Robust hypoxia-selective regulation of a retinal pigment epithelium-specific adeno-associated virus vector.
OBJECTIVE
To develop an hypoxia-regulated retinal pigment epithelium (RPE)-specific adeno-associated virus (AAV) gene transfer platform that exploits hypoxia as a physiologic trigger for an early antiangiogenic treatment strategy directed at arresting neovascularization in the eye.


METHODS
Tissue-specific and hypoxia-regulated expression vectors were constructed with tandem combinations of hypoxia responsive elements and aerobically silenced elements (HRSE) that together induce gene expression in hypoxia and suppress it in normoxia. For RPE-specific expression, the HRSE and a (6x) HRE (hypoxia responsive element) oligomer were ligated upstream of the Rpe65 promoter in a pGL3 firefly luciferase plasmid (pGL3-HRSE-6xHRE-Rpe65). The cell specificity of this novel hybrid promoter was tested in human RPE (ARPE-19), human glioblastoma, rat C6 glioma, mouse hippocampal neurons, and human embryonic kidney cell lines. Expression of all cell types in normoxia, and following 40 h hypoxia, was analyzed by dual luciferase assay. After confirmation of its tissue-specificity and hypoxia-inducibility, the hybrid promoter construct was integrated into a replication-deficient AAV delivery system and tested for cell-selectivity and hypoxia-inducible green fluorescent protein (GFP) reporter expression.


RESULTS
The HRSE-6xHRE-Rpe65 promoter was highly selective for RPE cells, strongly induced in hypoxia, and similar in activation strength to the cytomegalovirus (CMV) promoter. The AAV.HRSE.6xHRE.Rpe65 vector induced robust GFP expression in hypoxic ARPE-19 cells, but elicited no GFP expression in hypoxia in other cell types or in normoxic ARPE-19 cells.


CONCLUSIONS
The hypoxia-regulated, aerobically-silenced RPE-targeting vector forms a platform for focal autoregulated delivery of antiangiogenic agents in hypoxic regions of the RPE. Such autoinitiated therapy provides a means for early intervention in choroidal neovascularization, when it is most sensitive to inhibition.
Dougherty
Christopher J
CJ
Charles E. Schmidt College of Biomedical Science, Florida Atlantic University, Boca Raton, FL 33431, USA.
Smith
George W
GW
Dorey
C Kathleen
CK
Prentice
Howard M
HM
Webster
Keith A
KA
Blanks
Janet C
JC
D000818:Animals; D015687:Cell Hypoxia; D002460:Cell Line; D000229:Dependovirus / Q000235:genetics*; D005136:Eye Proteins / Q000235:genetics; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D009928:Organ Specificity; D010857:Pigment Epithelium of Eye / Q000821:virology*; D020218:Response Elements / Q000235:genetics; D014161:Transduction, Genetic / Q000379:methods*; D014162:Transfection
D005136:Eye Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases