Lack of enantioselectivity of herpes virus thymidine kinase allows safer imaging of gene delivery.
Herpes simplex virus thymidine kinase (HSV-TK) is widely used in gene therapy. The enzymatic activity of HSV-TK may be traced in vivo by specific radiopharmaceuticals in order to image transgene expression. However, most of these radiopharmaceuticals are toxic per se or after activation by HSV-TK, and therefore do not represent ideal molecules for clinical applications and repeated imaging. Unlike human cytosolic TK, HSV-TK is not enantioselective and can efficiently phosphorylate both D and L enantiomers of beta-thymidine. Here we show that, after phosphorylation by HSV-TK, tritiated L-beta-thymidine (LT) is selectively retained inside the cells in vitro and in vivo. We used the in vivo accumulation of radioactive phosphorylated LT to image the HSV-TK-positive cells inside a transplantable murine brain tumour after inoculation of cells producing retroviruses carrying HSV-TK. Owing to their unnatural enantiomeric conformation, phosphorylated LT metabolites are very poorly processed by mammalian enzymes, thus leading to increased cellular retention and minimal toxicity. The ability to image cells expressing the HSV-TK gene by using radiolabelled LT, without damaging the cells accumulating the phosphorylated L-nucleoside, will be important to monitor the levels and spatial distribution of therapeutic vectors carrying HSV-TK.
Neurosurgery, Department of Surgery, University of Pavia, IRCCS Policlinico S. Matteo, Pavia, Italy.
L
L
Magrassi
G
G
Finocchiaro
G
G
Milanesi
F
F
Benvenuto
S
S
Spadari
F
F
Focher
D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D045744:Cell Line, Tumor; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007527:Isoenzymes; D051379:Mice; D008810:Mice, Inbred C57BL; D010766:Phosphorylation; D019275:Radiopharmaceuticals / Q000378:metabolism; D018139:Simplexvirus / Q000201:enzymology*; D013936:Thymidine / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics*; D019076:Transgenes; D014316:Tritium / Q000378:metabolism
D007527:Isoenzymes; D019275:Radiopharmaceuticals; D014316:Tritium; D013937:Thymidine Kinase; D013936:Thymidine