Robust hypoxia-selective regulation of a retinal pigment epithelium-specific adeno-associated virus vector

Christopher J. Dougherty, George W. Smith, C. Kathleen Dorey, Howard M. Prentice, Keith A. Webster, Janet C. Blanks

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

Purpose: 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.

Original languageEnglish (US)
Pages (from-to)471-480
Number of pages10
JournalMolecular vision
Volume14
StatePublished - Mar 7 2008

ASJC Scopus subject areas

  • Ophthalmology

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    Dougherty, C. J., Smith, G. W., Dorey, C. K., Prentice, H. M., Webster, K. A., & Blanks, J. C. (2008). Robust hypoxia-selective regulation of a retinal pigment epithelium-specific adeno-associated virus vector. Molecular vision, 14, 471-480.