Gene transfer to the trabecular meshwork and anterior segment of living rat eyes using lentiviral vectors.

Abstract

Abstract: : Purpose:To test the potential of using lentiviral vectors as a tool for gene transfer into the cells of the anterior chamber of living animals. Methods:Recombinant lentiviruses expressing the reporter gene GFP under the CMV promoter (lenti–CMV–GFP) were slowly injected at 109 pfu per eye using a 5ul syringe with a 30g needle in the anterior chamber of living adult Sprague–Dawley rats by a small transcorneal–scleral limbus puncture with the aid of an operating microscope. Two weeks after injection, the rats were euthanized and the eyes fixed in 4% paraformaldehyde. Cryosections of the eyes were analyzed under a fluorescent microscope to study the distribution of the reporter gene expression within the anterior segment. Results:Injections of recombinant lentiviruses lenti–CMV–GFP in the anterior chamber of living rats resulted in intense expression of the reporter gene in most cells of the trabecular meshwork as well as in the cells of the anterior surface of the iris and ciliary body. Only low levels of expression were observed in the cells of the corneal endothelium. No detectable changes were observed in the intraocular pressure of the infected eyes with respect to the contralateral controls. Conclusions:The use of lentiviral vectors in the anterior chamber of the eye of living rats provides a new tool for permanent transgene expression in living animals that can be potentially useful to study the mechanisms involved in outflow regulation, the generation of animal models for glaucoma, and the testing of gene therapy approaches for glaucoma.

Keywords: gene transfer/gene therapy • gene/expression • microscopy: light/fluorescence/immunohistochemistry