Effects of bicistronic lentiviral vector-mediated herpes simplex virus thymidine kinase/ganciclovir system on human lens epithelial cells.
Posterior capsule opacification (PCO) is the most common complication after phacoemulsification cataract surgery. Hyperplasia of the lens epithelial cell after phacoemulsification is thought to be an important feature contributing to PCO. In this study,we investigated the feasibility of killing the human lens epithelial cells (HLECs) by lentivirus-mediated herpes simplex virus thymidine kinase (HSV-tk) gene/ganciclovir (GCV) in HLECs and studied the bystander effect. HLECs were infected with lentiviral vectors coexpressing HSV-tk and enhanced green fluorescent protein (EGFP) or expressing EGFP alone and treated with ganciclovir. Infection efficiency was assessed by fluorescence microscopy, fluorescence-activated cell sorting, and reverse transcription PCR. The cytotoxicity of the HSV-tk/GCV suicide gene therapy system was assessed by DNA ladder and electron microscopy. The time effect and bystander effect of HLEC growth inhibition were evaluated with cell proliferation assay. Lentiviral vector-mediated stable integration and efficient expression of HSV-tk in HLECs, with infection efficiency exceeding 95% GCV at concentrations of 15 approximately 25 mug/ml, significantly induced apoptosis or necrosis of infected HLECs. GCV also killed normal cells mixed with HSV-tk infected cells. The bystander effect markedly increased the cytotoxicity of the HSV-tk/GCV system. Our results suggest that bicistronic lentiviral vectors can efficiently integrate several genes into HLECs and may be a gene therapy platform. Lentivirus-mediated suicide gene therapy might be a feasible treatment strategy to prevent capsule opacification.
Yang
Jin
J
Department of Ophthalmology, Eye and ENT Hospital, Fudan University. Shanghai, China.
Liu
Tian Jin
TJ
Lu
Yi
Y
D000998:Antiviral Agents / Q000627:therapeutic use*; D024201:Bystander Effect / Q000187:drug effects*; D018929:Cell Culture Techniques; D016923:Cell Death; D053938:DNA Fragmentation; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D015774:Ganciclovir / Q000627:therapeutic use*; D015870:Gene Expression; D041722:Genes, Transgenic, Suicide / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000201:enzymology*; D006801:Humans; D007908:Lens, Crystalline / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D013937:Thymidine Kinase / Q000235:genetics*
D000998:Antiviral Agents; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase; D015774:Ganciclovir