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 different kinds of tumours have been initiated, 9 including clinical trials for brain tumours using retroviruses and adenoviruses encoding HSV1-TK in combination with GCV. 10When 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, indicate that, at viral doses where tumour cells are sensitive to the enzyme/prodrug system, (1) there is no major cytotoxicity for either neurones or glial cells grown in primary cultures, (2) on its own the full-length HSV1-TK is more cytotoxic 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 administration. 11,12 In vitro, Maron and co-workers 13 recently described that RAd-TK plus GCV is cytotoxic for astrocytes grown in primary culture. Work from our laboratory has recently shown that both HSV1-TK and an Nterminus truncated version HSV1-⌬TK, encoded by recombinant adenoviruses are equally effective in inhibit...

Human embryonic kidney 293 cells contain the E1 region E1-E3 regions, in an unpackagable form. Investigation of of adenovirus type 5, and thus sustain, through transcompthe reason for massive cell death after cotransfection led lementation, the production of recombinant E1-deleted us to determine that 293 cells express the FasL receptor, adenovirus vectors. During attempts to produce recombiFas-Apo1 (CD95), and respond with apoptosis to the nant adenovirus expressing the apoptosis-inducing molcross-linking of Fas-Apo1 with either IgM monoclonal antiecule Fas ligand (FasL) under the control of a very strong bodies or FasL. Therefore, we decided to generate adenotruncated major immediate-early human cytomegalovirus viral vectors expressing FasL under the control of tissue-(MIEhCMV) promoter, we discovered that 293 cells were specific and/or -inducible promoter elements. Our findings not surviving the initial cotransfection with a shuttle plasmid can explain difficulties several groups have had in generatencoding the mouse FasL; and pJM17, a plasmid containing recombinant adenoviral vectors expressing FasL using ing the genome of adenovirus type 5 with deletions in the 293 cells, as well as the lower titres reported.

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We have designed a system in which to test gene transferWe encountered a distinctive staining of cells arranged in into gut neurons consisting of an organ culture of neonatal two concentric circles corresponding in location to the rat small intestine. The tissue was exposed to herpes simmyenteric and submucosal plexuses. Cells in these areas plex-and adenovirus-derived vectors: (1) a temperaturewere of similar size and morphology to neonatal enteric sensitive herpes simplex virus-1 (HSV1) vector (tsK-␤gal) neurons, as visualized by NADPH-diaphorase histochemicontaining the lacZ gene encoding ␤-galactosidase (␤-gal), stry and immunocytochemical staining with antibodies to under the transcriptional control of the HSV1 immediatethe neuronally expressed proteins PGP 9.5, or neurofilaearly 3 (IE3) promoter; (2) RAd35, an E1 − /E3 − replicationments. Double labelling with antibodies recognizing neurodeficient adenovirus expressing lacZ under the control of filaments and ␤-galactosidase revealed that most cells a truncated HCMV major IE promoter; and (3) RAd122, an infected by tsK were neurons, while the RAd35 and 122 E1 − /E3− replication-deficient adenovirus expressing the vectors only infected non-neuronal cells. We thus demonlacZ under the control of the RSV LTR. Forty-eight hours strate that both HSV1-and adenovirus-derived vectors can after the vector was added to the organ culture, we be used to transfer genes to the gut in vitro, but they transdetected ␤-gal using immunohistochemistry or X-gal histoduce different populations of target cells. chemistry in tissue sections examined by light microscopy.