- Email: [email protected]
44. Oncolytic Virus Therapy of Glioma Sensitizes Glioma Cells to Cilengitide Treatment
Cancer - Apoptosis and Suicide: Viral Vectors primary endpoint is OS. Secondary endpoints are CSS and time to distant metastases. Exploratory endpoints include the development of cellular and humoral anti-tumor immunity, and PSADT before and after the gene therapy.
42. Adenoviral-Mediated Delivery of Mutated IL-13 (IL13.E13.K) Fused to PE Toxin Exhibits Enhanced Efficacy and Reduced Neurotoxicity When Compared to the Protein Formulation (Cintredekin Besudotox) for the Treatment of Glioblastoma
M. Candolfi,1 W. Xiong,1 A. G. Muhammad,1 M. Puntel,1 C. Liu,1 K. M. Kroeger,1 S. Mondkar,1 J. Lerner,1 S. Lee,1 D. Foulad,1 W. Debinski,2 R. Rodriguez,3 P. R. Lowenstein,1 M. G. Castro.1 1 Gene Therapy Research Institute, Cedars Sinai Medical CenterUCLA, Los Angeles, CA; 2Wake Forest University, Salem, NC; 3 Johns Hopkins University, Baltimore, MD. Treatment of human glioblastoma (GBM) has been attempted using native human IL-13 fused to Pseudomonas exotoxin A (PE), (hIL-13-PE38QQR, Cintredekin Besudotox), that binds the GBMassociated IL13α2R. However, hIL-13-PE38QQR exhibits short half-life, requiring frequent and prolonged administration. Also, this chimeric toxin binds not only to the GBM-associated IL13α2R, but also to the physiological IL-13/IL-4R expressed in normal brain cells. We constructed an adenoviral vector (Ad) expressing a mutant IL13 (IL13.E13K) fused to PE (muIL-13-PE), which binds to GBMassociated IL13α2R with high affinity and has negligible binding for the physiological IL13/IL4R (Nat Biotechnol. 1998 16(5):449-453). We compared the efficacy and neurotoxicity of our novel Ad vs. hIL-13-PE38QQR in intracranial human GBM xenografts in nude mice as well as in the brain of naive immunocompetent Balb/c mice. We constructed the therapeutic Ad-muIL4-TRE-muIL13-PE that expresses the cytotoxin muIL-13-PE, and, as an extra safety feature, a mutated IL-4 (IL4.Y124D, muIL-4) that blocks the binding of IL13 to the physiological IL13/IL4R (Int J Oncol. 1999 15(3): 481-486). Efficacy of Ad-muIL4-TRE-muIL13-PE+Ad-TetON (4x107 pfu) and hIL-13-PE38QQR (0.2 and 1µg) was studied by intratumoral injection into intracranial human U251 GBM xenografts in nude mice. Ad-muIL4-TRE-muIL13-PE significantly increased the survival of tumor-bearing mice when compared to saline-treated mice, leading to ~80% survival for over 100 days. Although 0.2 and 1 µg of hIL13-PE38QQR improved the survival of tumor-bearing mice when compared with saline-treated mice, all hIL-13-PE38QQR-treated mice succumbed due to tumor burden by day 55. Then we compared the toxicity profile 3 and 7 days after the administration of Ad-muIL4TRE-muIL13-PE+Ad-TetON (4x107 pfu) and hIL-13-PE38QQR (0.2, 0.5 and 1 µg) in the striatum of naïve Balb/c mice. Mice that received 1 µg of hIL-13-PE38QQR exhibited severe neurological signs, requiring euthanasia 2-3 days after injection. Their brains exhibited extensive tissue damage, profuse infiltration of inflammatory cells, reduction in TH expression in the striatum, and local hemorrhages. Mice that received reduced doses of hIL-13-PE38QQR (0.5-0.2 µg), survived until the 7 day-time point, but their brains also exhibited severe neuropathological side effects. However, none of the mice injected with Ad-muIL4-TRE-muIL13-PE+Ad-TetON exhibited systemic or local side effects. These results suggest that Ad delivery of muIL13-PE toxin to GBM will lead to strong antitumoral effect with negligible adverse side effects to the surrounding non-neoplastic brain. Supported by NIH/NINDS R01 NS44556.01, R21-NSO54143.01; UO1 NS052465.01; RO3 TW006273-01 to M.G.C.; RO1 NS 054193.01; RO1 NS 42893.01; U54 NS045309-01, and R21 NS047298-01 to P.R.L; F32 NS058156.01 to M.C..
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
43. Enhancement of Oncolytic Properties of Genetically-Engineered Fusogenic Newcastle Disease Virus through Antagonism of Cellular Innate Immune Responses
Dmitriy Zamarin,1 Luis Martinez-Sobrido,1 Kaitlyn Kelly,2 Adam Vigil,1 Adolfo Garcia-Sastre,1 Peter Palese,1 Yuman Fong.2 1 Microbiology, Mount Sinai School of Medicine, New York, NY; 2 Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY. Newcastle Disease Virus (NDV) is a member of the Avulavirus genus in the Paramyxoviridae family, which has been shown to infect a number of avian species, but to cause no disease in humans. For the past 50 years, NDV has been shown to be an effective oncolytic agent, causing specific lysis of cancerous but not normal cells. The specificity of the oncolytic effect of NDV is presumed to result from its sensitivity to the antiviral effects of the type I interferon (IFN) system, which is impaired in a great number of tumors. Despite these findings, the in vivo oncolytic efficiency of naturally-occurring NDV strains can still be relatively low, as many tumors still possess an adequate immune response to suppress viral replication and spread. We hypothesized that the enhanced suppression of the innate immune responses by the NDV would allow for increased viral replication and enhanced oncolytic activity. We have previously described the establishment of a reverse-genetics system for genomic manipulation and construction of NDV vaccines and oncolytic virus vectors. Using this system, we first generated a recombinant NDV virus of Hitchner B1 strain with enhanced fusogenic properties, and showed its enhanced ability to form syncytia and lyse a variety of tumor cell lines, when compared to its wild-type counterpart. To further enhance the interferon-antagonistic properties of the virus, we generated a fusogenic NDV virus expressing influenza NS1 protein, which was previously characterized as an antagonist of innate immune responses in human and mouse cells. The resultant virus suppressed the induction of cellular IFN response and was enhanced in its ability to replicate, form syncytia, and lyse a variety of human and mouse tumor cell lines. Inoculation of C57/BL6 and nude mice with the NDV-NS1 virus resulted in no significant adverse effects, confirming its safety. Using the aggressive syngeneic murine B16-F10 footpad melanoma model, we show that the intratumoral injection with NDV-NS1 virus was more effective in clearing the established footpad tumors and resulted in higher overall long-term animal survival, when compared to its counterpart not expressing NS1. Tumors excised from the mice treated with NDV showed a higher degree of lymphocyte infiltration, when compared to the untreated control group. Moreover, mice treated with NDV developed melanoma-specific CTL responses, which were stronger in the NDV-NS1 group. Overall, these findings demonstrate that modulation of innate immune responses by NDV can be an effective strategy for enhancement of its oncolytic properties and leads to generation of effective adaptive immune response against the tumor cells.
44. Oncolytic Virus Therapy of Glioma Sensitizes Glioma Cells to Cilengitide Treatment
Kazuhiko Kurozumi,1 Alvarez-Breckenridge Christopher,1 Dmitrieva Nina,1 Jayson Hardcastle,1 E. A. Chiocca,1 Balveen Kaur.1 1 Neurological Surgery, The Ohio State University, Columbus, OH. Oncolytic HSV-1 derived viruses (OVs) are being investigated as treatment modalities for many cancers. However, a clear understanding of the action of this biological therapy in human patients is currently not feasible. We have been investigating OV therapy induced changes in the tumor extracellular matrix. Transcript profiling of secreted proteins involved in angiogenesis revealed a significant, induction of CYR61 gene expression (8.94 fold, P = 0.001) in tumors treated with OV in vivo. Further CYR61 mRNA and protein were induced upon S17
Cancer - Apoptosis and Suicide: Viral Vectors infection using different OVs in multiple human cancer cell lines and primary human tumor derived cells in vitro and in orthotopic human gliomas grown in nude mice in vivo. CYR61 is a secreted ECM protein that can bind to and activate integrins αvβ3 and αvβ5. Increased CYR61 in the ECM of breast cancer cells has also been shown to activate an autocrine loop,resulting in upregulation of its own receptor αvβ3 resulting in their increased sensitization to integrin anatgonists (Menendez JA, et al. Oncogene 2005). Consistent with this we found that glioma cells stably over expressing CYR61 were much more sensitive to the integrin antagonist Cilengetide, than control cells (LD50=146.5 ng/ml of overexpressing Vs control LD50=521.1 ng/ ml) (P<0.05). Hence we hypothesized that OV treatment induced secreted CYR61 would increase integrins αvβ3 on both infected and uninfected U87delta EGFR glioma cells, and sensitizing them to integrin antagonists such as Cilengitide. We tested the effect of OV treatment of glioma cells on expression of integrin receptors αvβ3. Immunofluorescent staining of glioma cells revealed increased staining of these integrin receptors on cell membranes of cells infected with OV, compared to uninfected control cells. Moreover we detected synergistic killing of glioma cells treated with OV and Cilengitide compared to cells treated with either agent alone (Chou – Talalay combination indices = 0.39). These results suggested that treatment of glioma with OV followed by treatment with Cilengitide would enhance therapeutic efficacy. We tested this hypothesis in SCID mice with intracranial tumors (U87∆EGFR). Briefly seven days after tumor cell implantation, mice were treated with OV or PBS by direct intratumoral injection. Five days after OV treatment mice were injected systemically with a single dose of cRGD (5mg/kg) or PBS. Mice treated with OV and Cilengitide survived significantly longer than mice treated with OV alone (median survival: OV: d16; cRGD + OV: d25) (P < 0.01 between OV and cRGD + OV). These results indicate OV is a sensitizer to Cilengitide in proportion to the levels of target integrin expression and the potential of combining OV treatment with Cilengitide to improve therapeutic outcome.
45. Suppression of Tumor Growth by Single Injection of Type 8 Self-Complementary AAV Vector Expressing Endostatin in Lymphoma Model Mice at Low Vector Dose Yizhuo Wang, Xiao Chen, Yuki Oue, Noriko Miyake, Koichi Miyake, Takashi Shimada. 1 Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan.
Since tumor growth and metastasis depends on the generation of new blood vessels, anti-angiogenic therapy was proposed to be effective for the treatment of a variety of malignancies. To examine the feasibility of adeno-associated viral (AAV) vector mediated systemic anti-angiogenic gene therapy for lymphoma, we constructed AAV vectors (AAV2/1, AAV2/2, AAV2/7, and AAV2/8) expressing secretable murine endostatin (mEnd) and examined the secreted therapeutic protein levels in vivo. When these vectors (5x1010 vg each) were injected into a quadriceps muscle of C57BL/6 mice, the highest serum level of therapeutic protein detected by ELISA was observed in AAV2/8 injected mice (8>7>1>2) one week after injection. Sustained expression of mEnd was detected at least for four months. For further development of AAV vector for systemic anti-angiogenic gene therapy, we generated self-complementary AAV vector (scAAV2/8) expressing mEnd and compared with single stranded AAV (ssAAV2/8). Four weeks after injection, about 100 times higher level of endostatin was detected in the plasma of the mouse administered with scAAV2/8 compared to ssAAV2/8 (1x109 vg each). To examine the effect of scAAV2/8 expressing mEnd, we established a lymphoma murine model in which the lymphoma cell line A20 expressing luciferase gene was inoculated into the caudal vein of BALB/C. Using this lymphoma murine model, we can detect S18
the tumor growth and metastasis by a real-time in vivo imaging analyze system (IVIS). After single injection of ssAAV2/8 expressing mEnd into the right quadriceps muscle of the lymphoma model mice, tumor cell growth was monitored by IVIS. Suppression of tumor growth was observed in scAAV2/8 expressing mEnd injected mice compared to control GFP expressing AAV injected mice (1.2x109 vs. 4.4x109 photon/sec; p<0.05). Survival effect was also detected in scAAV2/8 injected mice (47 vs. 30 days). These results demonstrated that scAAV2/8 mediated systemic expression of endostatin is useful for the gene therapy of lymphoma with only low dose administration of AAV vector.
46. The Combination of Oncolytic Adenoviruses and Ionizing Radiation Inhibits DNA Double Strand Break Detection Which Results in Autophagocytotic Prostate Cancer Cell Death
M. Rajecki,1 T. M. Kiviharju-af Hallstrom,2 T. Hakkarainen,1 P. Nokisalmi,1 S. Hautaniemi,3 A. I. Nieminen,4 M. Tenhunen,5 R. M. Desmond,6 D.-T. Chen,7 K. Guse,1 U.-H. Stenman,8 R. Gargini,9 M. Kapanen,5 J. Klefstrom,4 A. Kanerva,1 S. Pesonen,1 A. Hemminki.1 1 Molecular Cancer Biology Program & Transplantation Laboratory, University of Helsinki (UH), Helsinki University Central Hospital (HUCH), Helsinki, Finland; 2Molecular Cancer Biology Program, UH, Helsinki, Finland; 3Computational Systems Laboratory, UH, Helsinki, Finland; 4Cancer Cell Circuitry Laboratory, UH, Helsinki, Finland; 5Department of Oncology, HUCH, Helsinki, Finland; 6Biostatistics and Bioinformatics Unit, University of Alabama, Birmingham; 7Moffitt Cancer Center, University of South Florida, Tampa; 8Department of Clinical Chemisty, UH, Helsinki, Finland; 9Universidad Autonoma de Madrid, Madrid, Spain. Most advanced solid tumors, including hormone refractory prostate cancer, are relatively radiation resistant. Approaches that increase sensitivity to or synergize with radiation can be useful. Oncolytic adenoviruses (Ads) have shown efficacy against prostate cancer in preclinical and clinical studies. The combination of Ads and radiotherapy has shown better efficacy in cell killing than either agent alone but the mechanisms underlying this phenomenon remains unknown. We investigated the synergy between Ads and radiotherapy and found that infection with Ads 24 h after irradiation results in synergistic prostate cancer cell killing. Mechanistic studies revealed that combination treatment did not increase viral replication or caspase-3 activity (a sign of apoptosis), nor were specific pathways affected in microarray analysis. Instead, western blot analyses showed a reduction in Mre11 levels and in its ATM mediated down-stream component Chk2-Thr68, in combination treated cells. Another downstream component of ATM, γH2AX- Ser139, which is a marker of double strand breaks and DNA configurations misinterpreted as such, was increased in combination treated cells. Our data indicates that the thousands of short double stranded DNA genomes that result from Ad replication, are recognized by the DNA signalling pathway, which results in induction of the double strand break repair response. However, Ad proteins E3orf3, E4orf6 and E1b55K counteract this mechanism to maximize cellular takeover and productive Ad replication. In cells that have sustained radiation injury, these Ad proteins inhibit double strand break repair and thus DNA damage accumulates. Our findings suggest that this eventually leads to autophagy, which was demonstrated by LC3 immunofluorescence and FACS. In summary, we found that oncolytic adenoviruses are synergistic with ionizing radiation because they inhibit double strand break repair for subsequent induction of autophagy. These molecular mechanisms may have widespread importance if they are confirmed relevant with regard to cytopathic effect and autophagy in general.
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
42. Adenoviral-Mediated Delivery of Mutated IL-13 (IL13.E13.K) Fused to PE Toxin Exhibits Enhanced Efficacy and Reduced Neurotoxicity When Compared to the Protein Formulation (Cintredekin Besudotox) for the Treatment of Glioblastoma
M. Candolfi,1 W. Xiong,1 A. G. Muhammad,1 M. Puntel,1 C. Liu,1 K. M. Kroeger,1 S. Mondkar,1 J. Lerner,1 S. Lee,1 D. Foulad,1 W. Debinski,2 R. Rodriguez,3 P. R. Lowenstein,1 M. G. Castro.1 1 Gene Therapy Research Institute, Cedars Sinai Medical CenterUCLA, Los Angeles, CA; 2Wake Forest University, Salem, NC; 3 Johns Hopkins University, Baltimore, MD. Treatment of human glioblastoma (GBM) has been attempted using native human IL-13 fused to Pseudomonas exotoxin A (PE), (hIL-13-PE38QQR, Cintredekin Besudotox), that binds the GBMassociated IL13α2R. However, hIL-13-PE38QQR exhibits short half-life, requiring frequent and prolonged administration. Also, this chimeric toxin binds not only to the GBM-associated IL13α2R, but also to the physiological IL-13/IL-4R expressed in normal brain cells. We constructed an adenoviral vector (Ad) expressing a mutant IL13 (IL13.E13K) fused to PE (muIL-13-PE), which binds to GBMassociated IL13α2R with high affinity and has negligible binding for the physiological IL13/IL4R (Nat Biotechnol. 1998 16(5):449-453). We compared the efficacy and neurotoxicity of our novel Ad vs. hIL-13-PE38QQR in intracranial human GBM xenografts in nude mice as well as in the brain of naive immunocompetent Balb/c mice. We constructed the therapeutic Ad-muIL4-TRE-muIL13-PE that expresses the cytotoxin muIL-13-PE, and, as an extra safety feature, a mutated IL-4 (IL4.Y124D, muIL-4) that blocks the binding of IL13 to the physiological IL13/IL4R (Int J Oncol. 1999 15(3): 481-486). Efficacy of Ad-muIL4-TRE-muIL13-PE+Ad-TetON (4x107 pfu) and hIL-13-PE38QQR (0.2 and 1µg) was studied by intratumoral injection into intracranial human U251 GBM xenografts in nude mice. Ad-muIL4-TRE-muIL13-PE significantly increased the survival of tumor-bearing mice when compared to saline-treated mice, leading to ~80% survival for over 100 days. Although 0.2 and 1 µg of hIL13-PE38QQR improved the survival of tumor-bearing mice when compared with saline-treated mice, all hIL-13-PE38QQR-treated mice succumbed due to tumor burden by day 55. Then we compared the toxicity profile 3 and 7 days after the administration of Ad-muIL4TRE-muIL13-PE+Ad-TetON (4x107 pfu) and hIL-13-PE38QQR (0.2, 0.5 and 1 µg) in the striatum of naïve Balb/c mice. Mice that received 1 µg of hIL-13-PE38QQR exhibited severe neurological signs, requiring euthanasia 2-3 days after injection. Their brains exhibited extensive tissue damage, profuse infiltration of inflammatory cells, reduction in TH expression in the striatum, and local hemorrhages. Mice that received reduced doses of hIL-13-PE38QQR (0.5-0.2 µg), survived until the 7 day-time point, but their brains also exhibited severe neuropathological side effects. However, none of the mice injected with Ad-muIL4-TRE-muIL13-PE+Ad-TetON exhibited systemic or local side effects. These results suggest that Ad delivery of muIL13-PE toxin to GBM will lead to strong antitumoral effect with negligible adverse side effects to the surrounding non-neoplastic brain. Supported by NIH/NINDS R01 NS44556.01, R21-NSO54143.01; UO1 NS052465.01; RO3 TW006273-01 to M.G.C.; RO1 NS 054193.01; RO1 NS 42893.01; U54 NS045309-01, and R21 NS047298-01 to P.R.L; F32 NS058156.01 to M.C..
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
43. Enhancement of Oncolytic Properties of Genetically-Engineered Fusogenic Newcastle Disease Virus through Antagonism of Cellular Innate Immune Responses
Dmitriy Zamarin,1 Luis Martinez-Sobrido,1 Kaitlyn Kelly,2 Adam Vigil,1 Adolfo Garcia-Sastre,1 Peter Palese,1 Yuman Fong.2 1 Microbiology, Mount Sinai School of Medicine, New York, NY; 2 Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY. Newcastle Disease Virus (NDV) is a member of the Avulavirus genus in the Paramyxoviridae family, which has been shown to infect a number of avian species, but to cause no disease in humans. For the past 50 years, NDV has been shown to be an effective oncolytic agent, causing specific lysis of cancerous but not normal cells. The specificity of the oncolytic effect of NDV is presumed to result from its sensitivity to the antiviral effects of the type I interferon (IFN) system, which is impaired in a great number of tumors. Despite these findings, the in vivo oncolytic efficiency of naturally-occurring NDV strains can still be relatively low, as many tumors still possess an adequate immune response to suppress viral replication and spread. We hypothesized that the enhanced suppression of the innate immune responses by the NDV would allow for increased viral replication and enhanced oncolytic activity. We have previously described the establishment of a reverse-genetics system for genomic manipulation and construction of NDV vaccines and oncolytic virus vectors. Using this system, we first generated a recombinant NDV virus of Hitchner B1 strain with enhanced fusogenic properties, and showed its enhanced ability to form syncytia and lyse a variety of tumor cell lines, when compared to its wild-type counterpart. To further enhance the interferon-antagonistic properties of the virus, we generated a fusogenic NDV virus expressing influenza NS1 protein, which was previously characterized as an antagonist of innate immune responses in human and mouse cells. The resultant virus suppressed the induction of cellular IFN response and was enhanced in its ability to replicate, form syncytia, and lyse a variety of human and mouse tumor cell lines. Inoculation of C57/BL6 and nude mice with the NDV-NS1 virus resulted in no significant adverse effects, confirming its safety. Using the aggressive syngeneic murine B16-F10 footpad melanoma model, we show that the intratumoral injection with NDV-NS1 virus was more effective in clearing the established footpad tumors and resulted in higher overall long-term animal survival, when compared to its counterpart not expressing NS1. Tumors excised from the mice treated with NDV showed a higher degree of lymphocyte infiltration, when compared to the untreated control group. Moreover, mice treated with NDV developed melanoma-specific CTL responses, which were stronger in the NDV-NS1 group. Overall, these findings demonstrate that modulation of innate immune responses by NDV can be an effective strategy for enhancement of its oncolytic properties and leads to generation of effective adaptive immune response against the tumor cells.
44. Oncolytic Virus Therapy of Glioma Sensitizes Glioma Cells to Cilengitide Treatment
Kazuhiko Kurozumi,1 Alvarez-Breckenridge Christopher,1 Dmitrieva Nina,1 Jayson Hardcastle,1 E. A. Chiocca,1 Balveen Kaur.1 1 Neurological Surgery, The Ohio State University, Columbus, OH. Oncolytic HSV-1 derived viruses (OVs) are being investigated as treatment modalities for many cancers. However, a clear understanding of the action of this biological therapy in human patients is currently not feasible. We have been investigating OV therapy induced changes in the tumor extracellular matrix. Transcript profiling of secreted proteins involved in angiogenesis revealed a significant, induction of CYR61 gene expression (8.94 fold, P = 0.001) in tumors treated with OV in vivo. Further CYR61 mRNA and protein were induced upon S17
Cancer - Apoptosis and Suicide: Viral Vectors infection using different OVs in multiple human cancer cell lines and primary human tumor derived cells in vitro and in orthotopic human gliomas grown in nude mice in vivo. CYR61 is a secreted ECM protein that can bind to and activate integrins αvβ3 and αvβ5. Increased CYR61 in the ECM of breast cancer cells has also been shown to activate an autocrine loop,resulting in upregulation of its own receptor αvβ3 resulting in their increased sensitization to integrin anatgonists (Menendez JA, et al. Oncogene 2005). Consistent with this we found that glioma cells stably over expressing CYR61 were much more sensitive to the integrin antagonist Cilengetide, than control cells (LD50=146.5 ng/ml of overexpressing Vs control LD50=521.1 ng/ ml) (P<0.05). Hence we hypothesized that OV treatment induced secreted CYR61 would increase integrins αvβ3 on both infected and uninfected U87delta EGFR glioma cells, and sensitizing them to integrin antagonists such as Cilengitide. We tested the effect of OV treatment of glioma cells on expression of integrin receptors αvβ3. Immunofluorescent staining of glioma cells revealed increased staining of these integrin receptors on cell membranes of cells infected with OV, compared to uninfected control cells. Moreover we detected synergistic killing of glioma cells treated with OV and Cilengitide compared to cells treated with either agent alone (Chou – Talalay combination indices = 0.39). These results suggested that treatment of glioma with OV followed by treatment with Cilengitide would enhance therapeutic efficacy. We tested this hypothesis in SCID mice with intracranial tumors (U87∆EGFR). Briefly seven days after tumor cell implantation, mice were treated with OV or PBS by direct intratumoral injection. Five days after OV treatment mice were injected systemically with a single dose of cRGD (5mg/kg) or PBS. Mice treated with OV and Cilengitide survived significantly longer than mice treated with OV alone (median survival: OV: d16; cRGD + OV: d25) (P < 0.01 between OV and cRGD + OV). These results indicate OV is a sensitizer to Cilengitide in proportion to the levels of target integrin expression and the potential of combining OV treatment with Cilengitide to improve therapeutic outcome.
45. Suppression of Tumor Growth by Single Injection of Type 8 Self-Complementary AAV Vector Expressing Endostatin in Lymphoma Model Mice at Low Vector Dose Yizhuo Wang, Xiao Chen, Yuki Oue, Noriko Miyake, Koichi Miyake, Takashi Shimada. 1 Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan.
Since tumor growth and metastasis depends on the generation of new blood vessels, anti-angiogenic therapy was proposed to be effective for the treatment of a variety of malignancies. To examine the feasibility of adeno-associated viral (AAV) vector mediated systemic anti-angiogenic gene therapy for lymphoma, we constructed AAV vectors (AAV2/1, AAV2/2, AAV2/7, and AAV2/8) expressing secretable murine endostatin (mEnd) and examined the secreted therapeutic protein levels in vivo. When these vectors (5x1010 vg each) were injected into a quadriceps muscle of C57BL/6 mice, the highest serum level of therapeutic protein detected by ELISA was observed in AAV2/8 injected mice (8>7>1>2) one week after injection. Sustained expression of mEnd was detected at least for four months. For further development of AAV vector for systemic anti-angiogenic gene therapy, we generated self-complementary AAV vector (scAAV2/8) expressing mEnd and compared with single stranded AAV (ssAAV2/8). Four weeks after injection, about 100 times higher level of endostatin was detected in the plasma of the mouse administered with scAAV2/8 compared to ssAAV2/8 (1x109 vg each). To examine the effect of scAAV2/8 expressing mEnd, we established a lymphoma murine model in which the lymphoma cell line A20 expressing luciferase gene was inoculated into the caudal vein of BALB/C. Using this lymphoma murine model, we can detect S18
the tumor growth and metastasis by a real-time in vivo imaging analyze system (IVIS). After single injection of ssAAV2/8 expressing mEnd into the right quadriceps muscle of the lymphoma model mice, tumor cell growth was monitored by IVIS. Suppression of tumor growth was observed in scAAV2/8 expressing mEnd injected mice compared to control GFP expressing AAV injected mice (1.2x109 vs. 4.4x109 photon/sec; p<0.05). Survival effect was also detected in scAAV2/8 injected mice (47 vs. 30 days). These results demonstrated that scAAV2/8 mediated systemic expression of endostatin is useful for the gene therapy of lymphoma with only low dose administration of AAV vector.
46. The Combination of Oncolytic Adenoviruses and Ionizing Radiation Inhibits DNA Double Strand Break Detection Which Results in Autophagocytotic Prostate Cancer Cell Death
M. Rajecki,1 T. M. Kiviharju-af Hallstrom,2 T. Hakkarainen,1 P. Nokisalmi,1 S. Hautaniemi,3 A. I. Nieminen,4 M. Tenhunen,5 R. M. Desmond,6 D.-T. Chen,7 K. Guse,1 U.-H. Stenman,8 R. Gargini,9 M. Kapanen,5 J. Klefstrom,4 A. Kanerva,1 S. Pesonen,1 A. Hemminki.1 1 Molecular Cancer Biology Program & Transplantation Laboratory, University of Helsinki (UH), Helsinki University Central Hospital (HUCH), Helsinki, Finland; 2Molecular Cancer Biology Program, UH, Helsinki, Finland; 3Computational Systems Laboratory, UH, Helsinki, Finland; 4Cancer Cell Circuitry Laboratory, UH, Helsinki, Finland; 5Department of Oncology, HUCH, Helsinki, Finland; 6Biostatistics and Bioinformatics Unit, University of Alabama, Birmingham; 7Moffitt Cancer Center, University of South Florida, Tampa; 8Department of Clinical Chemisty, UH, Helsinki, Finland; 9Universidad Autonoma de Madrid, Madrid, Spain. Most advanced solid tumors, including hormone refractory prostate cancer, are relatively radiation resistant. Approaches that increase sensitivity to or synergize with radiation can be useful. Oncolytic adenoviruses (Ads) have shown efficacy against prostate cancer in preclinical and clinical studies. The combination of Ads and radiotherapy has shown better efficacy in cell killing than either agent alone but the mechanisms underlying this phenomenon remains unknown. We investigated the synergy between Ads and radiotherapy and found that infection with Ads 24 h after irradiation results in synergistic prostate cancer cell killing. Mechanistic studies revealed that combination treatment did not increase viral replication or caspase-3 activity (a sign of apoptosis), nor were specific pathways affected in microarray analysis. Instead, western blot analyses showed a reduction in Mre11 levels and in its ATM mediated down-stream component Chk2-Thr68, in combination treated cells. Another downstream component of ATM, γH2AX- Ser139, which is a marker of double strand breaks and DNA configurations misinterpreted as such, was increased in combination treated cells. Our data indicates that the thousands of short double stranded DNA genomes that result from Ad replication, are recognized by the DNA signalling pathway, which results in induction of the double strand break repair response. However, Ad proteins E3orf3, E4orf6 and E1b55K counteract this mechanism to maximize cellular takeover and productive Ad replication. In cells that have sustained radiation injury, these Ad proteins inhibit double strand break repair and thus DNA damage accumulates. Our findings suggest that this eventually leads to autophagy, which was demonstrated by LC3 immunofluorescence and FACS. In summary, we found that oncolytic adenoviruses are synergistic with ionizing radiation because they inhibit double strand break repair for subsequent induction of autophagy. These molecular mechanisms may have widespread importance if they are confirmed relevant with regard to cytopathic effect and autophagy in general.
Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy