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320. Adenovirus Expansion of Radioiodine Therapy to Non-Thyroid Cancer Treatment and Diagnostics
Recent Advances and Applications in Adenovirus Vectors and Other DNA Viruses II Recent Advances and Applications in Adenovirus Vectors and Other DNA Viruses II 319. Improving the Odds of AdenovirusMediated Gene Therapy By Upregulation of the Coxsackievirus and Adenovirus Receptor
Katherine J.D.A. Excoffon,1 Poornima L.N. Kotha,1 Abimbola O. Kolawole,1 Ran Yan,1 Mahmoud S. Alghamri,1 Brockman L. Brockman,1 Julian Gomez-Cambronero,1 Priyanka Sharma.1 1 Wright State University, Dayton, OH. Adenovirus-mediated gene therapy has been limited by the fact that the primary receptor for most adenovirus serotypes, the Coxsackievirus and adenovirus receptor (CAR), is inaccessible or not expressed on many cell types of interest. Few mechanisms have been discovered that regulate CAR expression and tissue specific localization. In the airway, CAR is mostly considered a cell-cell adhesion protein localized at the basolateral surface of polarized epithelia. Recently, an alternate isoform of CAR, CAREx8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that upregulation of cellular mechanisms that facilitate endogenous CAREx8 protein expression at the apical surface would enhance adenovirus gene transfer. Using polarized model epithelial cell lines and primary human airway epithelia, we found that IL8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAREx8 via activation of AKT/S6K and inhibition of GSK3β. IL-8- mediated upregulation of CAREx8 increased AdV5-β-Gal entry and transduction by approximately 5-fold. Moreover, we found that infiltrating neutrophils bind CAREx8 at the apical surface of a polarized epithelium and, surprisingly, neutrophils enhance AdV5-β-Gal entry into the epithelium by 2-3 fold. The effect of IL-8 and neutrophils on AdV infection could be blocked by fiber-knob from AdV5 but not AdV3, a non-CAR binding serotype, indicating the importance of CAR. These findings suggest that acute inflammation may enhance adenovirus infection. Moreover, therapeutics that stimulate the AKT/ S6K pathway or inhibit GSK3β may be able to augment adenovirusmediated gene therapy.
320. Adenovirus Expansion of Radioiodine Therapy to Non-Thyroid Cancer Treatment and Diagnostics
Ben Eidenschink,1 Jordan Sell,1 Kari Jacobsen,1 Miguel Trujillo,2 Martin Ferdandez-Zapico,2 John Morris,2 Masato Yamamoto,1 Julia Davydova.1 1 University of Minnesota, Minneapolis; 2Mayo Clinic, Rochester. Radioiodine therapy has successfully treated and diagnosed cancers over the last 50 years. The clinical scope of radioiodine is limited in thyroid cancers because of restricted iodine uptake. Our lab proposes to expand the clinical use of radioiodine into nonthyroid cancer theranostics by targeting cancers (e.g. pancreatic and prostate) with oncolytic adenovirus to over express the human sodium iodine symporter (NIS). The adenovirus expression of the symporter allows iodine to concentrate into the targeted cancer cells providing diagnostic imaging with I123 and therapy with I131. We designed infectivity-enhanced oncolytic Adenoviruses (NISOAd), Ad5/3_TSP_E3_ADP_NIS, to express NIS and adenovirus death protein (ADP) from the E3 region. The Ad5/3 fiber modification enables the infection of CAR negative cells. Vector replication is controlled through a tumor specific promoter (TSP, e.g. COX2 in pancreatic and Pb in prostate cancer). We have shown the specificity of our viruses with TSPs in vitro with multiple pancreatic and prostate cancer cell lines. The expression of ADP resulted in an increase in Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
oncolytic activity and transgene expression. The functionality of NIS-OAds was tested with in vitro radioiodine uptake assay and showed up to a 10-fold increase in radioiodine uptake in prostate and pancreatic cancer cell lines. This endorses that oncolytic viruses increase functional NIS expression in tumors. Our animal model experiments included nude mice xenographs to evaluate therapeutic and imaging affects with i.v. injections of NISOAds in A549 lung and LnCap prostate cancer cells. In A549 models, the combination of NIS-OAds and I131 had an increase in therapeutic effect over virus alone signifying functional NIS expression in solid tumors. LnCap xenographs had SPECT/CT images taken after NISOAds and non-replicating virus administration with I123. NIS-OAds had the highest tumor specific signal confirming that NIS-OAds can be diagnostic in tumors. Viral functionality in human tissue was evaluated by PCR in ex vivo tissue slices showing that virus replication was specific to cancerous tissues and did not occur in normal tissue. This study supports the high-level replication control of NIS-OAds in non-thyroid cancer tissues. A related non-replicating NIS expressing Ad is also being evaluated in prostate cancer by Dr. Morris, our collaborator, in a stage I clinical trial.Together the virus specificity, imaging, and therapeutic data support the potential of NIS-OAds as cancer theranostics. In sum, this data provides key functionality and safety support of NIS expressing Ad use in humans. Current projects include therapeutic and imaging animal studies with radioiodine in pancreatic cancer immuno-competent Syrian hamsters, and human xenographs in nude mice. Further studies will evaluate the distribution of NIS expression in vivo and cytotoxicity of virus with radiation. Ultimately the goal of our research is to form a multimodal therapy with radiation and oncolytic virus for diagnosis and therapy of cancers.
321. Oncolytic Adenovirus Expressing IFN as a Tool To Eliminate Pancreatic Cancer Stem Cells Amanda R. Oliveira,1 Christopher J. LaRocca,1 Julia Davydova,1 Masato Yamamoto.1 1 Department of Surgery, University of Minnesota, Minneapolis, MN.
Pancreatic cancer is the 4th leading cause of cancer related death in the US, and curative resection of tumors is the most effective treatment against the disease. Unfortunately, the majority of patients are diagnosed in the advanced stage of the disease, and palliative treatment with Gemcitabine is the therapy of choice. In both cases, curative resection of tumors or chemotherapy, effectiveness can be limited by the presence of pancreatic cancer stem cells. Cancer stem cells (CSC) are correlated with drug resistance, tumor recurrence, and metastasis. CSC are usually located in the hypoxic center of the tumors in a quiescent stage and are not affected by conventional chemotherapeutics that target highly replicating cells, such as Gemcitabine. CSC are also called tumor initiating cells (TIC), and as low as 100 cells are reported to form tumors in pre-clinical models. Inclusion of IFN alpha (IFN) in combination therapy protocols against pancreatic cancer can be highly beneficial to tackle this problem. IFN is reported to be cytotoxic to cancer cells, have antiangiogenic properties, stimulate anti-tumor immunity, and sensitize cancer cells to chemoradiation. In addition, IFN is reported to induce activation of quiescent CSC making them susceptible to chemotherapy drugs that target highly replicating cells. Phase II and III clinical trials combining IFN with 5-FU and radiation in an adjuvant therapy setting reported a 35% increase in the five year overall survival of pancreatic cancer patients. Stimulated by the promising results reported in IFN clinical trials we developed an oncolytic adenovirus expressing IFN (OAd-IFN). Our aim is to use the virus to improve IFN therapeutic effects in combination therapy by restricting high levels of IFN expression to the tumor. In vitro data testing triple combination of OAd-IFN + radiation+ 5-FU using MIA S129
Katherine J.D.A. Excoffon,1 Poornima L.N. Kotha,1 Abimbola O. Kolawole,1 Ran Yan,1 Mahmoud S. Alghamri,1 Brockman L. Brockman,1 Julian Gomez-Cambronero,1 Priyanka Sharma.1 1 Wright State University, Dayton, OH. Adenovirus-mediated gene therapy has been limited by the fact that the primary receptor for most adenovirus serotypes, the Coxsackievirus and adenovirus receptor (CAR), is inaccessible or not expressed on many cell types of interest. Few mechanisms have been discovered that regulate CAR expression and tissue specific localization. In the airway, CAR is mostly considered a cell-cell adhesion protein localized at the basolateral surface of polarized epithelia. Recently, an alternate isoform of CAR, CAREx8, has been identified at the apical surface of polarized airway epithelia and is implicated in viral infection from the apical surface. We hypothesized that upregulation of cellular mechanisms that facilitate endogenous CAREx8 protein expression at the apical surface would enhance adenovirus gene transfer. Using polarized model epithelial cell lines and primary human airway epithelia, we found that IL8, a proinflammatory cytokine and a neutrophil chemoattractant, stimulates the protein expression and apical localization of CAREx8 via activation of AKT/S6K and inhibition of GSK3β. IL-8- mediated upregulation of CAREx8 increased AdV5-β-Gal entry and transduction by approximately 5-fold. Moreover, we found that infiltrating neutrophils bind CAREx8 at the apical surface of a polarized epithelium and, surprisingly, neutrophils enhance AdV5-β-Gal entry into the epithelium by 2-3 fold. The effect of IL-8 and neutrophils on AdV infection could be blocked by fiber-knob from AdV5 but not AdV3, a non-CAR binding serotype, indicating the importance of CAR. These findings suggest that acute inflammation may enhance adenovirus infection. Moreover, therapeutics that stimulate the AKT/ S6K pathway or inhibit GSK3β may be able to augment adenovirusmediated gene therapy.
320. Adenovirus Expansion of Radioiodine Therapy to Non-Thyroid Cancer Treatment and Diagnostics
Ben Eidenschink,1 Jordan Sell,1 Kari Jacobsen,1 Miguel Trujillo,2 Martin Ferdandez-Zapico,2 John Morris,2 Masato Yamamoto,1 Julia Davydova.1 1 University of Minnesota, Minneapolis; 2Mayo Clinic, Rochester. Radioiodine therapy has successfully treated and diagnosed cancers over the last 50 years. The clinical scope of radioiodine is limited in thyroid cancers because of restricted iodine uptake. Our lab proposes to expand the clinical use of radioiodine into nonthyroid cancer theranostics by targeting cancers (e.g. pancreatic and prostate) with oncolytic adenovirus to over express the human sodium iodine symporter (NIS). The adenovirus expression of the symporter allows iodine to concentrate into the targeted cancer cells providing diagnostic imaging with I123 and therapy with I131. We designed infectivity-enhanced oncolytic Adenoviruses (NISOAd), Ad5/3_TSP_E3_ADP_NIS, to express NIS and adenovirus death protein (ADP) from the E3 region. The Ad5/3 fiber modification enables the infection of CAR negative cells. Vector replication is controlled through a tumor specific promoter (TSP, e.g. COX2 in pancreatic and Pb in prostate cancer). We have shown the specificity of our viruses with TSPs in vitro with multiple pancreatic and prostate cancer cell lines. The expression of ADP resulted in an increase in Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy
oncolytic activity and transgene expression. The functionality of NIS-OAds was tested with in vitro radioiodine uptake assay and showed up to a 10-fold increase in radioiodine uptake in prostate and pancreatic cancer cell lines. This endorses that oncolytic viruses increase functional NIS expression in tumors. Our animal model experiments included nude mice xenographs to evaluate therapeutic and imaging affects with i.v. injections of NISOAds in A549 lung and LnCap prostate cancer cells. In A549 models, the combination of NIS-OAds and I131 had an increase in therapeutic effect over virus alone signifying functional NIS expression in solid tumors. LnCap xenographs had SPECT/CT images taken after NISOAds and non-replicating virus administration with I123. NIS-OAds had the highest tumor specific signal confirming that NIS-OAds can be diagnostic in tumors. Viral functionality in human tissue was evaluated by PCR in ex vivo tissue slices showing that virus replication was specific to cancerous tissues and did not occur in normal tissue. This study supports the high-level replication control of NIS-OAds in non-thyroid cancer tissues. A related non-replicating NIS expressing Ad is also being evaluated in prostate cancer by Dr. Morris, our collaborator, in a stage I clinical trial.Together the virus specificity, imaging, and therapeutic data support the potential of NIS-OAds as cancer theranostics. In sum, this data provides key functionality and safety support of NIS expressing Ad use in humans. Current projects include therapeutic and imaging animal studies with radioiodine in pancreatic cancer immuno-competent Syrian hamsters, and human xenographs in nude mice. Further studies will evaluate the distribution of NIS expression in vivo and cytotoxicity of virus with radiation. Ultimately the goal of our research is to form a multimodal therapy with radiation and oncolytic virus for diagnosis and therapy of cancers.
321. Oncolytic Adenovirus Expressing IFN as a Tool To Eliminate Pancreatic Cancer Stem Cells Amanda R. Oliveira,1 Christopher J. LaRocca,1 Julia Davydova,1 Masato Yamamoto.1 1 Department of Surgery, University of Minnesota, Minneapolis, MN.
Pancreatic cancer is the 4th leading cause of cancer related death in the US, and curative resection of tumors is the most effective treatment against the disease. Unfortunately, the majority of patients are diagnosed in the advanced stage of the disease, and palliative treatment with Gemcitabine is the therapy of choice. In both cases, curative resection of tumors or chemotherapy, effectiveness can be limited by the presence of pancreatic cancer stem cells. Cancer stem cells (CSC) are correlated with drug resistance, tumor recurrence, and metastasis. CSC are usually located in the hypoxic center of the tumors in a quiescent stage and are not affected by conventional chemotherapeutics that target highly replicating cells, such as Gemcitabine. CSC are also called tumor initiating cells (TIC), and as low as 100 cells are reported to form tumors in pre-clinical models. Inclusion of IFN alpha (IFN) in combination therapy protocols against pancreatic cancer can be highly beneficial to tackle this problem. IFN is reported to be cytotoxic to cancer cells, have antiangiogenic properties, stimulate anti-tumor immunity, and sensitize cancer cells to chemoradiation. In addition, IFN is reported to induce activation of quiescent CSC making them susceptible to chemotherapy drugs that target highly replicating cells. Phase II and III clinical trials combining IFN with 5-FU and radiation in an adjuvant therapy setting reported a 35% increase in the five year overall survival of pancreatic cancer patients. Stimulated by the promising results reported in IFN clinical trials we developed an oncolytic adenovirus expressing IFN (OAd-IFN). Our aim is to use the virus to improve IFN therapeutic effects in combination therapy by restricting high levels of IFN expression to the tumor. In vitro data testing triple combination of OAd-IFN + radiation+ 5-FU using MIA S129