Retrovirus-mediated transfer of a suicide gene into lens epithelial cells in vitro and in an experimental model of posterior capsule opacification.
OBJECTIVE
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.
Couderc
B C
BC
Institut Claudius Regaud et Faculté des Sciences Pharmaceutiques, EA UPRES 2048 Toulouse, France.
de Neuville
S
S
Douin-Echinard
V
V
Serres
B
B
Manenti
S
S
Darbon
J M
JM
Malecaze
F
F
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology / Q000627:therapeutic use; D002386:Cataract / Q000235:genetics / Q000517:prevention & control*; D002452:Cell Count / Q000187:drug effects; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism; D005260:Female; D005697:Galactosides / Q000378:metabolism; D015774:Ganciclovir / Q000494:pharmacology / Q000627:therapeutic use; D015971:Gene Expression Regulation, Enzymologic; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D018259:Herpesvirus 1, Human / Q000201:enzymology / Q000235:genetics; D007211:Indoles / Q000378:metabolism; D007903:Lens Capsule, Crystalline / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D013194:Staining and Labeling; D013937:Thymidine Kinase / Q000235:genetics*; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D000998:Antiviral Agents; D005697:Galactosides; D007211:Indoles; D011993:Recombinant Fusion Proteins; D013937:Thymidine Kinase; D001616:beta-Galactosidase; D015774:Ganciclovir; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside