TK-GFP fusion gene virus vectors as tools for studying the features of HSV-TK/ganciclovir cancer gene therapy in vivo.
The fusion gene of herpes simplex virus thymidine kinase and green fluorescent protein (TK-GFP) was shown to be a versatile tool for examining the features of thymidine kinase/ganciclovir gene therapy in vitro. In this study, we used viral vectors carrying the fusion gene to characterize the aspects of this gene therapy form in rodent tumor models. Growth of subcutaneous 9L rat tumors transduced ex vivo with TK-GFP gene was prevented when ganciclovir (GCV) treatment was initiated immediately after tumor inoculation. Established tumors (>100 mm(3)), however, were untreatable despite the initial 55% proportion of TK-GFP positive cells. This was due to a rapid clearance of TK-GFP positive cells, but not GFP positive cells. Propidium iodide staining revealed that TK-GFP lentivirus vector was able to induce apoptosis/necrosis in 9L cells, as opposed to the respective GFP vector. Furthermore, when a subcutaneous nude mouse tumor model was used, the percentage of TK-GFP positive cells in vivo was maintained similarly as in cultured cells, suggesting contribution of a fully functional immune response to the disappearance of fusion gene positive cells. In vivo gene transfer studies: adenovirus TK-GFP vector injections resulted in about 25% gene transfer efficiency to 9L tumors and showed that their growth could be significantly reduced even when the tumor volumes were already >120 mm(3). Part of the effect was shown to be due to cytotoxicity of the vector. In summary, our results demonstrate the utility of TK-GFP fusion gene-carrying viral vectors in animal studies and show that readily detectable therapeutic genes can help us to understand the complicated nature of in vivo cancer gene therapy experiments.
Pasanen
Tiina
T
Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, PO Box 1627 (Neulaniementie 2), FIN-70211 Kuopio, Finland.
Hakkarainen
Tanja
T
Timonen
Pekka
P
Parkkinen
Jyrki
J
Tenhunen
Anni
A
Loimas
Sami
S
Wahlfors
Jarmo
J
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D015774:Ganciclovir / Q000494:pharmacology*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D009369:Neoplasms / Q000628:therapy*; D011419:Propidium / Q000494:pharmacology; D051381:Rats; D018139:Simplexvirus / Q000201:enzymology*; D013937:Thymidine Kinase / Q000235:genetics*; D013997:Time Factors; D019076:Transgenes
D000998:Antiviral Agents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D011419:Propidium; D013937:Thymidine Kinase; D015774:Ganciclovir