Retrovirus-mediated transfer of the suicide gene into retinal pigment epithelial cells in vitro.
OBJECTIVE
Human retinal pigment epithelial (HRPE) cells are a major cell component in proliferative vitreoretinopathy (PVR) membranes. We investigated the feasibility of killing HRPE cells by retroviral-mediated transfer of the herpes simplex virus-thymidine kinase (HSV-tk) gene, also known as the suicide gene, into HRPE cells followed by ganciclovir treatment, and to study the so-called bystander effect. Such a treatment plan might serve as a possible therapy for PVR.


METHODS
Transduction efficiency was determined using retroviral vectors encoding the beta-galactosidase reporter gene. To evaluate the efficacy of suicide gene therapy. HRPE cells were transduced with retroviral vectors encoding the HSV-tk gene (G1TkSvNa), with empty vectors or without vectors, and were treated with 5 micrograms/ml ganciclovir. Sensitivity of HSV-tk positive HRPE cells to various concentrations of ganciclovir was evaluated. To demonstrate the bystander effect, HSV-tk positive cells were cultured with HSV-tk negative cells at varying proportions.


RESULTS
Transduction efficiency in vitro was 15.1 +/- 4.8%. Cell growth was significantly inhibited after transduction with G1TkSvNa followed by ganciclovir treatment (P < 0.01). Ganciclovir showed dose- and time-dependent cytotoxicity only on HSV-tk positive cells. The concentration that resulted in 50% inhibited was 0.1 micrograms/ml. In terms of the bystander effect, after ganciclovir treatment the viability of co-cultured cells decreased with increasing populations of HSV-tk positive cells.


CONCLUSIONS
HRPE cells were successfully transduced with the HSV-tk gene via retroviral vectors and displayed a strong bystander effect after treatment with ganciclovir. These results suggest that retrovirus-mediated suicide gene therapy might be a feasible treatment strategy for PVR.
Schubert
C A
CA
Doheny Eye Institute, Los Angeles, CA 90033, USA.
Kimura
H
H
Spee
C
C
Hinton
D R
DR
Gordon
E M
EM
Anderson
W F
WF
Ryan
S J
SJ
D000998:Antiviral Agents / Q000009:adverse effects / Q000494:pharmacology; D016923:Cell Death; D002478:Cells, Cultured; D018920:Coculture Techniques; D015774:Ganciclovir / Q000009:adverse effects / Q000494:pharmacology; D018014:Gene Transfer Techniques; D006801:Humans; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D012190:Retroviridae / Q000235:genetics*; D018139:Simplexvirus / Q000201:enzymology*; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism
D000998:Antiviral Agents; D013937:Thymidine Kinase; D015774:Ganciclovir