Construction of Tissue Specific and Hypoxia Regulated rAAV Gene Transfer Vectors to Retinal Pigment Epithelium Cells

Abstract

Purpose: : Hypoxia that elicits neovascularization (NV) forms a mechanistic basis for a novel therapeutic approach to arrest new vessel growth and associated vascular permeability. The lab is currently designing and testing several tissue specific and hypoxia specific rAAVs for delivery of anti–angiogenesis vectors to prevent neovascularization in animal models of AMD. These vectors will initially be tested in ARPE–19 and HEK–293 cell lines to verify hypoxia responsiveness and tissue specificity.

Methods: : A HRES (Hypoxia Responsive Element – Silenced) was integrated into a pGL3 Luciferase Reporter Vector (Promega) with the addition of an additional tandem HRE oligomer enhancer designed to increase the hypoxia responsiveness of a transgene. To this new construct the RPE₆₅ promoter was added to create pGL3–HRES–6xHRE–RPE₆₅. This reporter construct was tested for hypoxia responsiveness by co–cationic lipid transfection with pGL3–TK–Renilla (control) into ARPE–19 cells and exposure to normoxia (pO₂=20.8%) or either 24 or 48 hours of hypoxia (pO₂=0.5%). Cells were isolated from either normoxia or hypoxia, lysed and assayed for dual luciferase activity per the manufacturer’s protocol (Dual Luciferase Assay Kit, Promega).

Results: : As expected the HRES silenced the normoxic activity of the RPE₆₅ promoter by 59%. Although this is preliminary data, it reflects the type of response expected from this promoter in a normal oxygenated environment where expression of our transgene is not required. This demonstrates steps to stringently regulate the delivery of our therapeutic gene product only during a time where it would be therapeutic. The hypoxia responsiveness of our promoter constructs was also tested in hypoxic ARPE–19 cells at 24 and 48 hours culture at an oxygen tension of 0.5%. Preliminary data demonstrates a 10 fold activation of the promoter at 24 hours and an expanded 44 fold activation by 48 hours hypoxia.

Conclusions: : Initial research has successfully demonstrated the ability to regulate a promoter with varying oxygen tension and tissue specificity. Other cell type specific promoters are also being testing . Surrounding tissue that may be transfected with our new viral vector after sub–retinal injection will not express a transgene. Use of these cell specific gene therapy vectors will function in hypoxia/ischemia while the surrounding normoxic tissue remains unaffected.

Keywords: gene transfer/gene therapy • hypoxia • retinal pigment epithelium