Purchase this article with an account.
Howard M. Prentice, Manas R Biswal, C. Kathleen Dorey, Janet C. Blanks; Hypoxia-Regulated Retinal Glial Cell–Specific Promoter for Potential Gene Therapy in Disease. Invest. Ophthalmol. Vis. Sci. 2011;52(12):8562-8570. doi: https://doi.org/10.1167/iovs.10-6835.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Retinal Müller cells span the retina and secrete several trophic factors and represent the functional link between blood vessels and neurons, making them attractive targets for gene therapy. Therefore, a hypoxia-regulated, retinal glial cell–specific vector was constructed and tested for its response to hypoxia.
A hybrid promoter containing domains of human glial fibrillary acidic protein (GFAP) and several hypoxia-responsive and aerobically silenced elements (HRSE) was incorporated separately into plasmid vectors for generation of self-complementary adeno-associated virus. Müller cells trasfected with plasmids or virus were compared with other cell lines using standard methods. The mouse model of oxygen-induced retinopathy (OIR) was used to analyze retinas from mice exposed to high oxygen or room air to evaluate the induction of the regulated promoter.
The regulated promoter was silenced under aerobic conditions in comparison with unregulated promoter in Müller cells. Hypoxia induced a 12-fold and 16-fold increase in promoter activity in primary Müller cells and human Müller cell lines, respectively. In the OIR model, intravitreal injection of the regulated promoter at postnatal day 7 (P7) resulted in high levels of green fluorescent protein expression only in retinal Müller cells at P17. GFP expression was absent in retinas of mice only exposed to room air. In vivo studies confirm normoxia silencing, hypoxic induction, and cell specificity of the regulated promoter in the mouse retina.
This hypoxia-regulated, retinal glial cell–specific AAV vector provides a platform for gene therapy within regions of retinal hypoxia which are found in diabetic retinopathy and age-related macular degeneration.
This PDF is available to Subscribers Only