title: Using HSV-TK/GCV suicide gene therapy to inhibit lens epithelial cell proliferation for treatment of posterior capsular opacification.
abstract: OBJECTIVE
  To establish a novel, targeted lentivirus-based HSV-tk (herpes simplex virus thymidine kinase)/GCV (ganciclovir) gene therapy system to inhibit lens epithelial cell proliferation for treatment of posterior capsular opacification (PCO) after cataract surgery.
  
  
  METHODS
  An enhanced Cre recombinase (Cre/loxP) system with a lentiviral vector expressing Cre under the control of the lens-specific promoter LEP503 (Lenti-LEP503-HSVtk-Cre [LTKCRE]) was constructed, as well as another lentiviral vector containing a switching unit. The latter vector contains a stuffer sequence encoding EGFP (Lenti-hPGK-Loxp-EGFP-pA-Loxp-HSVtk [PGFPTK]) with a functional polyadenylation signal between two loxP sites, followed by the herpes simplex virus thymidine kinase (HSV-tk) gene, both under the control of the human phosphoglycerate kinase (hPGK) promoter. Expression of the downstream gene (HSV-tk) is activated by co-expression of Cre. Human lens epithelial cells (HLECs) or retinal pigmental epithelial cells (RPECs) were co-infected with LTKCRE and PGFPTK. The inhibitory effects on HLECs and RPECs infected by the enhanced specific lentiviral vector combination at the concentration of 20 µg/ml GCV were assayed and compared.
  
  
  RESULTS
  The specific gene expression of Cre and HSV-tk in HLECs is activated by the LEP503 promoter. LTKCRE and PGFPTK co-infected HLECs, but not RPECs, expressed high levels of the HSV-tk protein. After 96 h of GCV treatment, the percentage of apoptotic HLECs infected by the enhanced specific lentiviral vector combination was 87.23%, whereas that of apoptotic RPECs was only 10.12%. Electron microscopy showed that GCV induced apoptosis and necrosis of the infected HLECs.
  
  
  CONCLUSIONS
  The enhanced specific lentiviral vector combination selectively and effectively expressed HSV-tk in HLECs. A concentration of 20 µg/ml, GCV is effective against the proliferation of HLECs in vitro. This cell-type-specific gene therapy using a Cre/loxP lentivirus system may be a feasible treatment strategy to prevent PCO.
authors.affiliation: Department of Ophthalmology, Eye and ENT Hospital, Fudan University, 83 Fenyang Road, Shanghai, China.
authors.forename: Yong-Xiang
authors.initials: YX
authors.lastname: Jiang
authors.forename: Yi
authors.initials: Y
authors.lastname: Lu
authors.forename: Tian-Jing
authors.initials: TJ
authors.lastname: Liu
authors.forename: Jin
authors.initials: J
authors.lastname: Yang
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.forename: Yan-Wen
authors.initials: YW
authors.lastname: Fang
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D017209:Apoptosis; D002386:Cataract / Q000235:genetics* / Q000628:therapy*; D002387:Cataract Extraction / Q000379:methods*; D002460:Cell Line; D049109:Cell Proliferation; D005434:Flow Cytometry; D015774:Ganciclovir / Q000494:pharmacology*; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D019426:Integrases / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics; D008957:Models, Genetic; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic; D018139:Simplexvirus / Q000201:enzymology*; D013937:Thymidine Kinase / Q000235:genetics*
chemical_list: D000998:Antiviral Agents; D013937:Thymidine Kinase; C045073:Cre recombinase; D019426:Integrases; D015774:Ganciclovir