted proteins is to transduce cells on the outside of the eye, provided the gene product can gain access to the eye and have the desired effect. In this study, we investigated the feasibility of this approach. Periocular injection of an adenoviral vector encoding β-galactosidase (AdLacZ.10) resulted in LacZ-stained cells throughout the orbit and around the eye. Compared to periocular injection of 5 × 109 particles of control vector, periocular injection of 5 × 109 or 1 × 109 particles of an adenoviral vector expressing pigment epithelium-derived factor (PEDF) regulated by a CMV promoter (AdPEDF.11) resulted in significantly elevated intraocular levels of PEDF and suppression of choroidal neovascularization. Periocularly injected recombinant PEDF was also found to diffuse through the sclera into the eye. Although similar experiments are needed in an animal with a human-sized eye, these data suggest that periocular gene transfer deserves consideration for the treatment of choroidal diseases.

Periocular gene transfer of sFlt-1 suppresses ocular neovascularization and vascular endothelial growth factor-induced breakdown of the blood-retinal barrier

Vascular endothelial growth factor (VEGF) is a critical stimulus for both retinal and choroidal neovascularization, and for diabetic macular edema. We used mouse models for these diseases to explore the potential of gene transfer of soluble VEGF receptor-1 (sFlt-1) as a treatment. Intravitreous or periocular injection of an adenoviral vector encoding sFlt-1 (AdsFlt-1.10) markedly suppressed choroidal neovascularization at rupture sites in Bruchs membrane. Periocular injection of AdsFlt-1.10 also caused significant reduction in VEGF-induced breakdown of the blood-retinal barrier, but failed to significantly inhibit ischemia-induced retinal neovascularization. Periocular delivery of an adenoviral vector encoding pigment epithelium-derived factor (PEDF), another secreted protein, resulted in high levels of PEDF in the retinal pigmented epithelium and choroid, but not in the retina. This may explain why periocular injection of AdsFlt-1.10 inhibited choroidal, but not retinal neovascularization. Periocular delivery offers potential advantages over other routes of delivery and the demonstration that sFlt-1 enters the eye from the periocular space in sufficient levels to achieve efficacy in treating choroidal neovascularization and retinal vascular permeability is a novel finding that has important clinical implications. These data suggest that periocular gene transfer of sFlt-1 should be considered for treatment of choroidal neovascularization and diabetic macular edema.

993. Activation of Gene Expression from Silenced Adenovector Genomes

Top of pageAbstract 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. 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. 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. 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.