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682. Development of a Virus-Associated RNA-Deleted Adenovirus Vector
ADENOVIRUS AND OTHER DNA VIRUS VECTORS III TβRII∆cyt-treated rats compared to cirrhotic group (AST; 490±126.4 versus 180±58.1 p<0.001 and ALT 469±290.2 versus 102±60.4 p<0.05) at day 28. Conclusion: Expression of TGF-β-truncated receptor II prevents fibrosis progression in the liver and improves liver function reducing gene expression of pro-fibrogenic molecules (TGF-β, Col-1, PAI and CB1). Significant reduction in steatosis was also observed when Ad-TβRII∆cyt therapy was employed and there was a tendency of CB1 to decrease, which suggest that the two phenomena might be related.
682. Development of a Virus-Associated RNADeleted Adenovirus Vector
Mitsuhiro Machitani,1 Kazufumi Katayama,1 Fuminori Sakurai,1 Hayato Matsui,1 Tomoko Yamaguchi,2 Takayuki Suzuki,2 Kenji Kawabata,1,2 Hiroyuki Mizuguchi.1,2,3 1 Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan; 2Laboratory of Stem Cell Regulation, National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan; 3The Center for Advanced Medical Engineering and Informatics, Osaka University, Suita, Osaka, Japan. A major limitation of the use of adenovirus (Ad) vectors is the innate immune response, which causes inflammatory cytokine production and tissue damage. To overcome this limitation, it is necessary to develop safer Ad vectors that are less likely to induce this response. The adenoviral genome encodes two noncoding small RNAs, virusassociated (VA)-RNA I and VA-RNA II, which are transcribed by RNA polymerase III and promote adenovirus amplification. Recently, we reported that VA-RNAs are produced in the cells transduced with conventional first-generation (E1-deleted) Ad vector (FG-Ad) and trigger innate immune responses through intracellular nucleic acid sensors. In this study, we have developed a VA-RNA-deleted Ad (Ad∆VR) vector from which the transcriptional control elements of the VA-RNA-expression were deleted. First, pAd∆VR-EGFP, a plasmid encoding the PacI-flanked Ad∆VR vector genome with an EGFP-expression cassette in place of the E1 region, was digested with PacI and transfected into 293 cells according to the conventional method for preparing Ad vectors. However, no propagation of the Ad vector was observed. Next, we prepared 293 cells inducibly expressing VA-RNA I under the control of a tetracycline-dependent H1 promoter (VR293 cells). VR293 cells with appropriate induction of VA-RNA I-expression allowed the propagation of the Ad∆VREGFP vector. The Ad∆VR-EGFP vector showed efficient transduction in the cultured cells, comparable to that of the conventional FG-Ad vector expressing EGFP. Furthermore, although the FG-Ad vector shows E1-independent replication in some cultured cells, the Ad∆VR vector exhibited significantly lower levels of viral replication than the FG-Ad vector. Given these results, the Ad∆VR vector may be a safer alternative to the FG-Ad vector.
683. Pre-Clinical Characterization of Endothelial Cells as Carriers of Oncolytic Adenoviruses Bart Thaci,1 Ilya V. Ulasov,1 Maciej S. Lesniak.1 Neurosurgery, The University of Chicago, Chicago, IL.
1
Background Cancer remains the second most common cause of death in the developed world. New targeted therapies are being tested in clinical trials. Oncolytic adenoviruses are one of the promising new agents, but delivery to tumors is still unsatisfactory. Systemic delivery of oncolytic adenoviruses to tumors via cell based carriers is very attractive and needs to be optimized. In this work, we show that endothelial cells can deliver fiber modified, survivin driven adenovirus to glioma cells. Material and methods Human umbilical vein endothelial cells were screened for surface receptors via flow cytometry. Transduction efficacy of fiber modified viruses (Ad. S262
Luc-Pk7; Ad.Luc-RGD; Ad.Luc-5/3) was compared to wild-type fiber adenovirus by measuring luciferase expression. Survivin and CXCR4 activity was analyzed via qRT-PCR. Adenoviral replication was quantified by measuring E1A region copy numbers and progeny titer after infection with replication competent CRAd.Survivin.5/3. To show that endothelial cells can deliver adenovirus to tumor cells: endothelial cells were infected with CRAd.Survivin.5/3 and washed thoroughly before plated in different ratios with U87 glioma cells. Viability was visualized via Crystal Violet assay. Results Fiber modifications increased adenoviral transduction 2.5-3.5 folds. Longer incubation time (4h vs. 1 h) during infection enhanced transduction four folds. Survivin expression in endothelial cells was 20 fold higher than CXCR4 and was dependent on the presence of growth factors. It dropped four fold when using starved media instead of growth factor replenished media. The same did progeny titer levels, while E1A copy numbers were affected even more (up to 10 fold). Endothelial cells were able to deliver oncolytic adenovirus and effectively kill glioma cells after co-culture. Conclusion Endothelial cells can be used as cell carriers for oncolytic adenoviruses. Increasing expression of survivin in endothelial cell can enhance adenovirus delivery.
684. miR122 Targets Insertion in Wild-Type Serotype 5 Adenovirus To Ablate Liver Replication Sergio Lavilla-Alonso,1 Erkko Ylösmäki,1 Kalle Saksela.1 Virology, Haartman Institute - University of Helsinki, Helsinki, Finland.
1
Adenovirus -based oncolytic therapies present important limitations regarding tissue selectivity and toxicity, an issue well-documented for the commonly used adenovirus serotype 5 (Ad5). Basic research has shown a specific micro-RNA (miR) pattern for many tissues, like the virtually exclusive expression of miR122 by liver. We have shown that this feature can be utilized to selectively ablate adenovirus replication in cells of hepatic origin by genomic insertion of miR122 targets to different reduce E1A gene expression. In this study we have examined in more detail the optimal number of miR122 targets, studied the cell type-specific inhibitory potential of miR122 via careful cell viability analyses, and extended these studies to more relevant experimental systems. These results will provide a rational basis for proceeding towards ex vivo and in vivo studies to test the applicability of this system for clinical gene therapy purposes.
685. TRAIL-Expressing Oncolytic Viral DNA/ Liposome Hybrid Vector for Lung Cancer Gene Therapy
Oh-Joon Kwon,1 Eunah Kang,1 Jaerim Kim,1 Kyungmin Nam,1 Sung Wan Kim,2,3 Chae-Ok Yun.1 1 Brain Korea 21 Project for Medical Sciences, Institute for Cancer Research, Yonsei Cancer Center, Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Republic of Korea; 22Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT; 3Department of Bioengineering, College of Engineering, Hanyang University, Seoul, Republic of Korea. Adenovirus (Ad) as cancer molecular therapeutics has been extensively exploited with modification of Ad genetic information, and administration methods in vivo for effective delivery. However, delivery efficacy in vivo has been limited due to Ad envelopment by pre-existing neutralizing antibody, the liver uptake, and hepatotoxicity of Ad. In this study, as an alternative approach of cancer virotherapy, oncolytic viral DNA delivery via lipid envelopment was investigated for orthotopic lung cancer gene therapy. For synergistic therapeutic effect, multifunctional oncolytic Ad DNA expressing TRAIL as a model of therapeutic protein was generated. Lipid hybrid vectors encapsulating oncolytic Ad DNA, DOTAP:DOPE/H5mT-Rd19/ Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
682. Development of a Virus-Associated RNADeleted Adenovirus Vector
Mitsuhiro Machitani,1 Kazufumi Katayama,1 Fuminori Sakurai,1 Hayato Matsui,1 Tomoko Yamaguchi,2 Takayuki Suzuki,2 Kenji Kawabata,1,2 Hiroyuki Mizuguchi.1,2,3 1 Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka, Japan; 2Laboratory of Stem Cell Regulation, National Institute of Biomedical Innovation, Ibaraki, Osaka, Japan; 3The Center for Advanced Medical Engineering and Informatics, Osaka University, Suita, Osaka, Japan. A major limitation of the use of adenovirus (Ad) vectors is the innate immune response, which causes inflammatory cytokine production and tissue damage. To overcome this limitation, it is necessary to develop safer Ad vectors that are less likely to induce this response. The adenoviral genome encodes two noncoding small RNAs, virusassociated (VA)-RNA I and VA-RNA II, which are transcribed by RNA polymerase III and promote adenovirus amplification. Recently, we reported that VA-RNAs are produced in the cells transduced with conventional first-generation (E1-deleted) Ad vector (FG-Ad) and trigger innate immune responses through intracellular nucleic acid sensors. In this study, we have developed a VA-RNA-deleted Ad (Ad∆VR) vector from which the transcriptional control elements of the VA-RNA-expression were deleted. First, pAd∆VR-EGFP, a plasmid encoding the PacI-flanked Ad∆VR vector genome with an EGFP-expression cassette in place of the E1 region, was digested with PacI and transfected into 293 cells according to the conventional method for preparing Ad vectors. However, no propagation of the Ad vector was observed. Next, we prepared 293 cells inducibly expressing VA-RNA I under the control of a tetracycline-dependent H1 promoter (VR293 cells). VR293 cells with appropriate induction of VA-RNA I-expression allowed the propagation of the Ad∆VREGFP vector. The Ad∆VR-EGFP vector showed efficient transduction in the cultured cells, comparable to that of the conventional FG-Ad vector expressing EGFP. Furthermore, although the FG-Ad vector shows E1-independent replication in some cultured cells, the Ad∆VR vector exhibited significantly lower levels of viral replication than the FG-Ad vector. Given these results, the Ad∆VR vector may be a safer alternative to the FG-Ad vector.
683. Pre-Clinical Characterization of Endothelial Cells as Carriers of Oncolytic Adenoviruses Bart Thaci,1 Ilya V. Ulasov,1 Maciej S. Lesniak.1 Neurosurgery, The University of Chicago, Chicago, IL.
1
Background Cancer remains the second most common cause of death in the developed world. New targeted therapies are being tested in clinical trials. Oncolytic adenoviruses are one of the promising new agents, but delivery to tumors is still unsatisfactory. Systemic delivery of oncolytic adenoviruses to tumors via cell based carriers is very attractive and needs to be optimized. In this work, we show that endothelial cells can deliver fiber modified, survivin driven adenovirus to glioma cells. Material and methods Human umbilical vein endothelial cells were screened for surface receptors via flow cytometry. Transduction efficacy of fiber modified viruses (Ad. S262
Luc-Pk7; Ad.Luc-RGD; Ad.Luc-5/3) was compared to wild-type fiber adenovirus by measuring luciferase expression. Survivin and CXCR4 activity was analyzed via qRT-PCR. Adenoviral replication was quantified by measuring E1A region copy numbers and progeny titer after infection with replication competent CRAd.Survivin.5/3. To show that endothelial cells can deliver adenovirus to tumor cells: endothelial cells were infected with CRAd.Survivin.5/3 and washed thoroughly before plated in different ratios with U87 glioma cells. Viability was visualized via Crystal Violet assay. Results Fiber modifications increased adenoviral transduction 2.5-3.5 folds. Longer incubation time (4h vs. 1 h) during infection enhanced transduction four folds. Survivin expression in endothelial cells was 20 fold higher than CXCR4 and was dependent on the presence of growth factors. It dropped four fold when using starved media instead of growth factor replenished media. The same did progeny titer levels, while E1A copy numbers were affected even more (up to 10 fold). Endothelial cells were able to deliver oncolytic adenovirus and effectively kill glioma cells after co-culture. Conclusion Endothelial cells can be used as cell carriers for oncolytic adenoviruses. Increasing expression of survivin in endothelial cell can enhance adenovirus delivery.
684. miR122 Targets Insertion in Wild-Type Serotype 5 Adenovirus To Ablate Liver Replication Sergio Lavilla-Alonso,1 Erkko Ylösmäki,1 Kalle Saksela.1 Virology, Haartman Institute - University of Helsinki, Helsinki, Finland.
1
Adenovirus -based oncolytic therapies present important limitations regarding tissue selectivity and toxicity, an issue well-documented for the commonly used adenovirus serotype 5 (Ad5). Basic research has shown a specific micro-RNA (miR) pattern for many tissues, like the virtually exclusive expression of miR122 by liver. We have shown that this feature can be utilized to selectively ablate adenovirus replication in cells of hepatic origin by genomic insertion of miR122 targets to different reduce E1A gene expression. In this study we have examined in more detail the optimal number of miR122 targets, studied the cell type-specific inhibitory potential of miR122 via careful cell viability analyses, and extended these studies to more relevant experimental systems. These results will provide a rational basis for proceeding towards ex vivo and in vivo studies to test the applicability of this system for clinical gene therapy purposes.
685. TRAIL-Expressing Oncolytic Viral DNA/ Liposome Hybrid Vector for Lung Cancer Gene Therapy
Oh-Joon Kwon,1 Eunah Kang,1 Jaerim Kim,1 Kyungmin Nam,1 Sung Wan Kim,2,3 Chae-Ok Yun.1 1 Brain Korea 21 Project for Medical Sciences, Institute for Cancer Research, Yonsei Cancer Center, Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Republic of Korea; 22Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT; 3Department of Bioengineering, College of Engineering, Hanyang University, Seoul, Republic of Korea. Adenovirus (Ad) as cancer molecular therapeutics has been extensively exploited with modification of Ad genetic information, and administration methods in vivo for effective delivery. However, delivery efficacy in vivo has been limited due to Ad envelopment by pre-existing neutralizing antibody, the liver uptake, and hepatotoxicity of Ad. In this study, as an alternative approach of cancer virotherapy, oncolytic viral DNA delivery via lipid envelopment was investigated for orthotopic lung cancer gene therapy. For synergistic therapeutic effect, multifunctional oncolytic Ad DNA expressing TRAIL as a model of therapeutic protein was generated. Lipid hybrid vectors encapsulating oncolytic Ad DNA, DOTAP:DOPE/H5mT-Rd19/ Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy