Gene Therapy (2000) 7, 679–685  2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt VIRAL TRANSFER TECHNOLOGY BRIEF COMMUNICATION Central nervous system toxicity of two adenoviral vectors encoding variants of the herpes simplex virus type 1 thymidine kinase: reduced cytotoxicity of a truncated HSV1-TK C Cowsill 1 , TD Southgate 1,* , G Morrissey 1,* , RA Dewey 1 , AE Morelli 1 , TC Maleniak 1 , Z Forrest 1 , D Klatzmann 2 , GWG Wilkinson 3 , PR Lo ¨ wenstein 1 and MG Castro 1 1 Molecular Medicine and Gene Therapy Unit, School of Medicine, University of Manchester, Manchester, UK; 2 Laboratoire de Biologie et Therapeutique des Pathologies Immunitaires, Universite ` Pierre et Marie Curie, CNRS, Ho ˆpital de la Pitie ´-Salpe ´trie `re, Paris, France; and 3 Department of Medicine, University of Wales College of Medicine, Heath Hospital, Heath Park, Cardiff, UK Herpes simplex virus type 1-thymidine kinase (HSV1-TK) in combination with ganciclovir is an efficient and widely used strategy in brain tumour gene therapy. Recently, we have shown effective inhibition of glioma growth in a syngeneic rat model using recombinant adenoviruses expressing the full-length HSV1-TK and an N-terminus truncated variant, HSV1-TK in the presence of ganciclovir. We also showed active chronic brain inflammation in the long-term survivors (3 months) treated with HSV1-TK plus GCV. Furthermore, our results indicated loss of myelinated fibres, oedema and indices of ongoing axonal degeneration. In this study, we assessed the cytotoxicity of both HSV1-TK variants in the presence or absence of ganciclovir, in primary cultures of neurones and glia, and in the rat brain in vivo. Our results Keywords: thymidine kinase; brain tumours; flow cytometry; neurological; cytotoxicity Retroviral, adenoviral, and herpes simplex virus type 1- mediated transfer of the HSV1-TK gene to mammalian brain tumour cells in combination with the peripheral administration of the nucleoside analogue ganciclovir (GCV) has been shown to be efficient as an antitumoral strategy in several preclinical studies. 1–8 Using this approach, several clinical trials for the treatment of differ- ent kinds of tumours have been initiated, 9 including clini- cal trials for brain tumours using retroviruses and adeno- viruses encoding HSV1-TK in combination with GCV. 10 When recombinant adenoviruses (RAds) encoding HSV1-TK are administered into the brain, they will also infect (and might be cytotoxic for) peritumoral normal neurones and other non-tumoral cells in the CNS (eg glia, endothelium, microglia, meningeal cells). High-dose adenovirus-mediated HSV1-TK gene transfer to baboon (1.5 × 10 9 total plaque forming units (p.f.u.)) and rhesus monkey brain (1.5 × 10 11 total p.f.u.) led to gliosis, Correspondence: MG Castro or PR Lo ¨wenstein, Molecular Medicine and Gene Therapy Unit, Room 1.302 Stopford Building, School of Medicine, University of Manchester, Oxford Road, Manchester M13 9PT, UK *These authors contributed equally to the work Received 17 June 1998; accpeted 8 December 1999 indicate that, at viral doses where tumour cells are sensitive to the enzyme/prodrug system, (1) there is no major cytotox- icity for either neurones or glial cells grown in primary cul- tures, (2) on its own the full-length HSV1-TK is more cyto- toxic than its truncated version HSV1-TK for a population of non-neuronal and non-glial cells within neocortical primary cultures, and (3) in vivo, when delivered into the striatum, RAds encoding HSV1-TK are more cytotoxic than RAds encoding HSV1-TK, after administration of ganciclovir. The effectiveness of HSV1-TK in preventing brain tumour growth in vivo, combined with its reduced cytotoxicity, both in vivo and in primary cultures of CNS cells, could represent an advantage for treatment of brain tumours using gene therapy. Gene Therapy (2000) 7, 679–685. necrosis, meningitis and even death upon GCV adminis- tration. 11,12 In vitro, Maron and co-workers 13 recently described that RAd-TK plus GCV is cytotoxic for astro- cytes grown in primary culture. Work from our labora- tory has recently shown that both HSV1-TK and an N- terminus truncated version HSV1-TK, encoded by recombinant adenoviruses are equally effective in inhibiting the growth of CNS-1 glioma cells in Lewis rats. 1 We also showed active brain inflammation, demyel- ination and indices of ongoing axonal degeneration, 3 months after successful inhibition of glioma growth after treatment with the recombinant adenovirus expressing the full-length HSV1-TK. 1 We therefore considered it of critical importance to study the cytotoxicity of HSV1-TK and HSV1-TK within infected cells from the rat central nervous system in vitro, and in the rat brain in vivo. Our results show that neither neurones nor glial cells in primary cultures were killed by HSV1-TK or HSV1-TK alone, or by GCV on its own. However, HSV1-TK was more toxic, both on its own and in the presence of GCV, to non-neuronal/non-glial cells present in primary CNS cell cultures, when compared with HSV1-TK. Similarly, the nonspecific toxicity, as well as the activation of astroglia and infiltration of