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300. Adenoviral Delivery of the Gene Encoding Secretable Trimeric TRAIL Induces Apoptosis and Suppresses Human Malignant Glioma In Vivo
CANCER-APOPTOSIS
AND
SUICIDE
Vector sequences were detected in all tumors, with 30% to 85% positive distribution-cores from each gland, even in a specimen removed 15 weeks after vector injection. Pathological analyses demonstrated zones of regional tumor necrosis and inflammation of tumor tissue associated with significant CD8+ T-cell infiltration. Biological activity of the vector was evidenced by a spike in circulating PSA levels shortly after injection followed by an overall decrease from starting PSA concentration to the level observed just prior to surgery in seven of ten patients. No differences were observed between the two prodrugs. Patient follow-up has continued for an average of 52.8 months (range: 38 to 64 months) with encouraging results. These data demonstrate the safety and distribution-efficacy of simple outpatient procedure and oral prodrug for AdV-tk in prostate cancer.
Conclusion: Capsid modifications were transductionally beneficial both in vitro and in vivo. Detailed in vivo data with CRAds will be presented and discussed.
299. Increasing Gene Transfer and Oncolytic Potency of Adenoviruses in Orthotopic Models of Gastric Cancer
Malignant gliomas are most common human primary brain tumors. Despite of intensive research, current treatments have not significantly improved patient survival over last three decades. TNFrelated apoptosis-inducing ligand (TRAIL) induces apoptosis in a wide range of tumor cells without fatal effect on normal cells. In previous study, we designed a replication deficient adenovirus (Ad) to encode secretable trimeric TRAIL (stTRAIL). This adenovirus (Ad-stTRAIL) potently induced apoptosis in vivo and in vitro by stTRAIL secreted from infected tumor cells. As an approach to AdstTRAIL mediated cancer gene therapy, we found out the tumoricidal effects of Ad-stTRAIL in several human malignant glioma cell lines in vitro. In murine xenograft model bearing human malignant glioma U-87MG cell line, the intratumoral administration of Ad-stTRAIL exerted the significant suppression of tumor growth and histological analysis revealed that Ad-stTRAIL regressed tumor growth by inducing apoptotic cell death. In murine brain tumor model implanted U87-MG intracranially through stereotactics, the intratumoral and intracranial administration of Ad-stTRAIL prolonged the survival of mice and reduced the tumor volume in the absence of any acute and delayed neurotoxicity. Contrary to rapid clearance of systemically delivered recombinant TRAIL protein, Ad-stTRAIL showed the persistent expressed stTRAIL level and suppressed the tumor more significantly. Furthermore, we found out that Ad-stTRAIL and combination with chemotherapeutic agents reinforced the potential of Ad-stTRAIL overcoming the TRAIL resistance of certain glioma cell lines. These results suggest that Ad-stTRAIL shows its potential feature as a mean of treating human malignant glioma
Lotta Kangasniemi,1 Anna Kanerva,1 Mari Raki,1 Tuuli Ranki,1 Merja Sarkioja,1 Tuula Kiviluoto,2 Ulf-Hakan Stenman,3 Henrik Alfthan,3 Hongju Wu,4 David T. Curiel,4 Akseli Hemminki.1 1 Rational Drug Design, University of Helsinki, Helsinki, Finland; 2 Department of Surgery, Helsinki University Central Hospital, Helsinki, Finland; 3Department of Clinical Chemistry, Helsinki University Central Hospital, Helsinki, Finland; 4Division of Human Gene Therapy, University of Alabama, Birmingham, AL. Background: Gene therapy with adenoviral (Ad) vectors is a promising modality for treatment of cancer. The primary limiting factor for Ad vectors is their dependence on the coxsackie and adenovirus receptor (CAR), which is down-regulated in many cancers. However, this has not been studied in the context of gastric cancer, the fifth most common cause of cancer mortality worldwide. Native Ad5 tropism can be modified to circumvent CAR deficiency. For instance, Ad5/3luc1 with serotype 3 fiber knob binds to a receptor distinct from CAR. The fiber of Ad5lucRGD is modified with an integrin-targeted arginine-glycine-aspartic acid motif. Polylysine motifs pK7 and pK21 bind to heparan sulphates. The oncolytic potency of replicating agents is partly determined by their capability for infecting cells. Conditionally replicating adenoviruses (CRAds) are replication competent in tumor but not in non-cycling normal cells. ∆24 based CRAds with a 24 bp deletion in E1A region take advantage of tumor specific changes in the Rb pathway. Our hypothesis was, that capsid modified adenoviruses possess enhanced ability for transduction of gastric cancer cells and primary tumor tissues, and that this targeting would also benefit the oncolytic potency of CRAds. Methods: Gastric cancer cell lines were infected with transductionally modified replication incompetent viruses with 5/3, RGD and/or polylysine modifications. The same panel of viruses was used to study clinical samples with known histopathology. Transduction efficacy of these viruses was also tested in nude mice with intraperitoneal carcinomatosis, thus closely resembling the patient group in the most dire need of novel therapeutic interventions. ∆24 type CRAds were tested in vitro for their capability for killing gastric cancer cells, and in vivo experiments with CRAds will also be reported. Further, for non-invasive in vivo longitudinal evaluation of anti-tumor efficacy, we utilized a panel of tumor markers secreted by gastric cancer cells. Results: Capsid modifications benefited gene transfer efficiency in cell lines and clinical samples. In vivo Ad5/3luc1 was significantly more efficient in transducting gastic cancer tumors compared to the wild type capsid. Some of the tumor markers were found useful for following orthotopic tumor xenograft growth.
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
300. Adenoviral Delivery of the Gene Encoding Secretable Trimeric TRAIL Induces Apoptosis and Suppresses Human Malignant Glioma In Vivo Moonsup Jeong,1 In-Ho Kwon,1 Chae-Young Kim,1 Dai-Wu Seol,2 Paul D. Robbins,3 Byong-Moon Kim.1 1 Research Laboratories, Dong-A Pharmacetical Co., Ltd., Youngin, Kyunggi, Korea; 2Surgery, University of Pittsburgh School of Medicine, Piitsburgh, PA; 3Molecular Genetics & Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA.
301. Down-Regulation of bcl-xl Gene Expression with bcl-xl siRNA Vectors In Vitro and In Vivo Xiaobo X. Cao,1 Philip Rascoe,2 Jonathan Daniel,2 Charles Rodarte,1 W. Roy Smythe.1 1 Department of Surgery, Texas A&M University College of Medicine, Scott & White Hospital, Temple, TX; 2Department of Surgery, University of Texas M.D. Anderson Cancer Center, Houston, TX. Introduction BCL-XL, an anti-apoptotic member of the bcl-2 gene family, is highly expressed in human lung cancer. Over-expression of BCLXL has been correlated with conventional clinical treatment resistance. We previously demonstrated that down-regulation of BCL-XL expression by antisense will induce apoptosis and sensitize lung cancer cells to subsequent chemotherapy. Materials and Methods AN H1 promoter driven bcl-xl siRNA was designed according to the bcl-xl cDNA sequence. The human lung cancer line H1299 was utilized. Western blot and real-time RT-PCR were used to measure bcl-xl expression. In vitro apoptosis and cell growth were assessed following siRNA exposure alone and with cisplatin in a dose response S117
AND
SUICIDE
Vector sequences were detected in all tumors, with 30% to 85% positive distribution-cores from each gland, even in a specimen removed 15 weeks after vector injection. Pathological analyses demonstrated zones of regional tumor necrosis and inflammation of tumor tissue associated with significant CD8+ T-cell infiltration. Biological activity of the vector was evidenced by a spike in circulating PSA levels shortly after injection followed by an overall decrease from starting PSA concentration to the level observed just prior to surgery in seven of ten patients. No differences were observed between the two prodrugs. Patient follow-up has continued for an average of 52.8 months (range: 38 to 64 months) with encouraging results. These data demonstrate the safety and distribution-efficacy of simple outpatient procedure and oral prodrug for AdV-tk in prostate cancer.
Conclusion: Capsid modifications were transductionally beneficial both in vitro and in vivo. Detailed in vivo data with CRAds will be presented and discussed.
299. Increasing Gene Transfer and Oncolytic Potency of Adenoviruses in Orthotopic Models of Gastric Cancer
Malignant gliomas are most common human primary brain tumors. Despite of intensive research, current treatments have not significantly improved patient survival over last three decades. TNFrelated apoptosis-inducing ligand (TRAIL) induces apoptosis in a wide range of tumor cells without fatal effect on normal cells. In previous study, we designed a replication deficient adenovirus (Ad) to encode secretable trimeric TRAIL (stTRAIL). This adenovirus (Ad-stTRAIL) potently induced apoptosis in vivo and in vitro by stTRAIL secreted from infected tumor cells. As an approach to AdstTRAIL mediated cancer gene therapy, we found out the tumoricidal effects of Ad-stTRAIL in several human malignant glioma cell lines in vitro. In murine xenograft model bearing human malignant glioma U-87MG cell line, the intratumoral administration of Ad-stTRAIL exerted the significant suppression of tumor growth and histological analysis revealed that Ad-stTRAIL regressed tumor growth by inducing apoptotic cell death. In murine brain tumor model implanted U87-MG intracranially through stereotactics, the intratumoral and intracranial administration of Ad-stTRAIL prolonged the survival of mice and reduced the tumor volume in the absence of any acute and delayed neurotoxicity. Contrary to rapid clearance of systemically delivered recombinant TRAIL protein, Ad-stTRAIL showed the persistent expressed stTRAIL level and suppressed the tumor more significantly. Furthermore, we found out that Ad-stTRAIL and combination with chemotherapeutic agents reinforced the potential of Ad-stTRAIL overcoming the TRAIL resistance of certain glioma cell lines. These results suggest that Ad-stTRAIL shows its potential feature as a mean of treating human malignant glioma
Lotta Kangasniemi,1 Anna Kanerva,1 Mari Raki,1 Tuuli Ranki,1 Merja Sarkioja,1 Tuula Kiviluoto,2 Ulf-Hakan Stenman,3 Henrik Alfthan,3 Hongju Wu,4 David T. Curiel,4 Akseli Hemminki.1 1 Rational Drug Design, University of Helsinki, Helsinki, Finland; 2 Department of Surgery, Helsinki University Central Hospital, Helsinki, Finland; 3Department of Clinical Chemistry, Helsinki University Central Hospital, Helsinki, Finland; 4Division of Human Gene Therapy, University of Alabama, Birmingham, AL. Background: Gene therapy with adenoviral (Ad) vectors is a promising modality for treatment of cancer. The primary limiting factor for Ad vectors is their dependence on the coxsackie and adenovirus receptor (CAR), which is down-regulated in many cancers. However, this has not been studied in the context of gastric cancer, the fifth most common cause of cancer mortality worldwide. Native Ad5 tropism can be modified to circumvent CAR deficiency. For instance, Ad5/3luc1 with serotype 3 fiber knob binds to a receptor distinct from CAR. The fiber of Ad5lucRGD is modified with an integrin-targeted arginine-glycine-aspartic acid motif. Polylysine motifs pK7 and pK21 bind to heparan sulphates. The oncolytic potency of replicating agents is partly determined by their capability for infecting cells. Conditionally replicating adenoviruses (CRAds) are replication competent in tumor but not in non-cycling normal cells. ∆24 based CRAds with a 24 bp deletion in E1A region take advantage of tumor specific changes in the Rb pathway. Our hypothesis was, that capsid modified adenoviruses possess enhanced ability for transduction of gastric cancer cells and primary tumor tissues, and that this targeting would also benefit the oncolytic potency of CRAds. Methods: Gastric cancer cell lines were infected with transductionally modified replication incompetent viruses with 5/3, RGD and/or polylysine modifications. The same panel of viruses was used to study clinical samples with known histopathology. Transduction efficacy of these viruses was also tested in nude mice with intraperitoneal carcinomatosis, thus closely resembling the patient group in the most dire need of novel therapeutic interventions. ∆24 type CRAds were tested in vitro for their capability for killing gastric cancer cells, and in vivo experiments with CRAds will also be reported. Further, for non-invasive in vivo longitudinal evaluation of anti-tumor efficacy, we utilized a panel of tumor markers secreted by gastric cancer cells. Results: Capsid modifications benefited gene transfer efficiency in cell lines and clinical samples. In vivo Ad5/3luc1 was significantly more efficient in transducting gastic cancer tumors compared to the wild type capsid. Some of the tumor markers were found useful for following orthotopic tumor xenograft growth.
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
300. Adenoviral Delivery of the Gene Encoding Secretable Trimeric TRAIL Induces Apoptosis and Suppresses Human Malignant Glioma In Vivo Moonsup Jeong,1 In-Ho Kwon,1 Chae-Young Kim,1 Dai-Wu Seol,2 Paul D. Robbins,3 Byong-Moon Kim.1 1 Research Laboratories, Dong-A Pharmacetical Co., Ltd., Youngin, Kyunggi, Korea; 2Surgery, University of Pittsburgh School of Medicine, Piitsburgh, PA; 3Molecular Genetics & Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA.
301. Down-Regulation of bcl-xl Gene Expression with bcl-xl siRNA Vectors In Vitro and In Vivo Xiaobo X. Cao,1 Philip Rascoe,2 Jonathan Daniel,2 Charles Rodarte,1 W. Roy Smythe.1 1 Department of Surgery, Texas A&M University College of Medicine, Scott & White Hospital, Temple, TX; 2Department of Surgery, University of Texas M.D. Anderson Cancer Center, Houston, TX. Introduction BCL-XL, an anti-apoptotic member of the bcl-2 gene family, is highly expressed in human lung cancer. Over-expression of BCLXL has been correlated with conventional clinical treatment resistance. We previously demonstrated that down-regulation of BCL-XL expression by antisense will induce apoptosis and sensitize lung cancer cells to subsequent chemotherapy. Materials and Methods AN H1 promoter driven bcl-xl siRNA was designed according to the bcl-xl cDNA sequence. The human lung cancer line H1299 was utilized. Western blot and real-time RT-PCR were used to measure bcl-xl expression. In vitro apoptosis and cell growth were assessed following siRNA exposure alone and with cisplatin in a dose response S117