July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Development of a glial cell specific and hypoxia responsive gene therapy vector for use in models of retinal neovascularization.
Author Affiliations & Notes
  • James Sullivan
    Complex Systems, Florida Atlantic University, Lake Worth, Florida, United States
  • Janet C Blanks
    Complex Systems, Florida Atlantic University, Lake Worth, Florida, United States
  • Howard M Prentice
    Complex Systems, Florida Atlantic University, Lake Worth, Florida, United States
  • Footnotes
    Commercial Relationships   James Sullivan, None; Janet Blanks, None; Howard Prentice, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 2920. doi:
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      James Sullivan, Janet C Blanks, Howard M Prentice; Development of a glial cell specific and hypoxia responsive gene therapy vector for use in models of retinal neovascularization.. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2920.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Muller cells which comprise the primary glial cell type in the retina are known to secrete angiogenic factors under hypoxic conditions similar to those found in the mouse oxygen induced retinopathy (OIR) model and clinically in diabetic retinopathy. Previously, we constructed adeno-associated virus (AAV) vectors containing a hypoxia responsive domain together with the glial fibrillary acidic protein (GFAP) promoter to drive transgene expression specifically in Muller cells and astrocytes in culture and in the mouse OIR model.. In this study we tested a hypoxia regulated and glial cell specific vector for driving expression of the anti-angiogenic factor decorin in the retina.

Methods : AAV vectors were constructed with the ubiquitously active CMV promoter, the GFAP promoter or a regulatory region containing the GFAP promoter plus a hypoxia responsive domain (HRE). Vectors contained the human decorin coding sequence. Vectors were used to infect rat brain astrocytes, PC12 cells (neuronal cell line) or ARPE-19 cells (retinal epithelium cell line). Cells were then exposed to hypoxia (72 h) or normoxia. Decorin mRNA levels were determined using real time RT-PCR.

Results : In primary astrocytes infected with AAV-HRE-GFAP-decorin and exposed to hypoxia, an approximate 5 fold increase in decorin mRNA was observed relative to normoxic cells. Infection with AAV-GFAP-decorin resulted in high levels of decorin mRNA expression in both hypoxic and normoxic astrocytes. In the control ARPE19 cells and PC12 cells there was no significant decorin mRNA transcription in either hypoxia or normoxia. Infection with AAV-CMV-decorin resulted in high levels of expression of decorin mRNA in astrocytes, PC12 cells and ARPE19 cells in both normoxia and hypoxia.

Conclusions : Our study indicates that AAV-HRE-GFAP-Decorin expresses decorin mRNA in a hypoxia inducible and glial cell dependent manner. The glial specific AAV-GFAP decorin vector expresses decorin mRNA in astrocytes in both normoxic and hypoxic conditions. A positive control vector, AAV-CMV-decorin, expresses decorin mRNA in astrocytes as well as in PC12 cells and ARPE19 cells in both hypoxia and normoxia. The AAV-HRE-GFAP decorin vector may be applicable for use as a hypoxia inducible and glial cell specific vector for preventing neovascularization in models of diabetic retinopathy.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

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