title: Lentiviral transduction of green fluorescent protein in retinal epithelium: evidence of rejection.
abstract: This paper demonstrates lentiviral transduction of the humanized form of the Aequoria victoria gene for green fluorescent protein (GFP) into human fetal retinal pigment epithelium (RPE) in vitro and rabbit RPE in vivo. In vitro GFP expression of cultured human fetal RPE begins within two to three days after 12-16 h of maintained exposure to the virus at titers of 10(8)-10(9) infectious units (IU)/ml. Both stationary and dividing cells are transduced using a lenti viral vector with a cytomegalovirus (CMV) promoter. Expression remains stable for at least three to four months without evidence of toxicity and continues through cell division. In vivo expression is followed non-invasively in rabbit eye using a scanning laser ophthalmoscope (SLO), which can detect single fluorescing retinal cells. In vivo expression begins within a few days after a viral solution is introduced into the subretinal space. A solution of 10(9) IU/ml produces fluorescence within three to four days. Less concentrated solutions lead to slower and less expression. No expression is detectable at concentrations of 10(6) IU/ml. Within one to two weeks after introduction of the viral solution, there is evidence of rejection seen by SLO as a loss of GFP fluorescence and disruption of the RPE. Histology shows damage to the RPE layer and monocytic cell infiltrates in the choroid and subretinal space within the area receiving the viral solution. Strong GFP expression leads to rejection within two weeks. With less expression, rejection is delayed and in some cases undetectable for at least six months. If the GFP gene is not included in the viral vector or if the viral concentration is insufficient to produce detectable GFP expression, rejection is not seen. Using a rhodopsin promoter or injecting the virus intra rather than subretinally produces weak expression and no rejection. Lentivirus can induce expression of a foreign gene in the RPE. Viral induced transduction and GFP expression have no effect on the viability of the RPE in vitro. Continued expression of GFP after cell division implies chromosomal integration of the gene. In vivo expression of GFP in RPE encounters rejection. Rejection may not occur with low GFP expression. The latter occurs with low viral titers, a rhodopsin promoter or intra-retinal injection of viral solution. The results are relevant to gene therapy in retina when gene transduction leads to the expression of foreign proteins.
authors.affiliation: Department of Ophthalmology, Columbia University, 630 W 168 Street, New York, NY 10032, USA.
authors.forename: K
authors.initials: K
authors.lastname: Doi
authors.forename: J
authors.initials: J
authors.lastname: Hargitai
authors.forename: J
authors.initials: J
authors.lastname: Kong
authors.forename: S H
authors.initials: SH
authors.lastname: Tsang
authors.forename: M
authors.initials: M
authors.lastname: Wheatley
authors.forename: S
authors.initials: S
authors.lastname: Chang
authors.forename: S
authors.initials: S
authors.lastname: Goff
authors.forename: P
authors.initials: P
authors.lastname: Gouras
mesh_terms: D000818:Animals; D002455:Cell Division; D046508:Culture Techniques; D015870:Gene Expression; D005822:Genetic Vectors / Q000008:administration & dosage*; D006084:Graft Rejection; D049452:Green Fluorescent Proteins; D006801:Humans; D007267:Injections; D016086:Lentivirus / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics* / Q000276:immunology; D018613:Microscopy, Confocal; D010857:Pigment Epithelium of Eye / Q000196:embryology / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits; D013997:Time Factors; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins