Experimental Studies On Therapy For Gastrointestinal Malignant Tumor With Oncolytic Adenovirus Driven By HTERT Promoter And Carrying Mouse Interferon Gamma Gene

BackgroundGeneral treatment has become a basic principle in the therapy of malignant neoplasms,while single method can not obtain a satisfactory result at present.So it is necessary to explore new methods which must have novel mechanisms of action and thereby lack cross-resistance with currently available treatments.Virotherapy with this characteristic is paid more and more attention to in recent years.The adenovirus has several features that make it be studied best. Adenenovirus is a mildly pathogenic human virus, rapidly infect a broad range of human cells including proliferation and nonproliferating cells and tend to yield high levels of adenoviral doses. In addition adenoviral vectors are relatively easy to manipulate, purificate and manufacture using recombinant DNA techniques, and inserted foreign genes are generally maintained without change through successive rounds of viral replication.In the other hand, Adenenovirus vectors have poor therapeutic index and low efficiency of gene transfer. Therefore it is indispensable to reconstruct adenenovirus. Abnormal biological characters shared by multiple cancers have made it possible for us to optimize virus so that the geneticallly-engineered virus(we called it replication-selective oncolytic adenoviral vector) replicates exclusively in tumor cells while leaving normal cells intact by using the following two broad approaches, one is to complement loss-of-function mutations in cancers with loss-of-function mutations within the virus genome,another, regulate the expression of important gene products in the viral life circle by utilizing tissue/tumor specific promoters. ONYX-015 (d11520) is studied earliest and the best victory of such a kind geneticallly-engineered virus at present.ONYX-015,whose E1b-55kD(encoding E1B protein which can bind and inactivate p53) has been deleted,is limited in replication in normal cells;cancer cells with malfunction p53,however, should support virus replication and cell lysis, The p53 is lost or mutated in most of all human cancers,so ONYX-015 can exclusively kill those tumor cells. Combining with chemotherapy(5-Fu/cisplatin), ONYX-015 led to objective clinical responses in 63% of 30 evaluable patients with neck and head tumors, which is one of the best efficiency in the knubbly therapy.Now ONYX-015 has been applied in 15 clinical center and more than 10 kinds of tumors including neck and head tumors,hepatocellular carcinoma, pancreatic cancer, colonal cancer and ovarian cancer. However,being a single agent(approximately 20%)and used in a phase III trial, ONYX-015 is disappointing.In theory ONYX-015 can replicate in tumor cells,lyse them and then cause a further infection in adjacent tumor cells, finally spread in tumor tissues and kill all tumor cells. But in fact biological characters of tumors and virues are very complicated, and the treatment efficacy is influenced by many factors:①The proliferous selectivity is not as expected.The early studies showed that ONYX-015 can selectively replicate in tumors with lost P53 function while cannot replicate in wide type p53 and normal cells.But the secondary studies indicated that ONYX-015 could not replicate in all tumors with lost P53 function and could replicate in part wide type p53 and normal cells.②The p53 lost or mutated is only 34-70% in adenocarcinoma cells.③There are many adverse factors for viral spreading such as difference of heterogeneity in tumor cells, haemorrhage,necrosis, fibrosis or normal cells in tumor.④Adenoviral neutralization antibodies exsiting in 70-80% normal people influences the dispersing and reinfecting of the virus.⑤The gene transfer in this virus is still not high.So the reconstructed adenoviral vectors can be made in two aspects:1.Improve the selectivity and universality of viral replication in tumor cells. It is reported that telomerase is the most extensive tumor molecular marker to date and accounts for the ability of cancer cells to proliferate in a manner that is out of control. Telomerase is highly active in more than 85% of human cancers and activity was detected in 85-100% of Hepatocarcinoma, according to the documents,but inactive in most somatic cells. Human telomerase reverse transcriptase(htert) is a key determinant of the telomerase activity and highly correlated with telomerase activity. Because the expression of hTERT gene is regulated at the transcription level, we hypothesized that the hTERT promoter may be used for tumor-specific expression of gene necessary for viral replication in cancer cells.So the adenovirus can proliferate and lead to amplification of the input dose at the tumor site whose cells are telomorase postive, while a lack of replication in normal tissues can result in efficient clearance and reduced toxicity under the control of htert promotor.2.Combine with other therapeutic gene and kill tumor cells insensitive to these viruses utilizing gene therapy. Extensive research over the past several years has demonstrated that Interferon-gamma(IFN-y) is vital to the promotion of tumor surveillance in immunocompetent hosts and activates macrophages to nonspecifically lyse neoplastic cells through various mechanisms.IFN-yis capable of upregulating the expression of tumor-associated antigens in vitro and in vivo,thereby increasing the susceptibility of tumors to MHC-restricted CD8+cytotoxic T cell-mediated lysis. IFN-y is reguired for robust IL-12 secretion by antigen-presenting cells(APC) and can elicit the production of new capillary endothelium from the host. Furthermore, IFN-y can abrogate the development of neutralizing antibodies against adenoviral vectors by promoting the differentiation of Th2 cells to Thl cells, so that the efficacy of gene delivery can be further improved.The efficacy of IFN-y in tumor clinical experiment is not as expected in past years.The reason is that high doses of IFN-y was required when obtaining certain effect because of short half-life and lead to severe side effect. In our studies we use human htert promotor to control replicating of adenovirus in cells and carry mouse Interferon gamma (mifn-γ) as therapeutic gene. The therapeutic gene inserted into the genome of the virus will specifically and highly express in the tumor cells with the virus selectively proliferating in the tumor cells. Implementation of procedures, therefor, allowing high local production of ifn-γat the site of tumor foci without increasing systemic levels of the cytokine, would probably achieve therapeutic effect without toxicity.The new approach, defined as gene-viral therapy, can exploit the virtues of gene therapy in coordination with virotherapy and overcome the disadvantages of traditional gene therapy.ObjectivesIn Our early study, we have constructed a specifically replicative recombinant adenoviral vector,name CNHK300-Mγ,that is driven by hTERT promoter and carring mouse IFN-ygene. In present study, the capability of oncolysis,comparing with ONXY-015,CNHK300 and AdEasy-Mγ,and mouse IFN-y expression were assessed both in gastrointestinal malignant tumor and in HCC xenografts. The effect and feasibility of treatment of gastrointestinal malignant tumor with gene-viral therapy will be studied. Our findings may provide new insights into cancer treatment with gene-viral therapy, antiangiogenesis gene therapy and protocols targeting to telomerase.Methods1,Effect of specifically replicative adenovirus carrying mouse IFN-y gene on gastrointestinal malignant tumor cells. For assessing the selectivity and safety features of CNHK300-M Y, we used Viral replication,cytopathologic effect (CPE) and Cell viability assay in comparison with CNHK300,ONYX-015, AdEasy-Mγ,in vitro. To quantify the expression of IFN-γ, we used Western blot examination of IFN-γand ELISA determination of IFN-γexpression in vitro.2,Animal experiments.Cancer cells in log phase were subcutaneously injected into the right flanks of BALB/c nude mice and BALB/c normal mice, with administration of 5 x 106 SMMC-7721 or Hepal-6 cells cells per mouse. When SMMC-7721 tumors reached about 50 mm3, Hepal-6 tumors reached about 150 mm3, mice were allotted randomly into five groups:CNHK300-M Y, CNHK300, ONYX-015, AdEasy-M Y and control groups, n=16mice/group for SMMC-7721 models, with 6/16 mice per group prepared to sacrifice for detecting IFN-γexpression; and n=5 mice/group for Hepal-6 models. Mice underwent five intratumoral injections, once every other day, with a total dosage of 109 pfu per mouse in the virus-treated groups and with 100 Al viral preservation solution per mouse per injection in the control group. Tumor sizes were measured regularly. Tumor volume was estimated as a x b x b x 0.5, where a and b were the maximal and minimal diameters, respectively. At days 3 and 7 after treatment in SMMC-7721 models,3 mice per group per time point were sacrificed to collect serum by retroorbital puncture. The IFN-yexpression in mouse serum was measured using the Mouse IFN-y ELISA Kit.For Hepal-6 and SMMC-7721 models, mice were killed 28 and 42 days later, respectively. Tumors and organs were removed and fixed in 10% neutral formaldehyde for 6 h and paraffinembedded, and 5-Am-thick consecutive sections were cut for H&Estaining and immunohistochemistry. On sections of SMMC-7721 models, the expression of adenoviral capsid protein hexon was located using the mouse antiadenoviral hexon antibody. On sections of Hepal-6 models, the infiltrating lymphocytes were localized immunohistochemically with leukocyte common antigen (LCA), CD4, and CD8 antibodies. Cells positive for hexon, LCA, CD4, and CD8 and the MVDs were counted within four high-power fields under a light microscope.Results1. Viral replication assay:CNHK300-M Y can selectively proliferate in the tumor cells while not proliferate in the normal fibroblast BJ and MRC-5.The proliferous capability of CNHK300-M Y in normal fibroblast BJ and MRC-5 is the same as that of ONYX-015, but the proliferous capability in tumor cells much higher that of ONYX-015.2. The cytopathologic effect (CPE) and MTT:CNHK300-M Y could selectively kill cancer cells at very low multiplicity of infection (m.o.i.) and spare normal cells even at higher m.o.i.. Cell viability was below 40% in cancer cells infected with oncolytic adenoviruses at a m.o.i. of 1 pfu/cell, but more than 80% in normal cells infected with these viruses at a m.o.i. of 10 pfu/cell. The m.o.i. associated with 50% cell viability (IC50) for CNHK300-M Y ranged from 0.074 to 0.864 in cancer cells, but 281 to 428 in various normal cells. 3. The enzyme-linked immunoabsordent assay (ELISA) assay of mIFN-γprotein in cells show that CNHK300-M Y infection really caused the large expression of mIFN-γ,which is 141-1245 times(3 day)and 614-5150 times(7day)of that of traditional gene therapy (the replication-defected adenovirus AdEasy-Mγ) and increases largely as the time prolong in tumor cells. But in the normal cells the expression of mIFN-γwas not high and there was no obvious defference between CNHK300-Mγvirus and replication-defected adenovirus AdEasy-Mγ.4. Antitumor Efficacy of CNHK300-Mγin Mouse Models. In SMMC-7721 tumor models,6 weeks after treatment, the group injected with viruses exhibited excellent therapeutic efficacy compared with the control group。(P<0.001 for CNHK300-Mγ, ONYX-015,AdEasy-Mγand PBS groups, and P=0.029 for CNHK300-Mγ, ONYX-015). In Hepal-6 tumor models in immunocompetent mice,4 weeks after the first injection, the efficient antitumor activity was observed in each virus-treated group, with tumor inhibition rates of 90.3%,61.9%, and 57.7% in the CNHK300-Mγ, CNHK300, AdEasy-M Y, respectively, compared with the control group (P<0.001).5. Cells positive for the microvessel density(MVD) were counted within four high-power fields under a light microscope. MVD in tumor tissues was 81.0±6.2 in the CNHK300-Mγtreated group compared with 129.4±9.9 in the CNHK300 group,152.8±9.8 in the AdEasy-Mγgroup,183.3±15.3 in the control group (P <0.001).6. In immunocompetent mice, in addition to the extensive necrotic foci observed in tumor tissues,-more lymphocytes had infiltrated the tumor tissues of the CNHK300-M Y group compared with the control group. The LCA+, CD4+, and CD8±cell indices in the CNHK300-Mγgroup were 577.8±76.7,241.8±44.8, and 122.4±26.1, but were 343.3±51.3,124.8±40.3, and 74.3±17.3 in the AdEasy-M Y group and 207.8±19.2,100.8±12.4, and 74.0±9.7 in the control group. The numbers of LCA+(P<0.001), CD4+(P<0.001), and CD8+(P <0.01) cells were all increased in tumor tissues of the CNHK300-Mγcompared with the control group.ConclusionsThe tumor-specific replication-competent adenovirus driven by hTERT promoter carring IFN-y gene, can selectively replicate in and lyse gastrointestinal malignant tumor cells with telomerase activity. The mouse IFN-y gene cloned into the replicative adenovirus can copy and multiply to hundreds of times by the virus selectively proliferating in the tumor cells. Recombinant adenovirus CNHK300-Mγcan efficiently suppress gastrointestinal malignant tumor growth through virus direct killing effect and inhibiting tumor angiogenesis.