Purchase this article with an account.
Janet Blanks, James Sullivan, Manas Biswal, Howard Prentice, Shailaja Allani; Development of a hypoxia -regulated glial cell specific gene therapy vector encoding decorin.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1188.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Muller cells, the primary glial cells in the retina secrete factors that contribute significantly to neovascularization in response to retinal hypoxic conditions such as those found in the mouse oxygen induced retinopathy (OIR) model and those found clinically in diabetic retinopathy. We previously constructed an adeno-associated virus vector (AAV-HRE-GFAP-GFP) containing a hypoxia responsive domain together with the glial cell specific glial fibrillary acidic protein (GFAP) promoter to drive green fluorescent protein (GFP) reporter gene expression in Muller cells and astrocytes in culture and in the OIR model in vivo. In this study we have developed and tested foreign decorin mRNA expression from a new vector named AAV-HRE-GFAP-decorin containing the hypoxia responsive domain (HRE) plus the GFAP promoter plus the cDNA encoding decorin, an anti-angiogenic protein.
The human decorin cDNA was cloned into plasmids together with either the cytomegalovirus (CMV) promoter or the GFAP promoter or the hypoxia responsive domain (HRE) plus the GFAP promoter and then packaged into recombinant AAV. The hypoxia responsive vector named AAV-HRE-GFAP-decorin was tested for foreign decorin mRNA transcription in astrocyte cultures by quantitative reverse transcription PCR (Q-RT-PCR). Levels of decorin mRNA expression were compared to those for cells infected with AAV-GFAP-decorin or AAV-CMV-decorin or a capsid control using untreated cells or cells treated for 72h with 100 micromolar cobalt chloride.
Our findings indicate that AAV-CMV-decorin and AAV-GFAP-decorin vectors are capable of expressing foreign decorin mRNA in cultures astrocytes in the absence or presence of cobalt chloride treatment. Notably infection with AAV-HRE-GFAP-decorin did not result in significant foreign decorin mRNA expression in untreated cells but in cells treated with cobalt chloride there was highly significant foreign decorin mRNA expression.
In future experiments we plan to employ the mouse OIR model in vivo for testing the effectiveness of AAV-HRE-GFAP decorin and GFAP-decorin for inhibiting retinal neovascularization. These studies will determine the relative efficacy of the glial cell specific vector AAV-GFAP-decorin and the hypoxia responsive and glial cell specific vector AAV-HRE- GFAP-decorin for treatment of neovascularization in proliferative diabetic retinopathy.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
This PDF is available to Subscribers Only