Genetic Modification of Human Trabecular Meshwork with Lentiviral Vectors

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.

Ocular gene delivery using lentiviral vectors

“…[19][20][21] In a cultured intact human eye model, FIV or HIV-1 vectors efficiently transduce the TM but result in patchy CE transduction after intra-cameral delivery. 24,43 Femtosecond laser-assisted corneal stromal pocket creation permits direct vector delivery to this layer, significantly facilitating ex vivo HIV-1 mediated gene delivery to pig corneal stromal keratocytes. 23 Intra-vitreal delivery Intra-vitreal delivery of VSV-G-pseudotyped HIV-1, (refs 8,16,18) FIV 40 or SIV 28 vectors, or VSV-G or rabies-G-pseudotyped EIAV vectors, 30 in rodents or rabbits typically fails to produce efficient intraocular expression (Figure 2), although focal transduction of retinal pigment epithelium (RPE) or neurosensory retina cells within the vicinity of the injection site may occur.…”

“…5,6 OCULAR TRANSDUCTION PROFILES OF LENTIVIRAL VECTORS The transduction properties of HIV-1, (refs 7-24) HIV-2, (ref. 25) SIV, [26][27][28][29] EIAV, 21,[30][31][32][33][34][35] FIV, 24,[36][37][38][39][40][41][42][43] and BIV 44 vectors incorporating a diverse array of envelope pseudotypes and transgene promoters have been evaluated after in vivo and ex vivo delivery in a variety of animal species (Supplementary Tables 1 and 2). Most ocular studies have used vesicular stomatitis virus-G (VSV-G)-pseudotyped vectors incorporating ubiquitous promoters and these are described below unless otherwise stated.…”

FIV: from lentivirus to lentivector

“…FIV vectors have been used to target cells in the brain, eye, airway, hematopoietic system, liver, muscle and pancreas [8,15,16,19,[45][46][47][48][49][50][51][52][53][54][55][56][57]. Exceptional results in the central nervous system have been obtained [47].…”

“…Although controlled infectivity and efficacy comparisons on a per particle basis have been lacking, in several systems, FIV vectors have produced excellent levels of gene transfer and expression in cultured human organs [45,55,56]. In the one direct comparison using transducing unit-normalized preparations (trabecular meshwork transduction in an organ perfusion model used for preclinical glaucoma gene therapy research), FIV and HIV-1 vectors produced equivalent results in human eyes [55].…”

Lentiviral transgenesis of the leopard gecko, Eublepharis macularius

“…Development of Feline Immunodeficienty Virus (FIV) based lentiviral vectors began partly because FIV does not cause human infection since it is a virus that attacks the immune system of cats. FIV based vectors have been used to transduce nondividing and dividing cells of the brain, eye, airway, hematopoietic system, liver, muscle, and pancreas (Wang et al, 1999;Loewen et al, 2001;Derksen et al, 2002;Price et al, 2002;Stein and Davidson 2002).…”