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A clinically relevant ex vivo toxicity assay using human liver slices to determine specificity of oncolytic viruses
NOVEL CANCER TARGETED THERAPEUTICS considered to be the rate-limiting step in expression of genes delivered by nonviral methods. Presumably, nuclear delivery of plasmid DNA can be enhanced by the association of a nuclear localization sequence (NLS) with a nonviral vector. Ubiquitous NLSs have demonstrated inconsistent modulation of transgene expression in nonviral delivery systems. This class of NLSs, typically derived from viral proteins, is associated with inflammation and immune responses that may compromise the potential of in vivo applications. Furthermore, such NLSs have demonstrated no selectivity for tumor or other cellular targets, presumably modulating transgene expression in a uniform fashion among all cells. This work demonstrates the first discovery of ligands that differentially recognize nuclei of tumorigenic cells from nontumorigenic cell nuclei. The capacity for differential targeting of therapy to the nuclei of tumorigenic cells represents a new therapeutic strategy with the potential to improve efficacy of drug, gene, and radionuclide delivery. This work was funded by the US Army Medical Research and Materiel Command BCRP-CDMRP: BC023276 - “Nuclear Targeting of Drug and Gene Therapies to Breast Carcinoma.”
melanoma specific replicating adenovirus and wildtype adenovirus, which previously showed similar oncolytic activity in melanoma cells in vitro (Rots Mol Ther 2001). Medium samples taken 1 day after infection contained similar amounts of viruses as determined by a modified limiting dilution titer assay. Interestingly, samples taken after 7 days of incubation revealed a 6log higher concentration of wildtype virus compared to the cancer specific replicative virus. These data demonstrate that human liver slices are highly suitable to study replication of adenoviruses. Moreover, we showed that the melanoma specific virus is severely crippled for replication in the liver. In conclusion, the precision cut tissue slices represent a powerful tool for the preclinical screening on selectivity and efficacy of oncolytic viruses on human tissues.
28. A Clinically Relevant Ex Vivo Toxicity Assay Using Human Liver Slices To Determine Specificity of Oncolytic Viruses
Roisin M. Dwyer,1 Elizabeth R. Bergert,1 Michael K. O’Connor,2 Sandra J. Gendler,3 John C. Morris.1 1 Department of Endocrinology, Mayo Clinic, Rochester, MN; 2 Department of Nuclear Medicine, Mayo Clinic, Rochester, MN; 3 Department of Biochemistry and Molecular Biology, Mayo Clinic, Scottsdale, AZ.
Marianne G. Rots,1 Marieke GL Elferink,2 Dorenda Oosterhuis,1 Peter Olinga,2 David T. Curiel,3 Hidde J. Haisma,1 Geny M. M. Groothuis.2 1 Therapeutic Gene Modulation, Groningen University, Groningen, Netherlands; 2Pharmacokinetics and Drug Delivery, Groningen University, Groningen, Netherlands; 3Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL. Conditionally replicative viruses, based on tumor cell biology or tumor specific promoters, represent a powerful approach to treat cancer. However, several problems have been encountered including replication profiles which could not be explained by tumor cell biology and lack of specificity of the tumor specific promoters in viral context. As iv administrated viruses home to the liver, absence of aspecific replication is of crucial importance in clinical success of such agents. Animal studies still are the golden standard to study efficacy of this approach. However, replication of human (adeno)viruses is absent in non-human tissues. The use of animal models therefore does not provide information on the specificity of replication. Therefore the need for assays to determine lack of replication in healthy cells, especially liver cells, is obvious. To provide insight in specificity and efficacy of newly developed (adenoviral) oncolytic agents, we previously introduced the precision cut tissue slice technique as a well characterized tool to be used in gene therapy toxicity assays (Rots, Mol Ther 2003). Liver slices are about 15-cell layer thick organ sections (1x106 hepatocytes) in which the original architecture is conserved. In our institute, we use mouse, rat and human liver slices as well as slices prepared from heart, lung, kidney, and intestine. Slices have been extensively validated for the study of drug metabolism and toxicity. In addition they appeared valuable in studies on inflammatory reactions. Human and rat liver slices exposed to lipopolysaccharide show activation of the Kupffer cells resulting in cytokine release and in upregulation of iNOS gene expression in the hepatocytes and Kupffer cells. These data are in accordance with in vivo data. The release of cytokines by the Kupffer cells was inhibited by anti-inflammatory drugs such as dexamethasone (Elferink Hepatol 2000; Olinga J Hepatol 2001) Our studies on the use of liver slices to determine selectivity of retargeted adenoviruses prove that slices are a powerful model for the preclinical screening of newly developed viruses. In the present research we show that the specificity and efficiency of conditionally replicative adenoviruses can be determined extremely well using the slice technique. Human slices were infected with a S12
29. In Vivo Imaging and Therapy of Breast Cancer Xenografts Following MUC1 PromoterDriven Expression of the Sodium Iodide Symporter (NIS)
The sodium iodide symporter (NIS) is a transmembrane glycoprotein responsible for the uptake of iodide into cells. Robust NIS expression in the thyroid gland provides for effective imaging and non-invasive treatment of thyroid disease using radioactive iodide. Gene therapy involving selective induction of NIS expression in other tumors offers the possibility of imaging and treating a variety of tumor types with radioactive iodide. We have recently reported in vitro, plasmid-mediated expression of NIS under the control of the MUC1 promoter in a variety of tumor cell lines. Since MUC1 is overexpressed in many tumor types, particularly breast cancer, this promoter provides the ability to selectively target tumor tissue. In this study, a replication-deficient adenoviral construct containing the MUC1-NIS insert was used to induce iodide uptake in breast cancer xenografts. Subcutaneous T47D breast cancer xenografts were established on both flanks of athymic nude mice. When they had reached an appropriate volume, intratumoral injections of Ad5/ MUC1/NIS or control virus were administered. Mice were then given an intra-peritoneal injection of either 0.5mCi 123I (imaging) or 3mCi 131I (therapy). Scanning with a gamma camera revealed clear images of iodide uptake in the mouse stomach, thyroid and bladder, as well as at the site of the tumors injected with Ad5/MUC1/NIS. Tumors injected with the promoter-less Ad5/NIS did not take up iodide. Mice treated with 131I showed a marked regression in tumor size within one week of iodide administration, with tumor volume reduced by 50% within three weeks. Tumors that had been injected with control virus did not express NIS, and retained their original volume or increased in size. These preliminary in vivo experiments demonstrate that the Ad5/MUC1/NIS construct is capable of inducing robust NIS expression and iodide uptake in MUC1 positive breast tumor xenografts. This may have potential applications for the imaging and treatment of a variety of MUC1-positive tumor types.
Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy
melanoma specific replicating adenovirus and wildtype adenovirus, which previously showed similar oncolytic activity in melanoma cells in vitro (Rots Mol Ther 2001). Medium samples taken 1 day after infection contained similar amounts of viruses as determined by a modified limiting dilution titer assay. Interestingly, samples taken after 7 days of incubation revealed a 6log higher concentration of wildtype virus compared to the cancer specific replicative virus. These data demonstrate that human liver slices are highly suitable to study replication of adenoviruses. Moreover, we showed that the melanoma specific virus is severely crippled for replication in the liver. In conclusion, the precision cut tissue slices represent a powerful tool for the preclinical screening on selectivity and efficacy of oncolytic viruses on human tissues.
28. A Clinically Relevant Ex Vivo Toxicity Assay Using Human Liver Slices To Determine Specificity of Oncolytic Viruses
Roisin M. Dwyer,1 Elizabeth R. Bergert,1 Michael K. O’Connor,2 Sandra J. Gendler,3 John C. Morris.1 1 Department of Endocrinology, Mayo Clinic, Rochester, MN; 2 Department of Nuclear Medicine, Mayo Clinic, Rochester, MN; 3 Department of Biochemistry and Molecular Biology, Mayo Clinic, Scottsdale, AZ.
Marianne G. Rots,1 Marieke GL Elferink,2 Dorenda Oosterhuis,1 Peter Olinga,2 David T. Curiel,3 Hidde J. Haisma,1 Geny M. M. Groothuis.2 1 Therapeutic Gene Modulation, Groningen University, Groningen, Netherlands; 2Pharmacokinetics and Drug Delivery, Groningen University, Groningen, Netherlands; 3Human Gene Therapy, University of Alabama at Birmingham, Birmingham, AL. Conditionally replicative viruses, based on tumor cell biology or tumor specific promoters, represent a powerful approach to treat cancer. However, several problems have been encountered including replication profiles which could not be explained by tumor cell biology and lack of specificity of the tumor specific promoters in viral context. As iv administrated viruses home to the liver, absence of aspecific replication is of crucial importance in clinical success of such agents. Animal studies still are the golden standard to study efficacy of this approach. However, replication of human (adeno)viruses is absent in non-human tissues. The use of animal models therefore does not provide information on the specificity of replication. Therefore the need for assays to determine lack of replication in healthy cells, especially liver cells, is obvious. To provide insight in specificity and efficacy of newly developed (adenoviral) oncolytic agents, we previously introduced the precision cut tissue slice technique as a well characterized tool to be used in gene therapy toxicity assays (Rots, Mol Ther 2003). Liver slices are about 15-cell layer thick organ sections (1x106 hepatocytes) in which the original architecture is conserved. In our institute, we use mouse, rat and human liver slices as well as slices prepared from heart, lung, kidney, and intestine. Slices have been extensively validated for the study of drug metabolism and toxicity. In addition they appeared valuable in studies on inflammatory reactions. Human and rat liver slices exposed to lipopolysaccharide show activation of the Kupffer cells resulting in cytokine release and in upregulation of iNOS gene expression in the hepatocytes and Kupffer cells. These data are in accordance with in vivo data. The release of cytokines by the Kupffer cells was inhibited by anti-inflammatory drugs such as dexamethasone (Elferink Hepatol 2000; Olinga J Hepatol 2001) Our studies on the use of liver slices to determine selectivity of retargeted adenoviruses prove that slices are a powerful model for the preclinical screening of newly developed viruses. In the present research we show that the specificity and efficiency of conditionally replicative adenoviruses can be determined extremely well using the slice technique. Human slices were infected with a S12
29. In Vivo Imaging and Therapy of Breast Cancer Xenografts Following MUC1 PromoterDriven Expression of the Sodium Iodide Symporter (NIS)
The sodium iodide symporter (NIS) is a transmembrane glycoprotein responsible for the uptake of iodide into cells. Robust NIS expression in the thyroid gland provides for effective imaging and non-invasive treatment of thyroid disease using radioactive iodide. Gene therapy involving selective induction of NIS expression in other tumors offers the possibility of imaging and treating a variety of tumor types with radioactive iodide. We have recently reported in vitro, plasmid-mediated expression of NIS under the control of the MUC1 promoter in a variety of tumor cell lines. Since MUC1 is overexpressed in many tumor types, particularly breast cancer, this promoter provides the ability to selectively target tumor tissue. In this study, a replication-deficient adenoviral construct containing the MUC1-NIS insert was used to induce iodide uptake in breast cancer xenografts. Subcutaneous T47D breast cancer xenografts were established on both flanks of athymic nude mice. When they had reached an appropriate volume, intratumoral injections of Ad5/ MUC1/NIS or control virus were administered. Mice were then given an intra-peritoneal injection of either 0.5mCi 123I (imaging) or 3mCi 131I (therapy). Scanning with a gamma camera revealed clear images of iodide uptake in the mouse stomach, thyroid and bladder, as well as at the site of the tumors injected with Ad5/MUC1/NIS. Tumors injected with the promoter-less Ad5/NIS did not take up iodide. Mice treated with 131I showed a marked regression in tumor size within one week of iodide administration, with tumor volume reduced by 50% within three weeks. Tumors that had been injected with control virus did not express NIS, and retained their original volume or increased in size. These preliminary in vivo experiments demonstrate that the Ad5/MUC1/NIS construct is capable of inducing robust NIS expression and iodide uptake in MUC1 positive breast tumor xenografts. This may have potential applications for the imaging and treatment of a variety of MUC1-positive tumor types.
Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy