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.

Transient Immunosuppression Stops Rejection of Virus-Transduced Enhanced Green Fluorescent Protein in Rabbit Retina

“…Similar results have been reported with other reporter genes that express foreign proteins in the retina, such as LacZ (6,8,14). We have found that with relatively high retinal expression of EGFP, transduced by lentivirus, rejection will occur (7). GFP has been shown to be immunogenic (9) and will be rejected in mice (19).…”

“…4). The small fluorescent spots, seen in these retinas, have the dimensions of single retinal epithelial cells, as we have noted previously (7). It is possible to follow these single fluorescent cells and cell clusters over relatively long periods of time.…”

“…At 17 days after surgery, it became difficult to see the treated area because of slight edema, and EGFP fluorescence disappeared. At 20 days after surgery, there was disruption of the retinal pigment epithelium (RPE) layer, which we have found characteristic of rejection (7). The dark areas are due to piling up of these migrating, activated, retinal pigment epithelial cells.…”

“…The latter was used with and without a barrier filter for monitoring EGFP fluorescence. We used a subjective grading scale to evaluate the amount of EGFP-expressing cells (7). If there were more than 50 spots of fluorescence within the injected area, the grade was 3; if there were between 10 and 50, the grade was 2; if there were less than 10, the grade was 1; and the absence of fluorescence was graded as 0.…”

Simian immunodeficiency virus-based lentivirus vector for retinal gene transfer: a preclinical safety study in adult rats

“…Another hypothesis is a host immune response against the transgene products, including b-galactosidase and GFP, which can evoke CTL and cytotoxic antibodies. [26][27][28] Recent reports also suggested that the gradual reduction of GFP expression might be because of a host immune response against GFP itself when using the lentivirus vector. 22,25 However, these findings may not be sufficient to conclude these issues, because they used only one reporter gene (GFP).…”