[The effect of lentivirus-mediated suicide gene therapy on lens epithelial cells]
1Department of Ophthalmology, Eye and ENT Hospital, Fudan University, Shanghai 200031, China.
Objective:
To investigate the cytotoxicity of lentivirus-mediated herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) suicide gene therapy on human lens epithelial cell line and analyze the mechanism of cell death.
Methods:
a lentiviral containing the Lenti-HSVtk-EGFP as therapeutic vector and Lenti-EGFP as the control were used in the study. Transfection efficiency in vitro was assessed by fluorescence-activated cell sorting. Expression of HSV-tk in lens epithelial cells (LECs) mediated by lentivirus was examined by fluorescence microscope, genomic PCR and reverse transcription PCR. The cytotoxicity of HSV-TK/GCV suicide-gene system was assessed using DNA ladder and electron microscope. The time dependent transfection efficiency and bystander effect induced by the HSV-TK/GCV in LECs were evaluated.
Result:
the transduction efficiency was higher than 95%. When concentration of GCV was 15-25 microg/ml, apoptosis or necrosis was induced by Lenti-HSVtk-EGFP in HLE. The cytotoxicity was enhanced with increased time of transfection and concentration of GCV. Non transfected cells were also effectively killed by mixing the cell with GCV transfected cells (Bystander effect).
Conclusion:
GCV can effectively kill the LECs with the expressing of HSV-tk. Bicistronic lentiviral vectors can efficiently integrate multiple genes into LECs, therefore, it is a reliable vector for gene therapy; lentivirus mediated HSV-tk/GCV suicide gene therapy may provide an effective approach for the treatment of posterior capsule opacification.
Insights
Lentivirus-mediated herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) gene therapy effectively kills human lens epithelial cells, including neighboring cells. This approach shows promise for treating posterior capsule opacification.
Area of Science:
- Ophthalmology
- Gene Therapy
- Cell Biology
Context:
- Posterior capsule opacification (PCO) is a common complication after cataract surgery.
- Current treatments for PCO are limited and can have side effects.
- Novel therapeutic strategies are needed to prevent or treat PCO.
Purpose:
- To evaluate the efficacy and mechanism of lentivirus-mediated herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) suicide gene therapy in human lens epithelial cells (LECs).
- To assess the cytotoxicity and bystander effect of the HSV-TK/GCV system in LECs.
- To determine the potential of this gene therapy approach for treating PCO.
Summary:
- Lentiviral vectors achieved over 95% transduction efficiency in LECs.
- HSV-TK/GCV treatment induced apoptosis or necrosis in LECs at specific GCV concentrations (15-25 microg/ml).
- Cytotoxicity increased with transfection time and GCV concentration, demonstrating a significant bystander effect.
Impact:
- Lentivirus-mediated HSV-TK/GCV suicide gene therapy is a potent method for eliminating LECs.
- Bicistronic lentiviral vectors efficiently deliver multiple genes, offering a reliable platform for gene therapy.
- This approach holds significant potential as an effective treatment strategy for posterior capsule opacification.
