The herpes simplex virus type 1 thymidine kinase suicide idine. Using various murine and human cell lines expressgene (HSV1tk) together with ganciclovir (GCV) have been ing these viral tk, we show that HSV1-and EHV4tk are successfully used for in vivo treatment of various experithe more efficient suicide genes for the different nucleoside mental tumors, and many clinical trials using this system analogs tested. Moreover, EHV4tk expressing murine and have been launched. With the aim to improve this therahuman cells were three-to 12-fold more sensitive to GCV peutic system, we compared the potential efficacy of differthan HSV1tk expressing cells. This was correlated with the ent herpes virus derived thymidine kinases (HSV1, varpresence of five-fold higher amounts of the toxic triphosicella-zoster virus, equine herpes virus type-4 and Epsteinphated-GCV in EHV4-versus HSV1tk expressing cells. Barr virus) as suicide genes in association with the nucleoAltogether, these experiments underline the potential side analogs acyclovir, ganciclovir and bromovinyldeoxyuradvantages of the EHV4tk as a suicide gene. Keywords: nucleoside analogs; cancer; fusion proteinsThe herpes simplex virus type 1 thymidine kinase gene (HSV1tk) is the most widely used suicide gene, both in experimental settings and clinical trials. 1 Expression of the HSV1tk gene renders tumor cells sensitive to antiviral agents like acyclovir (ACV), ganciclovir (GCV) and bromovinyl deoxyuridine (BVDU). These nucleoside analogs are efficiently converted to their monophosphate form by HSV1tk, and are then converted to triphosphate compounds by host cellular kinases. Incorporation of these metabolites into elongating DNA blocks elongation leading to cell death. [2][3][4] The HSV1tk/GCV system has proved efficient for inducing the regression of transplanted tumors in various animal models, 5 as well as of carcinogen-induced tumors. 6 Based on these favourable results, several clinical gene therapy trials are in progress aimed at assessing safety and efficacy of this treatment for treating malignancies, and preliminary results have now been reported. 7,8 Despite the effectiveness of the HSV1tk/GCV system for killing tumor cells, it remains important to try to improve it, with the aim to maximize therapeutic efficacy and/or to facilitate treatment modalities. We thus looked for new suicide gene/prodrug combinations. Biochemical and in vivo studies have demonstrated different substrate specificity of herpes tk towards antiviral and cytostatic Correspondence: D KlatzmannReceived 8 January 1999; accepted 11 May 1999 nucleoside analogs. [9][10][11] We thus compared tk from HSV1, varicella-zoster virus (VZV), equine herpes virus type-4 (EHV4) and Epstein-Barr virus (EBV) for their capacity to sensitize tumor cells to ACV, GCV and BVDU.We generated expression vectors with each of these tk genes fused in frame with the Sh Ble gene conferring resistance to zeocin. 12 This should facilitate the selection of transduced cells and the detection of the chimeric proteins by immun...

Many antiviral drugs must be metabolized to their active form by cellular enzymes. Their antiviral activity may therefore be limited by an inefficient metabolism, leading to low intracellular concentration of the active form or to the accumulation of toxic intermediate metabolites. Gene transfer might be used to overcome such limitations by transducing a gene able to increase intracellular drug metabolism. To prove such a concept, we chose the well-studied paradigm of zidovudine (AZT) metabolism and anti-HIV activity. AZT-triphosphate is the active form of AZT, acting through inhibition of HIV reverse transcription. In human cells, the rate-limiting step for AZT phosphorylation is catalyzed by the thymidylate kinase. We thus tested the capacity of herpes simplex virus type 1 thymidine kinase, which possesses a thymidylate kinase activity, to improve AZT metabolism and antiviral activity. Our results show enhanced AZT phosphorylation in HSV-1 TK-expressing lymphoid and monoblastoid cells, which correlated with significantly improved antiviral activity against different strains of HIV-1. The antiviral activity of Foscarnet, another reverse transcriptase inhibitor that does not require phosphorylation, remained unchanged. These results suggest that gene transfer might be envisioned for genetic pharmacomodulation of antiviral drugs.

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