Lack of enantioselectivity of herpes virus thymidine kinase allows safer imaging of gene delivery

Abstract

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 b-thymidine. Here we show that, after phosphorylation by HSV-TK, tritiated L-b-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.

Noninvasive Molecular Neuroimaging Using Reporter Genes: Part II, Experimental, Current, and Future Applications

“…As an example of the latter, Majumdar et al 81 have tested modified dipeptide monoester ganciclovir prodrugs for their greater solubility and permeability. On the other hand, Ma- grassi et al 82 exploited the fact that HSV1-tk is not enantioselective and can therefore efficiently phosphorylate both D and L enantiomers of ␤-thymidine. Using autoradiography, they showed that tritiated l-␤-thymidine is selectively retained to a significant extent in experimental intracranial gliomas.…”