Construction of Tissue Specific and Hypoxia Regulated rAAV Gene Transfer Vectors to Müller Cells

Abstract

Purpose:: Hypoxia that elicits pathology may also be exploited as a physiological trigger in a novel therapeutic approach to gene delivery. Last year we introduced a Retinal Pigment Epithelium (RPE)-specific hypoxia-induced vector for anti-angiogenic therapy for Choroidal Neovascularization (CNV). We now present a Müller cell-specific and hypoxia-regulated rAAV to deliver an anti-angiogenic gene therapy to prevent intravitreal neovascularization. This vector is initially being tested in our Müller, MIO-M1, A-172, ARPE-19 and HEK-293 cell lines to verify hypoxia responsiveness and tissue specificity.

Methods:: A HRSE (Hypoxia Responsive Silenced Element) was ligated into a Luciferase Reporter Vector, coupled to an additional tandem HRE oligomer enhancer designed to increase the hypoxia responsiveness of the promoter. To this new construct we coupled the Glial Fibrillary Acid Protein (GFAP) promoter. This reporter construct was tested for hypoxia responsiveness by co-cationic lipid transfection with pGL3-TK-Renilla (control) into GFAP and non-GFAP expressing cells followed by exposure to normoxia (pO₂=20.8%) or 40 hours of hypoxia (pO₂=0.5%). Cells were then assayed for dual luciferase activity.

Results:: The HRSE silenced the normoxic activity of the GFAP promoter by 68%. This reflects the type of response expected from our promoter in a normal oxygenated environment where expression of a transgene is not required. This also demonstrates stringent regulation of our therapeutic gene product produced only during the therapeutic window associated with hypoxia. We tested the hypoxia-responsiveness of our promoter constructs in hypoxic cells expressing GFAP, cultured for 40 hours at 0.5% oxygen. Data demonstrates a 41 fold activation by 40 hours hypoxia. Non-GFAP cell lines show minimal activation.

Conclusions:: We have demonstrated in both RPE cells and Glial cells the ability to regulate a reporter gene with both tissue specificity and varying oxygen tension, this time to Müller cells expressing GFAP. This new HRSE-6xHRE-GFAP promoter will be recombined with the gene for Endostatin in our rAAV construction vector for propagation of our gene therapy vectors. Surrounding tissue that may be transfected with our new viral vector after sub-retinal injection should not express a transgene. Use in the clinic of hypoxia-regulated cell specific gene therapy vectors will function to reduce retinal NV in the disease state while the surrounding tissue remains unaffected.

Keywords: Muller cells • gene transfer/gene therapy • hypoxia