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Janet C Blanks, Howard Prentice, James Sullivan; Characterization of hypoxia–regulated, glial cell specific AAV vector for targeting retinal neovascularization.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4504.
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© ARVO (1962-2015); The Authors (2016-present)
The Muller cell is known to secrete angiogenic factors under conditions of hypoxia exposure similar to the mouse oxygen induced retinopathy (OIR) model or in diabetic retinopathy. Previously, we constructed adeno-associated virus vector (AAV) gene therapy vectors whose transgene expression is driven by a hypoxia responsive domain combined with the glial fibrillary associated protein (GFAP) promoter. Here we developed and tested a hypoxia regulated and glial cell specific vector encoding human decorin which functions as an antiangiogenic protein.
Plasmids for AAV generation were constructed with the human decorin cDNA together with the CMV promoter enabling generation of the virus AAV-CMV-Decorin or with the GFAP promoter giving virus AAV-GFAP-Decorin or with the GFAP promoter PLUS a hypoxia responsive domain (HRE) giving AAV-HRE-GFAP-Decorin. AAVs were then packaged using these plasmids for subsequent infection of primary rat brain astrocytes, PC12 neuronal cells or retinal epithelial ARPE-19 cells. After infection cells were subjected to 72 h of hypoxia. Levels of mRNA for human decorin were measured by quantitative real time RT-PCR.
Following infection of primary astrocytes with AAV-HRE-GFAP and hypoxia treatment there was a significant induction in decorin mRNA of greater than 5 fold relative to normoxic cells. Infection with AAV-GFAP-Decorin resulted in high level expression of decorin mRNA either in the absence or presence of hypoxia. Neither AAV-GFAP-Decorin nor regulated AAV-HRE-GFAP-Decorin infected into ARPE-19 cells or PC12 cells resulted in significant decorin mRNA expression with or without hypoxia. By contrast AAV-CMV-Decorin infection resulted in high level expression of decorin mRNA in ARPE19 cells and in PC12 cells with or without hypoxia.
Our data indicates that AAV-HRE-GFAP-Decorin drives hypoxia inducible, as well as glial cell specific expression, of decorin mRNA. AAV-GFAP-Decorin showed glial cell specific expression that was not further modified by hypoxic exposure. However, AAV-CMV-Decorin was ubiquitously expressing decorin mRNA in all cell types tested in both conditions of hypoxia and normoxia. These studies contribute to comparing the relative efficacy of hypoxia responsive and glial cell specific AAV-HRE- GFAP-decorin to glial cell specific vector AAV-GFAP-decorin for antiangiogenic treatment in diabetic retinopathy.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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