ABSTRACT
Glaucoma, a group of optic neuropathies, is the leading cause of irreversible blindness. Neuronal apoptosis in glaucoma is primarily associated with high intraocular pressure caused by chronically impaired outflow of aqueous humor through the trabecular meshwork, a reticulum of mitotically inactive endothelial-like cells located in the angle of the anterior chamber. Anatomic, genetic, and expression profiling data suggest the possibility of using gene transfer to treat glaucomatous intraocular pressure dysregulation, but this approach will require stable genetic modification of the differentiated aqueous outflow tract. We injected transducing unit-normalized preparations of either of two lentiviral vectors or an oncoretroviral vector as a single bolus into the aqueous circulation of cultured human donor eyes, under perfusion conditions that mimicked natural anterior chamber flow and maintained viability ex vivo. Reporter gene expression was assessed in trabecular meshwork from 3 to 16 days after infusion of 1.0 x 10(8) transducing units of each vector. The oncoretroviral vector failed to transduce the trabecular meshwork. In contrast, feline immunodeficiency virus and human immunodeficiency virus vectors produced efficient, localized transduction of the trabecular meshwork in situ. The results demonstrate that lentiviral vectors permit efficient genetic modification of the human trabecular meshwork when delivered via the afferent aqueous circulation, a clinically accessible route. In addition, controlled comparisons in this study establish that feline and human immunodeficiency virus vectors are equivalently efficacious in delivering genes to this terminally differentiated human tissue.
Subject(s)
Genetic Vectors/genetics , Glaucoma/genetics , Glaucoma/therapy , Lentivirus/genetics , Trabecular Meshwork/metabolism , Trabecular Meshwork/virology , Transduction, Genetic/methods , Aged , Animals , Aphidicolin/pharmacology , Aqueous Humor/metabolism , Cats , Cell Division , Cells, Cultured , Gene Expression , Genes, Reporter/genetics , HIV-1/genetics , Humans , Immunodeficiency Virus, Feline/genetics , Lac Operon/genetics , Leukemia Virus, Murine/genetics , Mice , Middle Aged , Organ Culture Techniques , Organ Specificity , Trabecular Meshwork/drug effects , Trabecular Meshwork/pathology , Transgenes/geneticsABSTRACT
PURPOSE: To determine the effect of human recombinant TIGR/myocilin (MYOC) protein on outflow resistance in the human anterior segment. METHODS: A cDNA for MYOC was inserted into a bacterial expression system and purified with nickel ion affinity chromatography. The anterior segments of 12 pairs of human eyes were placed in perfusion organ culture. One eye received an anterior chamber exchange with partially purified recombinant MYOC (25 microgram), whereas the other eye received either heat-denatured recombinant MYOC (25 microgram), partially purified ss-galactosidase (25 or 250 microgram), or partially purified control proteins isolated from a null expression lysate (25 microgram). Eyes were fixed up to 72 hours after infusion, and immunohistochemistry was performed using anti-MYOC polyclonal antibody. RESULTS: Recombinant MYOC caused an increase in IOP over 12 hours, increasing outflow resistance 94%, whereas the fellow eye infused with null expression sample increased 12% (n = 7; P = 0.0005). When compared with recombinant MYOC, neither heat-denatured MYOC, recombinant ss-galactosidase, bovine serum albumin, nor fetal calf serum caused an increase in outflow resistance. MYOC IOP remained above baseline levels for 48 to 72 hours. Immunohistochemistry results confirmed the presence of recombinant MYOC in the trabecular meshwork. CONCLUSIONS: Recombinant MYOC increased outflow resistance in human anterior segments, whereas control proteins did not. MYOC may increase outflow resistance by specific interactions within the trabecular meshwork.
