- Email: [email protected]
310 P53-DEPENDENT ANTIVIRAL RNA-INTERFERENCE FACILITATES TUMOR-SELECTIVE VIRAL REPLICATION
04e: MOLECULAR AND CELLULAR BIOLOGY − e) GENE TRANSFER/GENE THERAPY significantly compared to mice treated with Ad-LacZ-DC (p = 0.03). Flow cytometry of mIL12-DC-treated tumors revealed a significant tumorinfiltration with CD4+, CD8+-T-cells and NK-cells. Vaccination with AdmIL-12/Ad-mAFP co-transduced DC inhibited significantly the growth of AFP-positive tumors compared to controls: the mean tumor volume was 245.2±122.8 mm3 in IL-12/AFP-DC-vaccinated vs. 894.3±224.2 in AFPDC- or 1105±297.4 mm3 in LacZ-DC-vaccinated (p < 0.05). Conclusions: Transduction of DC with Ad-IL-12 increases significantly the stimulatory capacity of TAA-pulsed DC using tumor lysate or AFP. IL-12 -expressing DC can significantly inhibit the tumor-growth of subcutaneous hepatocellular tumors, activating both acquired and innate immunity. These results underline the potential of IL-12 for the use of TAA-pulsed DC for immunotherapy of hepatocellular tumors. 310 P53-DEPENDENT ANTIVIRAL RNA-INTERFERENCE FACILITATES TUMOR-SELECTIVE VIRAL REPLICATION E. G¨urlevik, A. Kloos, M. Manns, S. Kubicka, F. K¨uhnel. Medical School Hannover, Hannover, Germany E-mail: [email protected] Background and Aims: Oncolytic virotherapy is a promising and novel mean for treatment of solid tumors. A major challenge in virotherapy is tight restriction of viral replication to malignant cells combined with a large spectrum of potential target tumors. Inactivation of p53transcriptional activity is a crucial step of tumorigenesis and occurs in the majority of human cancers. Consequently, transcriptional p53deficiency represents an excellent broad-range target for tailored therapies by oncolytic viruses.Aim of this study was the generation of an adenovirus for conditional replication in p53-dysfunctional tumor cells that uses p53selective expression of a microRNA-network directed against essential adenoviral genes. Methods: We have developed the oncolytic adenovirus Ad-iREP-2 for selective replication in tumor cells without functional p53. To control its replication, Ad-iREP-2 contains a multiple miRNA-transcript that is selectively expressed in response to active p53 and then interferes with adenoviral genes essentially involved in the early onset of viral DNAreplication. Expression of this RNAi-network results in an antiviral transcript consisting of concatenated miRNAs against E1A, E1B/protein IX, E4, terminal protein and adenoviral polymerase. Selectivity and efficacy were characterized by luciferase assays, western blot analysis, replication kinetics in vitro and in vivo and oncolysis assays in vitro and in xenotransplanted mice in vivo. Results: We could demonstrate that RNAi was selectively exhibited in p53-active cells, but not in cells harboring either mutated or deleted p53. Compared to the control virus Ad-iREP-C that expressed a scrambled RNAi-network, antiviral RNAi of Ad-iREP-2 selectively attenuated viral replication in cells with transcriptionally active p53, but not in p53dysfunctional tumor cells where both viruses replicated equivalently. Since these results were confirmed by an in vivo comparison of both viruses after infection of p53-knockout and normal mice, we could demonstrate that attenuated replication was indeed a result of p53-selective expression of antiviral RNAi. Ad-iREP-2 further showed an improved selectivity for p53-inactivation compared to the p53-dependent reference virus ONYX015. Conclusions: In conclusion, Ad-iREP2 is an effective oncolytic virus that correctly recognizes and responds to the cellular p53 status. Our study shows for the first time that virus encoded RNAi is sufficient for targeted self control of replication.
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311 MULTIFUNCTIONAL CANCER GENE THERAPY DRIVEN BY TISSUE-SPECIFIC PROMOTER AND CANCERTARGETING TRANS-SPLICING RIBOZYME IN MOUSE LIVER CANCER MODEL J.-S. Jeong1 , S.-Y. Han2 , S.-J. Lee3 , S.-W. Lee4 , I.-H. Kim3 . 1 Pathology and Medical Research Center for Cancer Molecular Therapy, 2 Internal Medicine, Dong-A University College of Medicine, Busan, 3 Research Institute and Hospital, National Cancer Center, Goyang, 4 Molecular Biology and Institue of Nanosensor and Biotechnology, Dankook University, Yongin, South Korea E-mail: [email protected] Background and Aims: The development of control system of transgene expression combined with tissue-specific gene delivery systems leads to successful cancer gene therapy in clinical setting. Cancer-specific gene expression through transcriptional targeting is an important and valuable approach to reduce toxicity and potentially increase efficacy. We developed a new module for cancer specific therapeutic gene expression system composed of trans-splicing ribozymes (TSR) based on the group I intron of Tetrahymena thermophila, targeting and reprogramming cancer-specific human telomerase reverse transcriptase (hTERT), and therapeutic suicidal gene, Herpes Simplex Virus thymidine kinase (HSVtk). To restrict expression of the transgene to liver, we inserted liver-specific promoter, phosphoenolpyruvate carboxykinase (PEPCK). We expected increased specificity of therapy by hTERT targeting ribozyme and liver selective promoter only in hTERT expressing liver cancer cells and increased efficacy by removing hTERT RNAs and simultaneously triggering cytotixin RNA in targeted cells. Methods: We constructed adenoviral vectors encoding hTERT targeting TSR with downstream reporter genes including lacZ (Ad-PEPCKRibo.LacZ) or luciferase (Ad-PEPCK-Ribo.Luc), and therapeutic gene HSVtk (Ad-PEPCK-Ribo.Tk), driven by PEPCK promoter. We studied liver cancer cell-specific transgenic expression and MTT assay after transfection with viral vectors in hTERT (+) human hepatocellular carcinoma cells, Hep3B. In vivo study of specific transgenic expression and antitumor efficacy was performed with Intrahepatic multifocal HCC mouse model of Hep3B cells. Results: We observed liver and cancer specific transgenic expression of reporter genes in vitro, and in vivo normal and tumor-bearing mouse livers by imaging and histologic studies, through administration of AdPEPCK-Ribo.LacZ/Luc. We observed specific and efficient in vitro cytotoxicity of Ad-PEPCK-Ribo.Tk/ganciclovir and in vivo antitumor effect of intrahepatic multifocal HCC model, by systemic administration of AdPEPCK-Ribo.Tk/ganciclovir. As well, we administered adenoviral vectors in normal BALB/c mice, systemically, for evaluation of toxicity. We found least liver toxicity in Ad-PEPCK-Ribo.Tk treated group similar to AdMOCK group till 14 day. On contrary, Ad-PEPCK-Tk group showed increased liver enzymes and liver injury, and Ad-CMV-Tk group died before 7 day. Conclusion: These results demonstrate that the cancer-specific TSR expressed by liver-specific PEPCK promoter can be a promising tool as dual-targeting and dual-therapeutic multifunctional cancer gene therapy in HCC, preserving liver function.
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311 MULTIFUNCTIONAL CANCER GENE THERAPY DRIVEN BY TISSUE-SPECIFIC PROMOTER AND CANCERTARGETING TRANS-SPLICING RIBOZYME IN MOUSE LIVER CANCER MODEL J.-S. Jeong1 , S.-Y. Han2 , S.-J. Lee3 , S.-W. Lee4 , I.-H. Kim3 . 1 Pathology and Medical Research Center for Cancer Molecular Therapy, 2 Internal Medicine, Dong-A University College of Medicine, Busan, 3 Research Institute and Hospital, National Cancer Center, Goyang, 4 Molecular Biology and Institue of Nanosensor and Biotechnology, Dankook University, Yongin, South Korea E-mail: [email protected] Background and Aims: The development of control system of transgene expression combined with tissue-specific gene delivery systems leads to successful cancer gene therapy in clinical setting. Cancer-specific gene expression through transcriptional targeting is an important and valuable approach to reduce toxicity and potentially increase efficacy. We developed a new module for cancer specific therapeutic gene expression system composed of trans-splicing ribozymes (TSR) based on the group I intron of Tetrahymena thermophila, targeting and reprogramming cancer-specific human telomerase reverse transcriptase (hTERT), and therapeutic suicidal gene, Herpes Simplex Virus thymidine kinase (HSVtk). To restrict expression of the transgene to liver, we inserted liver-specific promoter, phosphoenolpyruvate carboxykinase (PEPCK). We expected increased specificity of therapy by hTERT targeting ribozyme and liver selective promoter only in hTERT expressing liver cancer cells and increased efficacy by removing hTERT RNAs and simultaneously triggering cytotixin RNA in targeted cells. Methods: We constructed adenoviral vectors encoding hTERT targeting TSR with downstream reporter genes including lacZ (Ad-PEPCKRibo.LacZ) or luciferase (Ad-PEPCK-Ribo.Luc), and therapeutic gene HSVtk (Ad-PEPCK-Ribo.Tk), driven by PEPCK promoter. We studied liver cancer cell-specific transgenic expression and MTT assay after transfection with viral vectors in hTERT (+) human hepatocellular carcinoma cells, Hep3B. In vivo study of specific transgenic expression and antitumor efficacy was performed with Intrahepatic multifocal HCC mouse model of Hep3B cells. Results: We observed liver and cancer specific transgenic expression of reporter genes in vitro, and in vivo normal and tumor-bearing mouse livers by imaging and histologic studies, through administration of AdPEPCK-Ribo.LacZ/Luc. We observed specific and efficient in vitro cytotoxicity of Ad-PEPCK-Ribo.Tk/ganciclovir and in vivo antitumor effect of intrahepatic multifocal HCC model, by systemic administration of AdPEPCK-Ribo.Tk/ganciclovir. As well, we administered adenoviral vectors in normal BALB/c mice, systemically, for evaluation of toxicity. We found least liver toxicity in Ad-PEPCK-Ribo.Tk treated group similar to AdMOCK group till 14 day. On contrary, Ad-PEPCK-Tk group showed increased liver enzymes and liver injury, and Ad-CMV-Tk group died before 7 day. Conclusion: These results demonstrate that the cancer-specific TSR expressed by liver-specific PEPCK promoter can be a promising tool as dual-targeting and dual-therapeutic multifunctional cancer gene therapy in HCC, preserving liver function.