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859. Considerations for Intravascular Administration of Oncolytic Herpes Virus for the Treatment of Multiple Liver Metastases
DNA VECTOROLOGY II lipofectamine reduced viral antigenicity, repeated administration could be allowed to attempt safer than virus alone. In the evaluation of IgM and IgE, there was no big difference between conjugated with or without. Moreover we repot the efficacy of HSV in the human and mice serum with or without HSV antibody, titer of the lipofectamineconjugated HSV did not so decreased in the serum with the HSV antibody, while the titer of the HSV without lipofectamine decreased to almost zero in the HSV antibody serum. In vivo study of the mice which had anti-HSV immunity and bearing metastatic liver tumor of mouse colon cancer (MC26), mice injected with lipofectamineconjugated HSV survive for long term more than virus alone with statistic difference. Those results encourage us to convince this new strategy using lipofectamine-conjugated HSV oncolytic virus holding high therapeutic potential for clinical trial.
858. Adapter-Mediated Tumor Targeting with HVEM-Restricted Herpes Simplex Virus
Hyunjung Baek,1,2 Hiroaki Uchida,3 Jae-Hong Kim,2 Masahide Kuroki,4 Justus B. Cohen,3 Joseph C. Glorioso,3 Heechung Kwon.1 1 Laboratory of Molecular Oncology, Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea; 2 School of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea; 3Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA; 4Molecular Oncology and First Department of Biochemistry, Fukuoka University School of Medicine, Fukuoka, Japan. Herpes simplex virus type 1 (HSV-1) is one of several viral systems under development for oncolytic virotherapy. Current designs of oncolytic HSV take advantage of the specific ability of tumor cells to complement certain viral functions required for virus replication. These conditionally replicating virus backbones can be armed with transgenes to ward off anti-viral responses. However, despite ongoing improvements, these designs have thus far failed to achieve full tumor eradication in vivo, largely due limited replication restricting spread throughout the tumor mass. HSV-1 infects cells mainly via the widely expressed entry receptor nectin-1, a cell adhesion molecule, and the less prevalent receptor HVEM expressed predominantly on cells of the immune system. Whereas HVEM has been detected on a variety of non-immune cell lines, we and others have shown that HVEM expression is often not sufficient for HSV-1 infection. We have developed two HSV-1 mutant viruses that are highly impaired for infection through nectin-1 and thus are largely restricted to susceptible HVEM-expressing cells. However, these constructs are only mildly impaired for nectin-1-mediated lateral spread. Accordingly, we hypothesized that these vectors could be used to selectively infect and destroy tumor cells in an HVEM-negative environment by directing the initial infection to a tumor antigen. We developed a soluble, bi-specific adapter that recognizes HVEM-selective HSV1 glycoprotein D (gD) at one end and carcinoembryonic antigen (CEA) on at the other end. We found that this adapter mediates efficient infection of HSV-resistant cells expressing ectopic CEA. We tested a gastric tumor cell line that is susceptible to wt HSV-1 infection, but resistant to infection by HVEM-restricted virus, and found that the adapter enabled infection as well as direct cell-tocell spread, albeit with lower efficiency than wt virus. Given the resistance of this and other tumor lines to infection via HVEM, we expect that the surrounding healthy tissue in such instances will also be resistant to HVEM-restricted viruses and thus that our strategy will provide significant tumor specificity. Since these viruses can spread via nectin-1 found on most tumor cells and are not impaired for replication, thus providing higher oncolytic activity than current oncolytic HSV, further development of this approach may provide a novel means for effective tumor treatment. S328
859. Considerations for Intravascular Administration of Oncolytic Herpes Virus for the Treatment of Multiple Liver Metastases
Naohiro Nomura,1 Hideki Kasuya,1 Toshio Shikano,1 Makoto Misawa,1 Akiyuki Kanzaki,1 Shin Takeda,1 Akimasa Nakao.1 1 Department of Surgery II, Nagoya University, Nagoya City, Japan.
Purpose. Oncolytic viral therapy is a newly-developed modality for treating tumors. Many clinical trials using oncolytic virus have been performed worldwide, but most of them have used local injection in the tumor. Determination of the effect and safety of intravascular virus injection instead of local injection is necessary for clinical use against multiple liver metastases and systemic metastases. Methods. To evaluate the efficacy and safety of intravascular virus therapy, mice bearing multiple liver metastases were treated by intraportal or intravenous administration of the herpes simplex virus type 1 (HSV-1) mutant, hrR3. Mice treated with hrR3 were killed and organs were harvested for lacZ staining and PCR analysis. Inactivation of oncolytic virus in bloodstream was assessed by neutralization assay in vitro. Infectious activity of hrR3 with vascular endothelial cells was evaluated by replication and cytotoxicity assay. Results. The survival rate of animals treated by hrR3 was significantly improved compared with the untreated group. lacZ staining and PCR analysis demonstrated detectable virus in the tumor but not in normal tissue or other organs except for the adrenal glands. We also showed that vascular endothelial cells allowed virus replication, while normal hepatocytes did not, and human anti-HSV antibody revealed attenuation of the infectious activity of hrR3. Conclusions. Intravascular delivery of hrR3 is effective in treating multiple liver metastases, however several points must be kept in mind at the time of human clinical trials using intravascular virus administration in order to avoid critical side effects.
DNA Vectorology II 860. Site-Directed piggyBac Transposon Integration in Human Cells
Claudia Kettlun,2,3 Aparna Kaja,2 Alfred L. George,4 Edward Rebar,5 Matthew H. Wilson.1,2,3 1 Michael E. DeBakey VA Medical Center, Houston; 2Department of Medicine, Baylor College of Medicine, Houston; 3Center for Cell and Gene Therapy, Baylor College of Medicine, Houston; 4 Vanderbilt University, Nashville; 5Sangamo Biosciences, Inc., Richmond. Gene addition studies for gene therapy impose the risk of insertional mutagenesis and genotoxicity. The ability to direct gene delivery to user-defined chromosomal locations would greatly improve the safety of gene therapy applications. We compared the efficiency of three different but widely used transposon systems (Sleeping Beauty, Mos1, and piggyBac) to mediate gene transfer after addition of a zinc-finger DNA binding domain (ZFP) to the N-terminus of each enzyme. We found that only piggyBac exhibited unaltered activity after ZFP fusion. We next evaluated the ability of our ZFP-piggyBac to target gene integration into a plasmid containing a ZFP target site. We observed ∼8 fold increased gene transfer using ZFP-piggyBac compared to native piggyBac and found that 70% of integrations occurred in a 500bp (250bp in either direction) surrounding the ZFP target site. In our plasmid based gene transfer assay, we did observe apparent “hot spots” for piggyBac integration giving potential insight into the required proximity of the integration site from the ZFP site. We then evaluated the ability of our ZFP-piggyBac to target gene integration near an engineered ZFP site in the human genome. When evaluating clonal cells, we observed that 38-50% of the clones exhibited a sitedirected integration event near the ZFP site. We have also developed Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy
858. Adapter-Mediated Tumor Targeting with HVEM-Restricted Herpes Simplex Virus
Hyunjung Baek,1,2 Hiroaki Uchida,3 Jae-Hong Kim,2 Masahide Kuroki,4 Justus B. Cohen,3 Joseph C. Glorioso,3 Heechung Kwon.1 1 Laboratory of Molecular Oncology, Korea Institute of Radiological and Medical Sciences, Seoul, Republic of Korea; 2 School of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea; 3Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA; 4Molecular Oncology and First Department of Biochemistry, Fukuoka University School of Medicine, Fukuoka, Japan. Herpes simplex virus type 1 (HSV-1) is one of several viral systems under development for oncolytic virotherapy. Current designs of oncolytic HSV take advantage of the specific ability of tumor cells to complement certain viral functions required for virus replication. These conditionally replicating virus backbones can be armed with transgenes to ward off anti-viral responses. However, despite ongoing improvements, these designs have thus far failed to achieve full tumor eradication in vivo, largely due limited replication restricting spread throughout the tumor mass. HSV-1 infects cells mainly via the widely expressed entry receptor nectin-1, a cell adhesion molecule, and the less prevalent receptor HVEM expressed predominantly on cells of the immune system. Whereas HVEM has been detected on a variety of non-immune cell lines, we and others have shown that HVEM expression is often not sufficient for HSV-1 infection. We have developed two HSV-1 mutant viruses that are highly impaired for infection through nectin-1 and thus are largely restricted to susceptible HVEM-expressing cells. However, these constructs are only mildly impaired for nectin-1-mediated lateral spread. Accordingly, we hypothesized that these vectors could be used to selectively infect and destroy tumor cells in an HVEM-negative environment by directing the initial infection to a tumor antigen. We developed a soluble, bi-specific adapter that recognizes HVEM-selective HSV1 glycoprotein D (gD) at one end and carcinoembryonic antigen (CEA) on at the other end. We found that this adapter mediates efficient infection of HSV-resistant cells expressing ectopic CEA. We tested a gastric tumor cell line that is susceptible to wt HSV-1 infection, but resistant to infection by HVEM-restricted virus, and found that the adapter enabled infection as well as direct cell-tocell spread, albeit with lower efficiency than wt virus. Given the resistance of this and other tumor lines to infection via HVEM, we expect that the surrounding healthy tissue in such instances will also be resistant to HVEM-restricted viruses and thus that our strategy will provide significant tumor specificity. Since these viruses can spread via nectin-1 found on most tumor cells and are not impaired for replication, thus providing higher oncolytic activity than current oncolytic HSV, further development of this approach may provide a novel means for effective tumor treatment. S328
859. Considerations for Intravascular Administration of Oncolytic Herpes Virus for the Treatment of Multiple Liver Metastases
Naohiro Nomura,1 Hideki Kasuya,1 Toshio Shikano,1 Makoto Misawa,1 Akiyuki Kanzaki,1 Shin Takeda,1 Akimasa Nakao.1 1 Department of Surgery II, Nagoya University, Nagoya City, Japan.
Purpose. Oncolytic viral therapy is a newly-developed modality for treating tumors. Many clinical trials using oncolytic virus have been performed worldwide, but most of them have used local injection in the tumor. Determination of the effect and safety of intravascular virus injection instead of local injection is necessary for clinical use against multiple liver metastases and systemic metastases. Methods. To evaluate the efficacy and safety of intravascular virus therapy, mice bearing multiple liver metastases were treated by intraportal or intravenous administration of the herpes simplex virus type 1 (HSV-1) mutant, hrR3. Mice treated with hrR3 were killed and organs were harvested for lacZ staining and PCR analysis. Inactivation of oncolytic virus in bloodstream was assessed by neutralization assay in vitro. Infectious activity of hrR3 with vascular endothelial cells was evaluated by replication and cytotoxicity assay. Results. The survival rate of animals treated by hrR3 was significantly improved compared with the untreated group. lacZ staining and PCR analysis demonstrated detectable virus in the tumor but not in normal tissue or other organs except for the adrenal glands. We also showed that vascular endothelial cells allowed virus replication, while normal hepatocytes did not, and human anti-HSV antibody revealed attenuation of the infectious activity of hrR3. Conclusions. Intravascular delivery of hrR3 is effective in treating multiple liver metastases, however several points must be kept in mind at the time of human clinical trials using intravascular virus administration in order to avoid critical side effects.
DNA Vectorology II 860. Site-Directed piggyBac Transposon Integration in Human Cells
Claudia Kettlun,2,3 Aparna Kaja,2 Alfred L. George,4 Edward Rebar,5 Matthew H. Wilson.1,2,3 1 Michael E. DeBakey VA Medical Center, Houston; 2Department of Medicine, Baylor College of Medicine, Houston; 3Center for Cell and Gene Therapy, Baylor College of Medicine, Houston; 4 Vanderbilt University, Nashville; 5Sangamo Biosciences, Inc., Richmond. Gene addition studies for gene therapy impose the risk of insertional mutagenesis and genotoxicity. The ability to direct gene delivery to user-defined chromosomal locations would greatly improve the safety of gene therapy applications. We compared the efficiency of three different but widely used transposon systems (Sleeping Beauty, Mos1, and piggyBac) to mediate gene transfer after addition of a zinc-finger DNA binding domain (ZFP) to the N-terminus of each enzyme. We found that only piggyBac exhibited unaltered activity after ZFP fusion. We next evaluated the ability of our ZFP-piggyBac to target gene integration into a plasmid containing a ZFP target site. We observed ∼8 fold increased gene transfer using ZFP-piggyBac compared to native piggyBac and found that 70% of integrations occurred in a 500bp (250bp in either direction) surrounding the ZFP target site. In our plasmid based gene transfer assay, we did observe apparent “hot spots” for piggyBac integration giving potential insight into the required proximity of the integration site from the ZFP site. We then evaluated the ability of our ZFP-piggyBac to target gene integration near an engineered ZFP site in the human genome. When evaluating clonal cells, we observed that 38-50% of the clones exhibited a sitedirected integration event near the ZFP site. We have also developed Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy