A hypoxia-responsive glial cell-specific gene therapy vector for targeting retinal neovascularization.
OBJECTIVE
Müller cells, the major glial cell in the retina, play a significant role in retinal neovascularization in response to tissue hypoxia. We previously designed and tested a vector using a hypoxia-responsive domain and a glial fibrillary acidic protein (GFAP) promoter to drive green fluorescent protein (GFP) expression in Müller cells in the murine model of oxygen-induced retinopathy (OIR). This study compares the efficacy of regulated and unregulated Müller cell delivery of endostatin in preventing neovascularization in the OIR model.


METHODS
Endostatin cDNA was cloned into plasmids with hypoxia-regulated GFAP or unregulated GFAP promoters, and packaged into self-complementary adeno-associated virus serotype 2 vectors (scAAV2). Before placement in hyperoxia on postnatal day (P)7, mice were given intravitreal injections of regulated or unregulated scAAV2, capsid, or PBS. Five days after return to room air, on P17, neovascular and avascular areas, as well as expression of the transgene and vascular endothelial growth factor (VEGF), were compared in OIR animals treated with a vector, capsid, or PBS.


RESULTS
The hypoxia-regulated, glial-specific, vector-expressing endostatin reduced neovascularization by 93% and reduced the central vaso-obliteration area by 90%, matching the results with the unregulated GFAP-Endo vector. Retinas treated with the regulated endostatin vector expressed substantial amounts of endostatin protein, and significantly reduced VEGF protein. Endostatin production from the regulated vector was undetectable in retinas with undamaged vasculature.


CONCLUSIONS
These findings suggest that the hypoxia-regulated, glial cell-specific vector expressing endostatin may be useful for treatment of neovascularization in proliferative diabetic retinopathy.
Integrative Biology PhD Program, Florida Atlantic University, Boca Raton, Florida, United States.
Manas R
MR
Biswal
Center for Complex Systems and Brain Sciences, Charles E. Schmidt College of Science, Florida Atlantic University, Boca Raton, Florida, United States Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, Florida, United States.
Howard M
HM
Prentice
Virginia Tech Carilion School of Medicine, Roanoke, Virginia, United States.
C Kathleen
CK
Dorey
Center for Complex Systems and Brain Sciences, Charles E. Schmidt College of Science, Florida Atlantic University, Boca Raton, Florida, United States.
Janet C
JC
Blanks
D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology*; D018076:DNA, Complementary; D003930:Diabetic Retinopathy / Q000503:physiopathology / Q000628:therapy*; D004195:Disease Models, Animal; D043169:Endostatins / Q000378:metabolism; D020868:Gene Silencing / Q000187:drug effects; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009457:Neuroglia / Q000502:physiology*; D015861:Retinal Neovascularization / Q000503:physiopathology / Q000628:therapy*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D018076:DNA, Complementary; D043169:Endostatins; D009419:Nerve Tissue Proteins; D042461:Vascular Endothelial Growth Factor A
GFAP; HIF-1 responsive element; Müller cell; OIR model; gene therapy; hypoxia