We have developed and validated a new tumor-targeting gene therapy strategy based upon the targeting and replacement of human telomerase reverse transcriptase (hTERT) RNA, using a trans-splicing ribozyme. By constructing novel adenoviral vectors harboring the hTERT-targeting trans-splicing ribozymes with the downstream reporter gene (Ad-Ribo-LacZ) or suicide gene (Ad-Ribo-HSVtk) driven by the cytomegalovirus (CMV) promoter, we demonstrated that this viral system selectively marks tumor cells expressing hTERT or sensitizes tumor cells to prodrug treatments. We confirmed that Ad-Ribo-LacZ successfully and selectively delivered a ribozyme that performed a highly specific trans-splicing reaction into hTERT-expressing cancer cells, both in vitro and in a peritoneal carcinomatosis nude mouse model. We also determined that the hTERT-specific expression of the suicide gene in the Ad-Ribo-HSVtk, and treatment with the corresponding prodrug, reduced tumor progression with almost the same efficacy as the strong constitutive CMV promoter-driven adenovirus, both in cancer cell lines and in nude mouse HT-29 xenografts. These observations provide the basis for a novel approach to cancer gene therapy, and demonstrate that trans-splicing ribozymes can be employed as targeting anti-cancer agents which recognize cancer-specific transcripts and reprogram them, thereby combating cancerous cells.

HSV-tk/ ganciclovir (GCV) gene therapy has been extensively studied in the setting of brain tumors and largely relies on the bystander effect. Large studies have however failed to demonstrate any significant benefit of this strategy in the treatment of human brain tumors. Since dexamethasone is a frequently used symptomatic treatment for malignant gliomas, its interaction with the bystander effect and the overall efficacy of HSV-TK gene therapy ought to be assessed.

Stable clones of TK-expressing U87, C6 and LN18 cells were generated and their bystander effect on wild type cells was assessed. The effects of dexamethasone on cell proliferation and sensitivity to ganciclovir were assessed with a thymidine incorporation assay and a MTT test. Gap junction mediated intercellular communication was assessed with microinjections and FACS analysis of calcein transfer. The effect of dexamethasone treatment on the sensitivity of TK-expressing to FAS-dependent apoptosis in the presence or absence of ganciclovir was assessed with an MTT test. Western blot was used to evidence the effect of dexamethasone on the expression of Cx43, CD95, CIAP2 and BclXL.

Dexamethasone significantly reduced the bystander effect in TK-expressing C6, LN18 and U87 cells. This inhibition results from a reduction of the gap junction mediated intercellular communication of these cells (GJIC), from an inhibition of their growth and thymidine incorporation and from a modulation of the apoptotic cascade.

The overall efficacy of HSV-TK gene therapy is adversely affected by dexamethasone co-treatment in vitro. Future HSV-tk/ GCV gene therapy clinical protocols for gliomas should address this interference of corticosteroid treatment.

Suicide gene therapy could be an attractive addition to the treatment of ovarian carcinomas, for which acquired chemoresistance frequently results in treatment failure. Here we show that transfection of the HSV-tk gene, followed by incubation with up to 1 mM ganciclovir fails to induce cell death in SKOV3 chemoresistant human ovarian carcinoma cells. However, co-transfection of HSV-tk with Cip1/Waf1 encoding the p21(cip1/waf1) inhibitor of cdks, allows 100 microM ganciclovir to eradicate the population of tumor cells. Potentiation of a drug by co-transfer of HSV-tk with Cip1/Waf1could thus represent another therapeutic approach for tumours that are resistant to conventional therapy.

Neuroblastoma arises as a direct result of genetic disorder; therefore, it should be well treated and conquered by gene therapy in future. In this study, neuroblastoma cell line SH-SY5Y experiments, in vitro and in nude mice in vivo, were subjected to research thymidine kinase suicide gene to treat neuroblastoma. The plasmid LXpsp-hytk and a plasmid LXSH were transduced separately by lipofectin into human neuroblastoma cell line SH-SY5Y. SH-SY5Y-hy and SH-SY5Y-hytk were selected by hygromycin B. Different ganciclovir (GCV) concentration was given to SH-SY5Y-hytk to determine optimal GCV concentration. The cytotoxic effect of GCV on SH-SY5Y-hytk, SH-SY5Y-hy, and SH-SY5Y cells was determined. Scapular subcutaneous tumors were established in nude mice by inoculating 2.5 x 10(6) SH-SY5Y-hytk on their left sides and 2.5 x 10(6) SH-SY5Y-hy cells on their right sides for every mouse of treatment group and control group, respectively. After 1 week, mass grew in both sides of all the mice, and from the eighth day on, every mouse in treatment group received daily intraperitoneal injection of GCV 50 mg/kg body weight for 14 days; every mouse in control group received daily intraperitoneal injection of 1 ml saline for 14 days. On day 22 tumors were excised and weighed on the left and right sides, respectively, and apoptosis was detected by TUNEL method. Apoptotic index was calculated on the left and on the right sides, respectively, for every mouse in treatment group and control group. The lowest concentration of hygromycin B was 60 microg/ml. The cytotoxic effect of GCV on SH-SY5Y-hytk cells was obvious (IC(50)=0.03 microM), whereas GCV showed almost no cytotoxic effect on SH-SY5Y and SH-SY5Y-hy cells (IC(50)>400 microM). SH-SY5Y-hytk was killed by concentrations of 30 microM GCV effectively and it obviously showed the bystander effect, when SH-SY5Y-hytk remained at least 18% in the mixture culture cells. The tumor on the left side was much smaller than that of the right side in control group (p<0.05), and apoptotic index of the left was higher than that of the right in control group (p<0.01). SH-SY5Y-hytk has the bystander effect over 18% SH-SY5Y-hytk of the mixture culture cells at the concentration of 30 microM GCV. The HSV-tk/GCV system was effective in treating SH-SY5Y neuroblastoma cell line in vivo as well. Our findings suggest that thymidine kinase gene therapy could be a potential method for treating neuroblastoma in the future.

Early passages of cultured cells derived from four spontaneous Balb/c murine adenocarcinomas were used to explore the feasibility of a nonviral HSVtk-based suicide gene therapy system. After lipofection with pCMVtk, the transiently HSVtk expressing P07 (lung), M3, M05, and M38 (mammary gland) cells were, respectively, about 130-, 30-, 120-, and 170-fold more sensitive to ganciclovir (GCV) in vitro than their respective controls. Eighty percent of Balb/c mice subcutaneously inoculated with ex vivo pCMVtk-lipofected P07 cells, followed by intraperitoneal GCV injection for 7 days, displayed a complete inhibition of tumor growth for over 70 days. Control animals started to display tumors 13 days after inoculation. We present evidence showing that early passages of cultured tumor cells can efficiently express lipofected genes and that they are sensitive to the lipoplex-mediated HSVtk/GCV system.

Intraperitoneal chemohyperthermia (IPCH) is a loco-regional treatment for intraperitoneal malignancies. This ultra-radical treatment combines complete cytoreduction of macroscopic peritoneal disease preceding perioperative intraperitoneal perfusion of a chemotherapeutic drug heated to 42 degrees to 44 degrees to treat microscopic residual disease. At present time, this approach is mainly indicated for isolated limited peritoneal carcinomatosis (PC) of colorectal origin and for treatment of low-grade pseudomyxoma peritonei. In selected patients, IPCH may lead to 27% five-year overall survival in cases of PC, and as high as 86% five-year survival in cases of pseudomyxoma peritonei. In the near future, this approach will become the standard treatment for selected cases of PC.

Several experimental approaches have been tested for suicide gene delivery into tumor cells, including viral and non-viral vectors. In this study, we compared the efficiency of Herpes Simplex Virus type 1 thymidine kinase gene (HSV-tk) delivery by retrovirus-producing cells and DNA/liposome complexes for the treatment of peritoneal carcinomatosis induced in syngeneic rats by DHD/K12 colorectal adenocarcinoma cells. After in vitro determination of the best transduction conditions, rats were treated with multiple intraperitoneal injections of plasmid DNA containing one or two copies of CMV-driven HSV-tk gene (pCMV-TK and p(CMV-TK)2, respectively) associated with LipofectAMINE, each injection being followed by a Ganciclovir (GCV) course. Animals treated by DNA/liposome complexes and GCV or with retrovirus-producing cells and GCV showed a similar increase of survival as compared to the control group. After DNA/ liposome injections, expression of the tk transgene was detected in tumor nodes (epiploon) and also in liver, lung, spleen, bowels and brain. The expression was not homogeneous throughout the different organs and most likely reflected the transfection of only a limited number of cells.

Carcinomatous peritonitis is characterized by massive malignant ascites, while peritoneally disseminated carcinomatosis is characterized by a large number of metastatic solid tumors in the peritoneal cavity. Although both are fatal end-stage manifestations of malignancies derived from the digestive system, the former is usually more serious than the latter due to massive malignant ascites. Although the effectiveness of gene therapy against peritoneally disseminated carcinomatosis has been shown in animal experiments, its effectiveness against carcinomatous peritonitis remains to be examined.

A carcinomatous peritonitis model was made by inoculating murine hepatocellular carcinoma cells, MH134, into the peritoneal cavity of syngeneic C3H/He mice, resulting in production of massive malignant ascites without development of intraperitoneal solid tumors. Model animals were injected intraperitoneally with retroviruses carrying the herpes simplex virus thymidine kinase (HSV-tk) gene followed by ganciclovir (GCV) treatment.

Retrovirus-mediated in vivo gene therapy with the HSV-tk/GCV system was shown to have a significant impact on survival of animals with carcinomatous peritonitis not only at an early stage, but also at an advanced stage. Furthermore, repeated injections of HSV-tk-carrying retroviruses significantly prolonged the survival of animals with carcinomatous peritonitis compared with a single injection protocol. When intraperitoneal administration of recombinant interleukin-2 (IL-2) was added to the HSV-tk/GCV system, levels of IL-1beta and IL-2 in malignant ascites were significantly increased, resulting in significantly reduced ascite volume and prolonged survival.

Our results indicate the feasibility of retrovirus-mediated in vivo gene therapy with the HSV-tk/GCV system plus IL-2 treatment against carcinomatous peritonitis.

We demonstrate that fusion proteins consisting of the herpes simplex virus (HSV) transport protein VP22 linked in frame to HSV thymidine kinase (tk) retain the ability to be transported between cells. In vivo radiolabelling experiments and in vitro assays show that the fusion proteins also retain tk activity. When transfected COS cells, acting as a source of the VP22-tk chimera, were co-plated on to gap junction-negative neuroblastoma cells, ganciclovir treatment induced efficient cell death in the recipient neuroblastoma cell monolayer. No such effect was observed with COS cells transfected with tk alone. Tumours established in mice with neuroblastoma cell lines expressing VP22-tk regressed upon administration of ganciclovir. Furthermore tumours established from 50:50 mixtures of VP22-tk transduced and nontransduced cells also regressed while no significant effect was observed in similar experiments with cells transduced with tk alone. VP22 mediated transport may thus have application in a clinical setting to amplify delivery of the target protein in enzyme-prodrug protocols.