Gene Therapy (2000) 7, 1483–1490  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt ACQUIRED DISEASES RESEARCH ARTICLE HSV vector cytotoxicity is inversely correlated with effective TK/GCV suicide gene therapy of rat gliosarcoma S Moriuchi 1,2 , DM Krisky 1 , PC Marconi 1,3 , M Tamura 1 , K Shimizu 2 , T Yoshimine 2 , JB Cohen 1 and JC Glorioso 1 1 Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA; and 2 Department of Neurosurgery, Osaka University Medical School Osaka, Japan Herpes simplex virus (HSV)-mediated delivery of the HSV thymidine kinase (tk) gene to tumor cells in combination with ganciclovir (GCV) administration may provide an effective suicide gene therapy for destruction of malignant glioblas- tomas. However, because HSV is a highly cytotoxic agent, gene expression from the virus is short-lived which may limit the effectiveness of HSVtk/GCV therapy. Using different rep- lication-defective HSVtk gene vectors, we compared HSV vector backgrounds for their cytotoxic activity on infection of 9L gliosarcoma cells in culture and brain tumors in rats and evaluated the impact of vector toxicity on the effectiveness of tk/GCV-mediated suicide gene therapy. As reported pre- viously for other cell lines, a vector deleted for both copies of the immediate–early (IE) gene ICP4 (SOZ.1) was highly toxic for 9L cells in culture while a vector deleted in addition for the ICP22 and ICP27 IE genes (T.1) reduced or arrested Keywords: herpes simplex virus; gene therapy; glioblastoma; cytotoxicity; HSV thymidine kinase; ganciclovir Introduction Despite recent advances in the treatment of malignant gliomas, the prognosis for patients suffering from this disease continues to be poor. 1–3 Current therapies involve the use of surgery, radiotherapy, and chemotherapy, but these measures are inadequate due to the significant mor- bidity associated with these procedures, the infiltrative nature of gliomas, and the development of drug resist- ance. 4–6 The challenges of malignant gliomas are unique given the immuno-privileged status of the central ner- vous system and the confined and sensitive nature of the tumor environment. A novel approach to the treatment of these tumors is the use of virus-based gene therapeutic vectors. The aim of these approaches is to transduce tumors with genes that can aid in the specific destruction of tumor cells. For example, an antitumor immune reac- Correspondence: JC Glorioso, Department of Molecular Genetics and Bio- chemistry, E1240 Biomedical Science Tower, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA 3 Current address: Department of Experimental and Diagnostic Medicine (Section of Microbiology) and Biotechnology Center, University of Fer- rara, Ferrara, Italy Received 15 March 2000; accepted 30 May 2000 9L cell proliferation with more limited cell killing. Neverthe- less, both vectors supported widespread killing of uninfected cells in the presence of GCV following low multiplicity infec- tions, indicating that vector cytotoxicity did not preempt the production of vector-encoded TK enzyme necessary for the killing of uninfected cells by the HSV-tk/GCV bystander effect. Although an SOZ.1-related vector (SHZ.2) caused tumor cell necrosis in vivo, injection of SHZ.2 at multiple coordinates thoughout the tumor followed by GCV adminis- tration failed to prolong markedly the survival of tumor- bearing rats. In contrast, a single injection of T.1 produced a life-extending response to GCV. These results indicate that vector cytotoxicity can limit the efficacy of HSV-tk/GCV treatment in vivo, which may be due to premature termin- ation of tk gene expression with attendant abortion of the bystander effect. Gene Therapy (2000) 7, 1483–1490. tion may be triggered upon delivery of immunostimula- tory gene products although immunosuppressive sub- stances commonly secreted by gliomas 7,8 may hinder this process. Another strategy involves blocking DNA syn- thesis through the delivery of ‘suicide genes’ such as those encoding herpes simplex virus thymidine kinase (HSV-TK), cytosine deaminase (CD), or cytochrome p450 2B1. The products of these genes have the ability to con- vert certain nontoxic pro-drugs to activated forms which interfere with DNA synthesis. Most cells in the central nervous system are nonreplicating and terminally differ- entiated suggesting that local gene therapy leading to the cessation of DNA synthesis should be largely tumor cell specific. Previous experiments using various viral vectors to deliver the HSVtk gene as part of a suicide gene therapy strategy have yielded generally positive results in a num- ber of glioma models. 9–12 The HSV-TK protein phos- phorylates the nontoxic pro-drug ganciclovir (GCV), which results in the generation of a defective nucleoside analogue. This product, when incorporated into cellular DNA during replication, leads to DNA chain termination and selective killing of dividing cells. Of particular inter- est in this strategy is the ability of the defective nucleo- side analogue to be passed to cells that are in physical