RESUMEN
The most common complication of cataract surgery is the development of posterior capsule opacification (PCO). Hyperplasia of the lens epithelium is one of the main cellular events following phacoemulsification, and has been found to be an important feature contributing to opacification of the posterior capsule. Adenoviral vector-mediated transfer is a suitable method for transducing the herpes simplex virus thymidine kinase gene (HSV-tk) into proliferating cells, allowing for the selective killing of these cells by ganciclovir (GCV) treatment. To determine the potential of gene transduction for lens epithelial cells, we studied the transduction of rabbit lens epithelial cells with adenoviral vectors containing either the Escherichia coli beta-galactosidase (lacZ) gene or the HSV-tk gene in vitro and in vivo in an experimental model of PCO. The efficiency of lacZ gene transfer in rabbit lens epithelial cells was at least 95% both in vitro and in vivo. In vivo transduction with HSV-tk adenoviral vector followed by GCV treatment significantly inhibited the development of PCO (p<0.001). These results suggest that adenoviral vector-mediated transfer of HSV-tk into the proliferating lens epithelial cells is feasible and may provide a novel therapeutic strategy for PCO.
Asunto(s)
Catarata/prevención & control , Terapia Genética , Cápsula del Cristalino/patología , Facoemulsificación/efectos adversos , Adenoviridae/genética , Animales , Antivirales/farmacología , Catarata/etiología , Catarata/patología , Células Epiteliales/metabolismo , Estudios de Factibilidad , Ganciclovir/farmacología , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Hiperplasia , Técnicas In Vitro , Microscopía de Contraste de Fase , Conejos , Simplexvirus/genética , Timidina Quinasa/genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
PURPOSE: The most common complication of cataract surgery is the development of posterior capsule opacification (PCO). Hyperplasia of the lens epithelium is one of the main cellular events following phacoemulsification and was found to be an important feature contributing to opacification of the posterior capsule. We investigated the feasibility of killing the residual lens epithelial cells by retroviral-mediated transfer of the herpes simplex virus-thymidine kinase (HSV-tk) gene, a well-studied suicide gene, into rabbit lens epithelial cells followed by ganciclovir (GCV) treatment. METHODS: The capacity of retroviral vectors to transfer genes into rabbit lens epithelial cells was determined either in vitro (culture of rabbit lens epithelial cells) or in vivo (experimental model of PCO in rabbits) using cDNA encoding the beta-galactosidase (LacZ) reporter gene. To evaluate the efficiency of suicide gene therapy (infection with retroviral vectors encoding the HSV-tk gene followed by GCV treatment) we determined the sensitivity of HSV-tk infected lens epithelial cells to different concentrations of GCV in vitro. Then, in an experimental model of PCO, rabbits were treated with HSV-tk retroviral vectors at the end of the surgery and they received repeated intracameral and intravitreal injections of GCV at the concentration determined by the in vitro experiments. RESULTS: Infection efficiency using LacZ retroviral vectors was about 29% in vitro and 10% in vivo. After infection of the HSV-tk cDNA in vitro, the cell killing effect of GCV was evaluated. A significant enhancement (four- to five-fold) of the cell sensitivity to GCV was shown in FLY-DFGtk as compared with mock infected (P < 0.01) cells even without selection of the HSV-tk positive cells. The GCV concentration leading to 50% reduction in cell number (IC50) was 50 microg/ml. In vivo infection with a HSV-tk vector led to the tk gene transfer into lens epithelial cells. Despite this local HSV-tk gene expression, we could not prevent capsule opacification. CONCLUSIONS: Lens epithelial cells were successfully infected both in vitro and in vivo by beta-galactosidase and HSV-tk genes via retroviral vectors. In vitro infected lens epithelial cells displayed a strong sensitivity to GCV treatment. In vivo, we could not prevent capsule opacification in the rabbit model, very likely due to the limited level of the HSV-tk gene expression. However, our results suggest that virus-mediated suicide gene therapy might be a feasible treatment strategy to prevent capsule opacification with a more powerful vector.