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1
TRIPODI, LORELLA. "INTESTINAL MICROBIOTA IS A MAJOR DETERMINANT IN THE RESPONSE TO ONCOLYTIC VACCINE IN A MOUSE MODEL OF MELANOMA." Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/884815.
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Cancer immunotherapy has achieved tremendous results, however the outcome of therapies
targeting immune inhibitory pathways, specifically CTLA-4 and the axis between programmed cell
death protein 1 (PD-1) and its ligand 1 (PD-L1) has many genetic and environmental sources of
variability. Many studies demonstrated the influence of gut microbiome on immune checkpoint
inhibitors (ICIs) outcome. Besides ICIs, oncolytic vaccines (OVs) are a promising therapeutic
alternative in cancer immunotherapy with possible relevant contribution to treatment of several
types of tumors; OVs are, in fact, able to convert immunologically “cold” tumors into “hot” ones.
OVs represent an optimum candidate to combine with ICIs, increasing their response blockade both
in immunogenic and poorly immunogenic tumors. We hypothesized that manipulation of intestinal
gut microbiota could also affect OVs therapeutic efficacy; at this aim, we determined whether
efficacy of the oncolytic adenovirus Ad5D24-CpG (Ad-CpG) therapy could be affected by the gut
microbiome in a syngeneic mouse model of melanoma. Sterilization of the gut microbiota with highdose vancomycin impaired efficacy of Ad-CpG therapy, reducing the tumor-infiltrating IFN-gamma
CD8 T-cell. Cohousing mice pre-treated with vancomycin and a control group, with consequent
microbiota restoration, prior to treatment with Ad-CpG, ablated the negative effect of antibiotic,
confirming that Ad-CpG-reduced efficacy was mediated by the intestinal microbiota.
Considering the ability of Bifidobacterium as a positive regulator of antitumor immunity in vivo, by
promoting pro-inflammatory signals in innate immune cells, we evaluated tumor regression in
syngeneic mouse model of melanoma treated with a combination of Ad-CpG and Bifidobacterium
spp. cocktail. The group receiving the combined regimen showed the best tumor control and an
enrichment of bacteria belong to Firmicutes phylum, evaluated by fecal microbiome profiling by 16S
rRNA. Our data indicates that gut microbiota affects the immune responses elicited by oncolytic
adenovirus Ad-CpG and Bifidobacterium supplementations maximize its activity.
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Leja, Justyna. "Oncolytic Adenovirus Therapy of Neuroendocrine Tumors." Doctoral thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-146966.
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Neuroendocrine tumors (NETs), originally described as carcinoids, represent a rare and heterogeneous group of neoplasms associated with intensive secretion of hormones, bioactive peptides and amines. Most of the patients are diagnosed at a late stage of disease, often with liver metastases. Surgery remains the main treatment to control metastatic disease, but is not curative. Oncolytic virotherapy represents a promising approach to treat cancer and different strategies have been exploited to restrict viral replication to tumor cells. We developed an oncolytic adenovirus based on serotype 5, Ad5[CgA-E1A], where the chromogranin A (CgA) promoter controls expression of the E1A gene and thereby virus replication. We found that Ad5[CgA-E1A], selectively replicates in NET cells and it is able to suppress fast-growing human BON carcinoid tumors in nude mice. The activity of Ad5[CgA-E1A] was not completely blocked in liver cells. We further repressed virus replication in hepatocytes by targeting E1A with miR122, an miRNA specifically expressed in the liver. miRNAs bind to mRNA and induce its cleavage or translational blockage. By insertion of tandem repeats of miR122 target sequences in 3’UTR of E1A gene, we observed reduced E1A protein expression and replication arrest in miR122 expressing liver cells. The oncolytic potency of the miR122-targeted virus was not affected in NET cells. Since some NET and neuroblastoma cells express high levels of somatostatin receptors (SSTRs), we introduced in the virus fiber knob cyclic peptides, which contain four amino acids (FWKT) and mimic the binding site of somatostatin for SSTRs. The FWKT-modified Ad5 transduces midgut carcinoid cells from liver metastases about 3-4 times better than non-modified Ad5. Moreover, FWKT-modified Ad5 overcomes neutralization in an ex vivo human blood loop model to a greater extent than Ad5, indicating that the fiber knob modification may prolong the systemic circulation time. NETs represent a huge therapeutic challenge and novel diagnostic markers are needed for early detection and effective treatment of NETs. We have profiled primary tumors and liver metastases of ileocaceal NETs, using Affymetrix microarrays and advanced bioinformatics. We have identified six novel marker genes and show high similarity between primary lesions and liver metastases transcriptome by hierarchical clustering analysis.
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Alqahtani, Ali Saeed. "Investigation of a potentially novel oncolytic adenovirus." Thesis, University of Bristol, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.687423.
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In order to investigate protein V binding partners and the cellular pathways modulated by protein V, affinity purification coupled with SILAC based quantitative proteomics was used to determine the viral and cellular protein(s) binding to adenovirus protein V. This analysis indicates that the viral protein IVa2 and three cellular proteins (C1QBP, KNPA4 and PARP1) may bind to adenovirus protein V. Furthermore, quantitative proteomics and RNAseq techniques were combined to examine the response of cellular proteins and genes in A549 and WI-38 cells infected by Ad5-dVrTSB compared to wild type Ad5 and rAd5-EGFP. Interestingly, I found that PARP1 and C1QBP were moderately increased by wild type Ad5 infection in both cell lines, but remained unchanged with Ad5-dVrTSB and rAd5-EGFP infection. Furthermore, PARP1 knockdown experiments indicated that PARP1 could make a small positive contribution to wild type Ad5 replication, but not for Ad5-dVrTSB replication. Finally, C1QBP knockdown experiments showed that C1QBP may play a role in inhibiting wild type Ad5 replication.
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Thoma, Clemens Matthias Manuel. "Improving intraperitoneal adenovirus virotherapy for ovarian cancer." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:841e4334-f408-4da3-b8e6-1d29350c5304.
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The use of intraperitoneal (i.p.) adenovirus virotherapy of ovarian cancer is currently limited by insufficient efficacy and high toxicity. Both factors are associated with adenovirus serotype 5 (Ad5) in this setting and may be serotype-specific. Low levels of uptake receptors (CAR and αV integrins) on ovarian tumour cells and widespread immunity against Ad5 among patients appear to restrict efficacy and intraperitoneal inflammatory responses against Ad5 were among the reasons for the termination of a phase II/III clinical trial in ovarian cancer. This thesis sought to overcome these obstacles by investigating the alternative adenovirus serotypes Ad3 and Ad11. For these viruses lower pre-existing antiviral immunity and utilisation of different uptake receptors have been reported. Furthermore, virus cloaking with novel polymers which could impart enhanced protection from neutralisation was examined. In vitro, wild-type Ad3, Ad5 and Ad11 displayed differential oncolytic activity in a panel of ovarian cancer cell lines which partly correlated to uptake receptor expression and virus internalisation. However, some cell lines displayed lysis resistance in a serotype-specific manner. While the inflammatory response six hours after i.p. administration of Ad11 in CD46-transgenic mice did not differ from Ad5, in long-term studies of repeated administration Ad5 induced significantly more severe pathologic effects in the form of adhesions and liver toxicity than Ad11 or mock-treatment. Oncolysis inhibition assays using malignant exudate samples demonstrated greater neutralisation of Ad3 and Ad5 in comparison to Ad11 at low concentrations of samples. Notably, 10-fold less Ad11 than Ad5 was required for oncolytic efficacy at a sample concentration of 10%. In an ex vivo model of ascites from ovarian cancer patients Ad5 modified with novel polymer formulations achieved at least 50% cell kill in six of eight samples, in contrast to two of eight samples for non-modified Ad5. These data suggest that virotherapy using Ad11 might be advantageous over Ad3 or Ad5. The lack of strong inflammation and the possibility to decrease treatment doses due to less neutralisation of Ad11 might result in considerably improved patient safety. Chemical modification of Ad with novel polymers presents an exciting advancement in overcoming treatment neutralisation in adenovirus virotherapy.
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Clarkin, Ryan Gregory. "Enhancing Oncolytic Adenovirus Vector Efficacy through Co-expression of the p14 Fusion-associated Small Transmembrane Protein and Adenovirus Death Protein." Thesis, Université d'Ottawa / University of Ottawa, 2018. http://hdl.handle.net/10393/38379.
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Conditionally-replicating adenoviruses (CRAds) have generally demonstrated only modest therapeutic efficacy in human clinical trials, in part due to their poor ability to spread throughout a tumor mass. In these studies, I first examined whether inclusion of an intact early region 3 (E3) and the p14 fusion-associated small transmembrane (FAST) protein in a CRAd vector can enhance oncolytic efficacy by improving viral spread. E3 encodes the adenovirus death protein (ADP), which enhances virus progeny release from infected cells, while p14 FAST can allow spread of the virus through cell-cell fusion. I generated viruses with (CRAdRC109) or without (CRAdRC111) an intact E3 region, which encoded the p14 FAST gene between the fiber coding sequence and E4 region of their viral genomes. In the A549 human lung cancer cell line, both CRAdRC109 and CRAdRC111 expressed p14 FAST at very low levels when compared to CRAdFAST, a similar virus that expressed the protein from within the E3 deletion, and thus had a relatively poor ability to mediate cell-cell fusion. Although inclusion of E3/ADP in CRAdRC109 did result in larger plaques and increased virus spread relative to CRAdRC111, neither virus showed improved oncolytic activity relative to CRAdFAST. I subsequently developed CRAdRC116, in which the E3 region of the viral genome was replaced with a bicistronic expression cassette containing the p14 FAST and ADP coding sequences separated by a self-cleaving 2A peptide sequence. This virus co-expressed p14 FAST and ADP and caused extensive cell-cell fusion in A549 cells. However, expression of ADP from CRAdRC116 did not increase cancer cell killing nor virus spread, and thus did not enhance oncolytic efficacy relative to CRAdFAST. These studies suggest that p14 FAST and ADP do not exhibit synergy when co-expressed from a CRAd vector. Future studies should instead focus on combining other methods of improving viral spread in conjunction with expression of ADP or FAST proteins from CRAd.
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Cooper, Lisa May. "Bioprocessing of oncolytic group B adenovirus for scalable production." Thesis, University of Oxford, 2014. https://ora.ox.ac.uk/objects/uuid:bc62bd13-f43f-4d35-8975-7fc341ce209c.
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The central aim of this thesis was to develop strategies to improve the manufacture of the group B chimeric oncolytic adenovirus, ColoAd1, which rapidly kills and lyses host cells. In attempting to improve the cellular yield of ColoAd1, this thesis therefore sought to identify host infection-related factors that limited ColoAd1 production. In the widely-used manufacturing cell line, HEK293, ColoAd1 replication depleted intracellular ATP earlier than Ad11p and activated the intracellular energy sensor, AMPK. This might have reflected earlier ATP depletion, or possibly the absence of the E4orf4 protein from ColoAd1 compared to Ad11p. Despite this difference in AMPK activation, both viruses appeared able to maintain mTORC1 activity, which may be essential particularly for protein synthesis in the early stages of virus infection. For production purposes, preventing intracellular ATP depletion was seen as an attractive mechanism of maintaining ColoAd1 infected host cell viability and was hypothesised to lead to increased virus yield. A range of strategies were explored to enhance depleting ATP levels. Even though none of these were dramatically successful, they indicated that perhaps the anabolic building blocks required for viral replication were more important than cellular energy levels. Finally, a screening methodology based on siRNA knockdown was used to identify kinases that affected ColoAd1 replication. Many hits were identified, and several candidate kinases indicated a role for intracellular calcium signalling limiting virus particle production. Overall, data presented in this thesis supports the manufacture of ColoAd1 in HEK293 cells and suggest that enhancing glycolysis may increase ColoAd1 yield. It also provides mechanistic insights into the replication of ColoAd1 and Ad11p that may inform the improved design of group B oncolytic adenoviruses.
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Silver, Jim. "Replication-competent adenovirus 11p vector as a new oncolytic agent." Doctoral thesis, Umeå universitet, Virologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-50773.
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Human adenoviruses (Ads) as vectors have been studied for cancer gene therapy for several decades due to their ability to shut down host cell replication and lyse tumour cells. Ad5 of species C is commonly used as a replication-defective or a replication-competent vector. However, many tumour cells are relatively refractory to infection by Ad5 since the cells lack the viral receptor CAR. Thus, species B Ads are becoming more important as alternative vectors since they use CD46 as primary receptor, the expression of which is up-regulated on many tumour cell surfaces, and they also have low seroprevalence in humans. Although Ad3, Ad7, Ad11 and Ad35 have been altered to become replication-defective vectors, investigations based on replicating adenovirus vectors are still warranted. The major aim of this thesis has been to characterize the transduction efficacy and oncolytic effect of the replication-competent adenovirus 11pGFP vector (RCAd11pGFP) in human solid tumour cell lines. Evaluation of the vector would ultimately help us to understand whether the tumour cells affect virus replication and whether the vector replicates differently in tumour cells and in untransformed diploid cells, and would eventually lead to development of more potent oncolytic adenoviruses for treatment of human cancers. The Ad11-based vector RCAd11pGFP consists of the entire Ad11p genome with a green fluorescence protein (GFP) expression cassette inserted. RCAd11pGFP shows all the characteristics of the wild-type virus and expresses GFP in cells four hours p.i. Antisera raised against Ad11p virions and hexons were able to neutralize RCAd11pGFP infection but antiserum raised against the Ad11p fibre knob could not. The infection is reduced by 90% but the fibre knob antiserum cannot completely block virus infection. Initial screening of the infection capacity of five wild-type adenoviruses in four colon cancer cell lines revealed that Ad11p, Ad11a and Ad35 of species B, showed similar replication kinetics but Ad5 showed delayed onset of virus replication in comparison to species B Ads. These data support the use of Ad11p as an alternative vector for treatment of colon cancer. The transduction efficiency of RCAd11pGFP in colon cancer and prostate cancer cell lines was studied using flow cytometry assay (FACS), and this showed that the cytolytic effect was not always in accordance with GFP expression. Toxicity assay and virus one-step replication assay showed that RCAd11pGFP replicates in highly tumorigenic cell lines (HT29, T84 and PC-3) to a greater extent than less tumorigenic cell lines (LS174T, HCT-8, DU145 and LNCaP cells), even though the latter showed relatively high GFP expression. This initial finding led to the subsequent discovery of CEACAM-family molecules, which were highly expressed in HT29 and T84 cells. Interestingly, the Ad5 wild-type virus did not manifest the same tumour-specific replication that RCAd11pGFP did in the cell lines studied. Furthermore, we investigated the influence of tumour markers for RCAd11pGFP replication in colon cancer cells. A double-staining FACS assay for detecting members of CEACAM-family molecules was established and we found that the levels of CEACAM6 were up-regulated in the cells infected by RCAd11pGFP or Ad11pwt relative to uninfected cells. However, this virus replication could not be suppressed by CEACEA6 siRNA. Our results indicate that several tumour markers or factors might be involved in promoting propagation of the virus. In vivo experiments showed significant growth inhibition of T84 and HT-29 tumours in xenograft mice treated with either RCAd11pGFP or Ad11pwt, compared to untreated controls. Furthermore, the role of the anti-tumour effect of RCAd11pGFP was also confirmed in PC3 prostate tumours in BALB/c mice. In conclusion, the novel RCAd11pGFP vector was shown to have an anti-tumour effect in vitro and in vivo. This tumour-killing effect could be enhanced in highly tumorogenic cells through virus replication. Consequently, RCAd11p may lead to development of a more potent and useful vector for human cancer therapy.
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Del, Papa Joshua. "Assessing the Oncolytic Capacity of Conditionally Replicating Adenovirus Armed with p14 Fusion Associated Small Transmembrane Protein and the Adenovirus Death Protein." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39485.
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Intratumoral injection of oncolytic viruses provides a direct means of tumor cell elimination for inoperable tumors. Unfortunately, oncolytic vectors based on human adenovirus (HAdV) typically do not spread efficiently throughout the tumor mass, reducing the efficacy of treatment. In this thesis, I explore the efficacy of conditionally replicating HAdV vectors expressing either the p14 Fusion Associated Small Transmembrane (FAST) protein (CRAdFAST) or p14 FAST protein in combination with the adenovirus death protein (CRAdFAST-ADP). The p14 FAST protein mediates cell-cell fusion, which may enhance spread of the virus-mediated, tumor cell-killing effect, while ADP aids in cell lysis and HAdV spread at late times in infection. I first explored the efficacy of CRAdFAST in the 4T1 immune competent mouse model of cancer. Treatment with CRAdFAST resulted in enhanced cell death compared to vector lacking the p14 FAST gene in vitro, but did not reduce the tumor growth rate in vivo. The 4T1 model was significantly resistant to HAdV infection and propagation, so I next explored CRAdFAST efficacy in human A549 cell culture and a xenograft mouse model of cancer. In the human A549 lung adenocarcinoma model of cancer, CRAdFAST showed significantly improved oncolytic efficacy in vitro and in vivo. In an A549 xenograft tumor model in vivo, CRAdFAST induced tumor cell fusion which led to the formation of large acellular regions within the tumor, and significantly reduced the tumor growth rate compared to control vector. Finally, to assess the use of a newly constructed CRAdFAST vector co-expressing the adenovirus death protein (ADP), a new model was explored comprised of CMT-64.6 mouse lung carcinoma cells which are syngeneic with Balb/C mice. This model was significantly more sensitive to HAdV infection and CRAdFAST induced fusion than the 4T1 cell line. In this model, expression of ADP and p14 FAST from a CRAdFAST-like vector (CRAdFAST-ADP) resulted in significant oncolytic synergy in vitro but not in vivo. My results indicate that expression of p14 FAST protein, and potentially ADP, from an oncolytic HAdV can improve vector efficacy for the treatment of cancer, but improved in vivo models will be required to analyze the full preclinical potential of these oncolytic HAdV vectors.
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Herod, Morgan Reece. "Oncolytic adenovirus vectors for nitroreductase suicide gene therapy of prostate cancer." Thesis, University of Birmingham, 2010. http://etheses.bham.ac.uk//id/eprint/957/.
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Prostate cancer is the most common male cancer in the UK and USA, with a 1/13 chance of diagnosis and a 1/30 lifetime risk of death from the disease. Current treatment options include radiotherapy, surgery and hormone therapy, however 1/3 patients escape from all therapies and novel therapies are urgently required for this patient group. The University of Birmingham gene therapy group constructed two oncolytic adenovirus vectors, CRAd-NTR and vNR6, both of which contained the E1B-55K deletion and expressed the transgene nitroreductase for combined oncolytic virotherapy and enzyme/prodrug gene therapy. The latter of these two vectors, vNR6, expressing nitroreductase from the pIX virus promoter demonstrated the greatest cytotoxicity at low virus concentrations however also showed some lytic activity to non-transformed human fibroblasts. Our collaborators at the Institut Català d'Oncologia designed a panel of oncolytic adenovirus vectors with the E1A CR2 24 deletion and the E1A promoter replaced by an insulated E2F-1 promoter. The latest two in this series of vectors, termed ICOVIR-5 and ICOVIR-7, provide potential oncolytic backbones for the introduction of the therapeutic transgene nitroreductase. The aim of this thesis was therefore to ‘arm’ the ICOVIR based vectors with nitroreductase for combined oncolytic virotherapy and enzyme/prodrug therapy. At the beginning of this study no reports were published with either ICOVIR-5 or ICOVIR-7 based vectors. It was therefore first decided to construct both vectors expressing the marker transgene eGFP. These vectors were characterised in terms of cytotoxicity, transgene expression, DNA replication and E1A expression. Furthermore, these vectors were compared to the vNR3, an E1B-55K deleted virus similar to vNR6, but the eGFP ORF replacing that of pIX. The ICOVIR-7 based vectors were identified as being the most tumour selective vectors and demonstrated no cytotoxicity to non-transformed human fibroblasts, and were therefore chosen for the introduction of the therapeutic transgenes. The new ICOVIR-7 based vectors were constructed to express either wildtype, double mutant or triple mutant nitroreductase. Double and triple mutant nitroreductase are two previously characterised mutant nitroreductases, which show enhanced catalytic activity for the prodrug CB1954. The new nitroreductase expressing ICOVIR-7 vectors were characterised in terms of virus mediated cytotoxicity, tumour selectivity, E1A and NTR expression and cytotoxicity with the prodrug CB1954. One vector, expressing double mutant nitroreductase, showed the highest tumour selectivity and greatest combined cytotoxicity with the prodrug CB1954. Furthermore, this vector showed greater tumour selectivity and combined cytotoxicity than the E1B-55K deleted vector vNR6.
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Weigert, Melanie. "Investigating the role of programmed necrosis in oncolytic adenovirus-induced death." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8054/.
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Oncolytic viruses are a group of viruses that preferentially replicate in cancer cells and are a promising cancer treatment. However, how these oncolytic adenoviruses kill cancer cells is not fully understood. It was long thought that DNA viruses utilize apoptosis to induce cell death but there is now evidence that adenovirus and vaccinia cytotoxicity displays features of necrosis-like programmed cell death. In order to investigate the role of necrosis in cell death as a result of oncolytic adenovirus infection, a panel of ovarian cancer cells with varying sensitivities to the oncolytic adenoviral mutant dl922-947 was used. Cells infected with dl922- 947 displayed key features of necrotic death. Using necrosis inhibitors necrostatin-1, necrosulfonamide, GSK2791840B, GSK2399872B and GSK2393843A, as well as RNAi-mediated knockdown of RIPK1, RIPK3 or MLKL, I showed that cells undergo RIPK3-dependent necrosis and that blockage of the downstream effector mixed lineage kinase domain-like (MLKL) attenuated cell death. While Tumour necrosis factor-α (TNF-α)-induced programmed necrosis(Laster, Wood and Gooding 1988) relies on the (RHIM)-dependent interaction of RIPK1 and RIPK3 (Li et al. 2012, Wu et al. 2014), RIPK1 seems to be redundant for adenovirus-induced death. Further, the addition of TNF-α blocking antibody to virus-infected cells showed no effect on either cell death. Using a RIPK3 overexpression model, I showed that the amount adenovirus- induced cell death correlated with the amount of RIPK3 expression and that RIPK3 expression did not affect virus production, infectivity or the expression of viral proteins. Further, in vivo experiments using human xenografts showed that expression of RIPK3 significantly improved anti-tumour activity following intra-tumoural injection of dl922-947.
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Marguerie, Monique. "Combining the Immunogenic Cancer Mutanome with Oncolytic Virus Therapy." Thesis, Université d'Ottawa / University of Ottawa, 2014. http://hdl.handle.net/10393/31409.
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Oncolytic viruses (OVs) are effective anti-cancer agents, however their abilities to induce anti-tumor immunity are not yet optimal. Mutanome epitopes are a novel source of tumor antigen formed as a result of mutations within the tumor genome. Within this project we attempted to combine B16F10 mutanome vaccination with OV therapy. We confirmed previous findings that significant immune responses to these epitopes can be generated. Furthermore, we designed and cloned a multi-epitope mutanome construct into MG1 Maraba virus and E1-/E3- deleted type 5 Adenovirus to use for heterologous prime-boost vaccination. While we demonstrated that these viruses induced T-cell responses to one mutanome epitope, we failed to detect responses to the other epitopes. Furthermore there was no effect seen on overall survival. This approach warrants further investigation because coupling mutanome vaccination with OV therapy has the potential to exploit the therapeutic effects of the OV while inducing anti-tumor immunity to tumor-unique antigens.
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Spurrell, Emma Louise. "The role of the innate immune system in oncolytic adenoviral therapy." Thesis, University of London, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.511359.
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Makkouk, Al Hassan. "The development and preclinical characterization of the glioblastoma-targeted ICOVIR-5 oncolytic adenovirus." [New Haven, Conn. : s.n.], 2008. http://ymtdl.med.yale.edu/theses/available/etd-12092008-133655/.
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Leung, Elaine Yee Ling. "The role of innate immune responses in oncolytic adenovirus therapy in ovarian cancer." Thesis, University of Glasgow, 2018. http://theses.gla.ac.uk/30608/.
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Epithelial ovarian cancer is the deadliest gynaecological cancer: most women die within five years of their diagnoses. Moreover, survival of women with ovarian cancer (OC) has not significantly improved in the past decade. Oncolytic viruses (OVs), a new class of anti-cancer agent, infect and replicate selectively within malignant cells, whilst sparing normal cells. OVs also induce profound immune responses, for example disruption of chemokine and cytokine networks, with potential influence on therapeutic effectiveness. On the other hand, Natural Killer cells (NK cells), a key immune population that surveillance against cancers and viruses, may hinder the spread of OVs or promote anti-tumoural effects of OVs. This work investigated the role of innate immune responses, in particular NK cells and interleukin (IL)-17F, on the efficacy of oncolytic adenovirus in OC. I demonstrated that NK cells were activated by adenovirus-infected OC cells. Activated NK cells then augmented oncolytic adenovirus in eliminating OC via contact-dependent interactions between activating NK receptor DNAM-1 and adenovirus-infected malignant cells. In addition, consistent changes in chemokines and cytokines were observed after wild-type and oncolytic adenovirus infection. In particular, IL-17F, but not IL-17A, was significantly upregulated in different established and primary OC lines after adenovirus infections. Moreover, a range of inflammatory chemokines, including CCL2, CXCL1, CXCL2 and CXCL5, were down-regulated after oncolytic adenovirus infection. This work also revealed the logistical and technical challenges of the use of primary patient materials. I identified that our primary culture method for expanding OC cells was suboptimal. I subsequently evaluated a simple immunohistochemical method to screen for successful primary expansion of malignant cells from OC ascites. I showed that PAX8, but not CK7, was a specific marker of successful ex vivo expansion of HGSOC.
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Ramachandran, Mohanraj. "Cancer Immunotherapy : Evolving Oncolytic viruses and CAR T-cells." Doctoral thesis, Uppsala universitet, Science for Life Laboratory, SciLifeLab, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-302891.
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In the last decade cancer immunotherapy has taken huge strides forward from bench to bedside and being approved as drugs. Cancer immunotherapy harnesses the power of patient’s own immune system to fight cancer. Approaches are diverse and include antibodies, therapeutic vaccines, adoptively transferred T-cells, immune checkpoint inhibitors, oncolytic viruses and immune cell activators such as toll-like receptor (TLR) agonists. Excellent clinical responses have been observed for certain cancers with checkpoint antibodies and chimeric antigen receptor (CAR)-engineered T-cells. It is however becoming evident that strategies need to be combined for broader effective treatment responses because cancers evolve to escape immune recognition. A conditionally replication-competent oncolytic adenovirus (Ad5PTDf35-[Δ24]) was engineered to secrete Helicobacter pylori Neutrophil Activating Protein (HP-NAP, a TLR-2 agonist) to combine viral oncolysis and immune stimulation. Treatment with Ad5PTDf35-[Δ24-sNAP] improved survival of mice bearing human neuroendocrine tumors (BON). Expression of HP-NAP in the tumor microenvironment promoted neutrophil infiltration, proinflammatory cytokine secretion and increased necrosis. We further studied the ability of HP-NAP to activate dendritic cells (DCs) a key player in priming T-cell responses. HP-NAP phenotypically matured and activated DCs to secrete the T-helper type-1 (Th-1) polarizing cytokine IL-12. HP-NAP-matured DCs were functional; able to migrate to draining lymph nodes and prime antigen-specific T-cell proliferation. CAR T-cells were engineered to secrete HP-NAP upon T-cell activation. Secreted HP-NAP was able to mature DCs, leading to a reciprocal effect on the CAR T-cells with improved cytotoxicity in vitro. Semliki Forest virus (SFV), an oncolytic virus with natural neuro-tropism was tagged with central nervous system (CNS)-specific microRNA target sequences for miR124, miR125 and miR134 to selectively attenuate virus replication in healthy CNS cells. Systemic infection of mice with the SFV4miRT did not cause encephalitis, while it retained its ability to replicate in tumor cells and cure a big proportion of mice bearing syngeneic neuroblastoma and gliomas. Therapeutic efficacy of SFV4miRT inversely correlated with type-I antiviral interferon response (IFN-β) mounted by tumor cells. In summary, combining immunotherapeutic strategies with HP-NAP is a promising approach to combat cancers and SFV4miRT is an excellent candidate for treatment of neuroblastomas and gliomas.
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Peerlinck, Inge D. L. "Development of a WNT-selective oncolytic adenovirus for imaging the therapy of colorectal cancers." Thesis, Queen Mary, University of London, 2008. http://qmro.qmul.ac.uk/xmlui/handle/123456789/1595.
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Introduction: The concept of oncolytic adenoviruses has been validated in preclinical studies but clinical trials have demonstrated that the virus spread remains limited and the virus fails to infect all cancer cells in a tumour. Arming an oncolytic virus with a therapeutic trans gene would enhance the antitumour effect of these viruses by killing adjacent non-infected cells. The aim of this thesis is to test armed oncolytic adenoviruses targeting a constitutive activation of the Wnt signalling pathway in pre-clinical models of colorectal cancer. Methods: The Nail symporter was inserted into the genome of Wnt-selective oncolytic adenoviruses to visualise adenoviral spread in the tumour and assess image-guided radiotherapy. Results: In vitro testing of the virus has demonstrated that the Wnt-selectivity of the virus remains intact. The virus we generated has an equal or greater cytopathic effect than wild type adenovirus in Wnt-expressing cancer cell lines. The ability of the infected cells to take up iodine has been confirmed by iodine uptake assays. The virus has been injected into subcutaneous human tumour implants in nude mice. Images obtained with a SPECT/CT camera have demonstrated that viral propagation can be visualised in vivo. Finally, we have used the imaging data to determine the correct timing for the administration of therapeutic doses of 131 I. Conclusion: We have validated a non invasive method to image viral propagation and transgene expression in a preclinical model of colonic cancer. Sequential imaging can provide information on the ideal time point for therapeutic intervention. In pilot experiments, the aim was to exploit the potential of the Nail symporter for the concentration of radioactive iodine, but it did not lead to increased therapeutic efficacy in vivo in preclinical models. There is strong evidence that if these experiments were repeated, therapeutic efficacy could he demonstrated.
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Calderon, Hugo. "Investigating the oncolytic properties of a group B adenovirus on cancer cells and its effects on the local immune response." Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:da89b317-5f76-4447-bbb1-26740db3b3ef.
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Oncolytic viruses are characterised by their ability to selectively infect and kill tumour cells. Recently it has emerged that they can exert an additional anticancer mechanism stimulating adaptive immune-mediated cancer cell killing. Enadenotucirev (EnAd, formerly known as ColoAd1), is a chimeric Ad11p/Ad3 virus group B oncolytic adenovirus that binds CD46 and is under development for the systemic treatment of metastatic carcinomas. The central aim of this thesis was to to assess whether EnAd provides an adjuvant effect on tumour-associated antigen presenting cells (APCs) that could drive TH1 polarisation for an effective anti-tumour immune response. This thesis describes the potent oncolytic properties, fast replication and high numbers of virus progeny production by EnAd in cancer cells. Recombinant EnAd variants were engineered to investigate the roles of the mutant regions in the genome of EnAd, and how these influence the modified phenotype. A chemical drug panel was used to identify pathways and cellular factors involved in cellular production of EnAd, finding that several mTOR inhibitors and microtubule inhibitors could improve virus replication. An in vitro system using partially matured human monocyte-derived dendritic cells (DCs), which displayed a similar phenotype to tumour-infiltrating DCs, was used to explore the effect of EnAd on APC responses. EnAd induced a strong adjuvant effect on these cells by up-regulating surface markers and secretion of pro-inflammatory factors. Further mechanistic experiments, alongside a CAR-binding group C adenovirus 5, indicated these adjuvant effects were virus particle-mediated and dependent on CD46 binding. To understand the functional implications downstream of these interactions, T cell activation and phenotype was assessed using a mixed lymphocyte reaction approach. The data indicated EnAd was a good candidate compared to other adenoviruses, that may steer the response of activated T-cells towards a TH1 phenotype, for an effective immune response. In conclusion, the potent oncolytic properties of EnAd virus may provide an adjuvant effect on tumour-associated APCs, helping to harness an adaptive immune response.
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Tookman, Laura. "The role of the DNA damage and repair pathways in the efficacy of oncolytic adenovirus for ovarian cancer." Thesis, Queen Mary, University of London, 2016. http://qmro.qmul.ac.uk/xmlui/handle/123456789/24648.
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Defects within the DNA damage response (DDR) pathways are common in human malignancies. This is especially true in high-grade serous ovarian cancer (HGSOC) where defects within the Homologous Recombination (HR) pathway may be present in up to 50% of tumours. Oncolytic adenovirus is a potential novel therapy for human malignancies. These viruses infect malignant cells and multiply selectively within them causing cell death and release of mature virions. Here, I have investigated the role of the DDR in determining the efficacy of the E1A-CR2 deleted adenovirus type 5 (Ad5) vector, dl922-947, in ovarian cancer. I show that infection with dl922-947 stimulates a robust DDR within the host cell, which the virus manipulates in order to ensure optimal viral replication. In a panel of HGSOC cell lines, the extent of overreplication of genomic DNA and the degree of genomic damage following infection with dl922-947 was shown to correlate closely with viral efficacy. Functional HR, however, promoted viral DNA replication and augmented overall anti-cancer efficacy. Mechanistically, both BRCA2 and RAD51 localised to viral replication centres within the infected cell nucleus. RAD51 co-localisation was also demonstrated in cells with defective HR and occurred independently of BRCA2. In addition, a direct interaction was identified between RAD51 and adenovirus E2 DNA binding protein. Using functional assays of HR competence, I show that Ad5 infection does not alter cellular ability to repair DNA double-strand break damage via HR. These data suggest that oncolytic adenoviral therapy may be most clinically relevant in tumours with intact HR function. Using a high-throughput siRNA DNA repair screen, potential novel targets have been identified that can increase the efficacy of dl922-947 (for example: NONO) and also result in increased resistance (RPA). These results highlight the complex interplay between adenovirus and host cell. Further understanding of these pathways is vital to increase efficacy, develop biomarkers and improve patient selection into clinical trials for these therapies.
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Farrera, Sal Martí. "Enhanced hyaluronidase and tumor neoepitope expression by oncolytic adenoviruses." Doctoral thesis, Universitat de Barcelona, 2020. http://hdl.handle.net/10803/671748.
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The oncolytic viruses (OVs) preferentially infect tumor and selectively replicate in cancer cells without harming normal tissues. OVs have been tested in clinical trials as monotherapy or combined with chemotherapy, radiotherapy, and immunotherapy. Nonetheless, the intratumoral spreading and the immune response hamper the treatment efficacy. In this thesis, these two challenges have been addressed in three separate chapters.
First, VCN-01, a hyaluronidase-expressing oncolytic adenovirus, was tested in a clinical trial in pancreatic cancer patients. We assessed the immune response triggered by VCN-01 as monotherapy or in combination with chemotherapy. We reported an early anti-viral immune response induction of IL-6, IL-10, IFNγ, IDO1, IP-10, and sLAG-3 in serum, independently of chemotherapy. We found a correlation between treatment toxicity and the IL-6 and IL-10. Furthermore, the triggered anti-viral immune response such as IFNγ, sLAG-3, and neutralizing antibodies anti-Ad5 was associated with better antitumor activity in patients.
The neoepitope vaccines have been tested in patients with limited clinical responses. We hypothesized that an oncolytic adenovirus (OAd) encoding for stroma.
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Cawood, Ryan. "Liver specific microRNA control of adenovirus serotype five." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:d97d80b5-6272-4aab-b555-d03d0016eeff.
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MicroRNAs are small non-coding RNA molecules that regulate mRNA translation by binding to complementary sequences usually within the 3’ un-translated region (UTR). By inserting four perfectly complementary binding sites for the hepatic specific microRNA mir122 into the 3’ UTR of adenovirus wild type 5 (Ad5 WT) E1A mRNA I show that the acute liver toxicity caused by Ad5 WT in mice can be significantly reduced. This virus, termed Ad5-mir122, is a promising virotherapy candidate and causes no obvious liver pathology whilst maintaining Ad5 WT replication in mir122 negative cells. Data shows that repeat intravenous administration of Ad5-mir122 (2x1010vp) to HepG2 tumour bearing mice mediated significant anti-cancer efficacy. RT-QPCR for E1A mRNA demonstrated a 29-fold reduction when compared to Ad5 WT in murine liver whilst western blot confirmed that all E1A protein variants were knocked down. Viral genomic replication was also reduced in mouse liver by 25-fold compared to Ad5 WT. This control of virus activity reduced alanine and aspartate transaminase release by >15-fold and histological analysis showed little to no pathology in Ad5-mir122 infected livers. Measurement of mature mir122 levels in Ad5-mir122 infected livers by RT-QPCR showed that the quantity of mir122 remained unaffected at therapeutic doses. Complete genome mRNA array profiling of infected livers showed that the transcript levels of >3900 different mRNAs were changed more than 2-fold following Ad5 WT infection whilst less than 600 were changed by Ad5-mir122. A non-replicating control adenovirus vector altered >550 mRNAs. No known mir122 target mRNAs were affected following infection with Ad5-mir122. Western blot analysis of a known mir122 regulated target (Aldolase A) confirmed these results, demonstrating no change in protein level despite infection with Ad5-mir122. These data combined demonstrate that the exploitation of microRNA mir122 regulation to control adenovirus replication is a safe method of control and does not alter the endogenous level or activity of the microRNA or its endogenous mRNA targets. Ad5-mir122 is a potent anti-cancer agent that replicates to wild-type levels in microRNA mir122 negative cells but is specifically and safely attenuated in hepatocytes.
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Young, Anna-Mary. "Development of an immunocompetent model of oncolytic adenoviral gene therapy for ovarian cancer." Thesis, Queen Mary, University of London, 2012. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8365.
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Oncolytic adenoviral gene therapy has potential as a novel anti-cancer agent for ovarian cancer. Host immune responses are thought to contribute to its therapeutic effects. However further evaluation has been hampered by the lack of an immunocompetent animal model. This is predominantly because human adenovirus is highly species-specific and replicates poorly in murine cells. The second generation human adenovirus (hAd5) type 5 mutant dl922-947 contains a deletion in the E1A CR2 region which allows it to replicate selectively in cells with Rb pathway abnormalities, a finding observed in >90% of human cancers. Previous work has shown that dl922-947 has considerable activity in ovarian cancer and is more potent than E1A wild-type adenoviruses and the E1B-55K mutant dl1520 (Onyx-015, H101). Unfortunately, like its wild-type counterpart, dl922-947 replicates poorly in murine cells and infectious virion progeny are not generated. Mechanisms for the failure of infectious virion formation remain unclear and have been investigated as part of this project. I have found that murine malignant cells can be infected readily with hAd5 vectors. Both early and late viral genes are transcribed and there is evidence of viral genome replication. However, a profound failure of infective virion production is observed together with low levels of late viral protein expression. Ribosome fractionation assays show reduced viral mRNA loading in murine cells, resulting in failure of translation, especially of late transcripts. Aberrant function of the non-structural L4 protein 100K has been identified as a major hurdle to successful viral replication in murine cells. Ectopic expression of L4 100K promotes translation of viral late mRNA and increases expression of late viral proteins and virion production. However, these increases are only partial.
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Samuel, Shirley Kulangara Tong Alex W. "Anti-tumor activity of an oncolytic adenoviral construct expressing a small interfering RNA transgene." Waco, Tex. : Baylor University, 2007. http://hdl.handle.net/2104/5117.
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Strauss, Robert. "Analysis of resistance of primary ovarian cancer cells to viral oncolysis." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2010. http://dx.doi.org/10.18452/16163.
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Auf Adenoviren (Ads) basierende Vektoren wurden als ein gezielter Anti-Krebs-Wirkstoff entwickelt, der erfolgversprechende Resultate in prä-klinischen Studien erzielen konnte. Solche onkolytischen Ads sind zwar in klinischen Studien generell als sicher eingestuft worden, konnten jedoch die therapeutischen Erwartungen nicht erfüllen. In dieser Doktorarbeit konnte, unter Verwendung der Genexpressionsprofile von Ovarialkarzinom-Zellen, der epitheliale Phänotyp als Hindernis für allgemein verwendete onkolytische Ads, die auf den Coxsackie- und Adenovirusrezeptor (CAR) oder CD46 ausgerichtet sind, identifiziert werden. Der Zugang zu den Virus-Rezeptoren war zwingend an Zelldepolarisation und den Verlust der epithelialen Zonulae occludens und adherens gekoppelt, was Merkmale der Epithelial-zu-Mesenchymal-Transition (EMT) darstellt. Bedeutsam ist in diesem Zusammenhang, dass Tumore in situ als auch Xenograft-Tumore zum größten Teil aus Epithelzellen oder epithelial/mesenchymalen (E/M) Hybrid-Zellen bestehen. Diese E/M Hybrid-Zellen sind die einzigen Zellen, welche an Zellkulturbedingungen adaptieren, wo sie durch EMT während weiterem Passagieren in Mesenchymzellen differenzieren. Bemerkenswert ist hierbei die Tatsache, dass nur Mesenchymzellen und E/M Hybrid-Zellen, die sich im EMT-Prozess befanden, sensitiv zu viraler Onkolyse waren. In Versuchen, die festgestellte Resistenz zu überwinden, wurde herausgefunden, dass bisher nur wenig erforschte Adenovirus-Serotypen (Ad3, Ad7, Ad11 und Ad14), welche einen anderen Rezeptor als CAR oder CD46 auf Zellen benutzen, besser geeignet sind, um polarisierte Epithelzellgewebe zu infizieren. Diese Ads induzierten EMT-ähnliche Prozesse in Ovarialkarzinom-Kulturen mit epithelialem Phänotyp, was zu deren effizienter Onkolyse führte. Die vorliegende Arbeit trägt somit zur Aufklärung der Diskrepanz zwischen der Virustherapie-Effizienz in vivo und in vitro bei und bietet Anhaltspunkte für die Konstruktion von zukünftigen onkolytischen Ads.Vectors based on adenoviruses have been designed as targeted anti-cancer therapeutics that showed promising results in pre-clinical applications. In clinical trials, these oncolytic adenoviruses have generally been proved safe in patients, but have fallen short of their expected therapeutic value. In this thesis the susceptibility of primary ovarian cancer cells to oncolytic adenoviruses was studied in order to identify cellular mechanisms that confer resistance to virotherapy. Using gene expression profiling of cancer cells either resistant or susceptible to viral oncolysis, it was discovered that the epithelial phenotype of ovarian cancer represents a barrier to infection by commonly used oncolytic adenoviruses targeted to coxsackie- and adenovirus receptor (CAR) or CD46. Accessibility to viral receptors was critically linked to depolarization and the loss of tight and adherens junctions, both hallmarks of epithelial-mesenchymal transition (EMT). Importantly, tumors in situ as well as xenograft tumors derived from primary ovarian cancer cells mostly contained epithelial cells and cells that are in an epithelial/mesenchymal (E/M) hybrid stage. These E/M cells are the only xenograft-derived cells that can be cultured and with passaging undergo EMT to differentiate into mesenchymal cells. Notably, only mesenchymal cells and E/M cells in the process of EMT were susceptible to viral oncolysis. In attempts to overcome the observed resistance, it was found that thus far little explored adenovirus serotypes (Ad3, Ad7, Ad11, and Ad14), which use cellular receptor(s) other than CAR and CD46, have superior oncolytic abilities on polarized epithelial tissue. This study therefore contributes to the clarification of observed discrepancies between virotherapy performances in vitro and in vivo and gives a rationale for the construction of future oncolytic adenoviruses.
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Danielsson, Angelika. "Adenovirus-mediated Gene Therapy of Prostate Cancer." Doctoral thesis, Uppsala universitet, Enheten för klinisk immunologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-114132.
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Adenovirus-mediated gene therapy is a potential complement to standard cancer treatments. Advantages are that vectors can be used to target tumors and that replicating viruses lead to increased therapeutic dosage. In this thesis, an oncolytic serotype 5 adenovirus (Ad5), Ad[i/PPT-E1A, E3], was developed where viral replication is controlled by the insulator-shielded (i) prostate-specific PPT promoter. The adenoviral E3 region was inserted for its immune regulatory and lysis functions. Ad[i/PPT-E1A, E3] had improved cytotoxic abilities both in vitro and in a prostate cancer xenograft mouse model compared to a virus lacking the E3 region. To further improve adenoviral vectors, the histone deacetylase inhibitor (HDACi) FK228 was studied. FK228 has been proposed to enhance the effect of adenoviral therapy by upregulation of CAR, the primary receptor for Ad5 infection. In the present study, we observed that FK228 promotes transgene expression even better when administered after viral transduction, indicating a post-transductional enhancement of transgene expression. Another interesting finding was that FK228 reduced transgene expression from the PPT promoter in the prostate cancer cell line LNCaP. This is explained by the fact that different HDACi have the ability to provoke a neuroendocrine phenotype of LNCaP. A potential drawback with adenoviral gene therapy is the rapid clearance of the virus from the circulation. Viral particles have been coated with polyethylene glycol (PEG) to evade immune recognition, a strategy that works well in mouse models. However, less is known about the effects of adenoviral PEGylation in human blood. We have studied cell interactions and immune responses to PEGylated and uncoated Ad5 vectors in human whole blood using a blood loop model with constant blood flow. Limited effects of PEGylation were observed in human blood, which were associated with the neutralizing ability of the donor blood. An important finding that donors with high neutralizing ability in whole blood do not necessarily have neutralizing antibodies against the virus strongly implies that neutralization should be measured in whole blood.
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Hall, Alexis K. "Harnessing the Heat Shock Response to Raise Refined Therapeutic Outcomes." Scholarly Repository, 2008. http://scholarlyrepository.miami.edu/oa_dissertations/102.
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Activated Heat Shock Transcription Factor 1 (HSF1) has received attention in recent literature as a therapeutic effector in diseases of protein misfolding, as an immune modulating adjuvant in tumor regression, and as a trigger for gene therapy transcription. In its normal function, activated HSF1 enhances heat shock protein (Hsp) expression when additional molecular chaperoning is required (i.e., in situations of proteotoxic stress, including thermal stress) in a process known as the heat shock (HS) response. Thus, HSF1 acts as an environmental sensor, and a harness based on the biology of this capability enables transcription of genes for engineered purposes. The hypothesis of this thesis is that a harness of the heat shock response, when paired with a therapeutic mechanism, will refine novel therapies. Extensions to the concept of deliberately activating HSF1's normal functions for therapeutic purposes are examined through in vitro trials and in vivo preliminary studies that feature the use of HSF1 as a regulator of therapy. Successful in vitro work translated to pioneering preclinical studies, launched at the University of Florida's Center for Environmental and Human Toxicology. Collaboration supported the development of an innovative project to treat solid tumors using a recombinant virus system. The system was designed to facilitate intratumoral delivery of a previously characterized molecular switch, which was newly engineered to control cytotoxic gene transcription that produced dramatic consequences in cells of human origin. Central to the targeting of the in vivo therapy, is a transient, initial trigger: a thermal dose, delivered to solid tumors, which localizes HSF1 activation (a constitutively active mouse HSF1 construct was also produced to aid clarification of physiological consequences associated with deliberately upregulating HSF1 activity in vivo). Gene transcription was expected to ensue to both cause and sustain tumor regression through other regulatory elements of the molecular switch. Results demonstrated practical potential to achieve a therapeutic outcome of solid tumor regression and define contemporary challenges that continuing research directions (e.g.: production of additional viral vectors, an improved animal model, and a refined heat system) now confront in order to target and safely regulate even more potent, novel therapeutic agents.
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Fajardo, Calderón Carlos Alberto. "Arming oncolytic adenoviruses with bi-specific T-cell engagers to improve antitumor efficacy." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/403492.
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Oncolytic adenoviruses that selectively replicate in cancer cells while sparing normal tissue have gained considerable attention as novel anticancer drugs. However, clinical trials with these viruses have identified the immune system as a major hurdle for their success in cancer patients. Despite the existence of a highly immunosuppressive tumor environment, adenovirus-infected cells can nonetheless be efficiently cleared by virus-specific infiltrating cytotoxic T lymphocytes without compromising tumor burden. We hypothesize that arming oncolytic adenoviruses with bi-specific T-cell engagers (BiTEs), a new class of antibodies that re-direct T-cells to cancer cells, might favor antitumor rather than anti-viral immune responses. We have engineered the oncolytic adenovirus ICOVIR-15K to express EGFR-targeting BiTEs under the control of the major late promoter. ICOVIR-15K armed with a BiTE targeting human CD3 and EGFR (ICOVIR-15K-cBiTE) was successfully rescued and it showed similar oncolytic properties as the parental virus. cBiTE expression and secretion was detected in supernatants from ICOVIR-15K-cBiTE-infected cells, and the secreted BiTEs bound specifically to both CD3+ and EGFR+ cells. In cell co-culture assays, ICOVIR-15K-cBiTE-mediated oncolysis resulted in robust T-cell activation, proliferation, and bystander cell-mediated cytotoxicity. Notably, intratumoral injection of this cBiTE-expressing adenovirus increased the persistence and accumulation of tumor-infiltrating T cells in vivo. Moreover, in two distinct tumor xenograft models, combined delivery of ICOVIR-15K-cBiTE with peripheral blood mononuclear cells or T cells enhanced the antitumor efficacy achieved by the parental counterpart. We also demonstrate that another oncolytic adenovirus expressing a chimeric BiTE targeting human EGFR and mouse CD3 (ICOVIR-15K-mcBiTE) induce robust mouse T-cell activation, proliferation, and cell-mediated cytotoxicity of cancer cells in vitro. Thus, ICOVIR-15K-mcBiTE is a promising surrogate of ICOVIR-15K-cBiTE that will aid in future pharmacological and toxicological preclinical studies of BiTE-armed oncolytic adenoviruses. Finally, we show that the combination of ICOVIR-15K-cBiTE with chimeric antigen receptor T-cell therapy can overcome some of the limitations encountered by both agents as monotherapies. The results described in this thesis demonstrate that BiTE-armed oncolytic adenoviruses hold properties with the potential of solving key limitations in oncolytic virotherapy, and encourage their further evaluation and development.Los virus oncolitos, capaces de infectar selectivamente células cancerosas sin afectar aquellas sanas, han despertado interés en los últimos años como nueva terapia contra el cáncer. Sin embargo, los ensayos clínicos con estos virus han demostrado que el sistema inmune supone un obstáculo para el éxito de los mismos en pacientes con cáncer. A pesar de la inmunosupresión que se observa en el ambiente tumoral, las células cancerosas infectadas por el adenovirus pueden ser eliminadas eficientemente por los linfocitos T anti-adenovirales sin comprometer la carga tumoral. La hipótesis de esta tesis es que adenovirus oncoliticos expresando bi-specific T-cell engagers (BiTEs por sus siglas en inglés) capaces de redirgirir los linfocitos T para atacar las células cancerosas, puede favorecer la respuesta inmune antitumoral sobre la antiviral. El genoma del adenovirus oncolitico ICOVIR-15K fue modificado genéticamente para expresar BiTEs contra el receptor del factor de crecimiento epidérmico (EGFR por sus siglas en inglés) bajo el control del promotor mayor tardío. El virus ICOVIR-15K expresando un BiTE que reconoce el EGFR y el CD3 humanos (ICOVIR-15K-cBiTE) fue generado y retuvo propiedades oncoliticas similares a la del virus parental in vitro. La expresión y secreción del cBiTE fue detectada en los sobrenadantes de células infectadas ICOVIR-15K-cBiTE, y sus propiedades de unión a células CD3+ o EGFR+ fueron confirmadas in vitro. En experimentos de cocultivos, la oncolisis generada por ICOVIR-15K-cBiTE indujo la activación y proliferación de los linfocitos T, y aumentó la citotoxicidad de células cancerosas. La inyección de este adenovirus aumentó la persistencia y la acumulación de linfocitos infiltrantes de tumor in vivo. Adicionalmente, experimentos en modelos murinos de cáncer basados en la administración combinada de ICOVIR-15K-cBiTE y linfocitos humanos demostraron un aumento en la eficacia antitumoral comparado con el virus parental. Por último, hemos demostrado que la combinación de ICOVIR-15K-cBiTE y linfocitos T con receptores de antígeno quiméricos (CAR por sus siglas en inglés) pueden superar muchas de las carencias que tienen ambas terapias. Los resultados de esta tesis demuestran que los adenovirus oncoliticos expresando BiTEs tienen propiedades que puede superar muchas de las limitaciones de la viroterapia del cáncer, y alienta a continuar su evaluación y desarrollo a nivel clínico.
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Aguirre, Hernandez Carmen. "The oncolytic adenoviral AdΔΔ mutant sensitizes prostate cancer cells to mitoxantrone by promoting apoptosis and attenuating autophagy." Thesis, Queen Mary, University of London, 2017. http://qmro.qmul.ac.uk/xmlui/handle/123456789/24530.
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Prostate cancer (PCa) is the second most common cause of cancer-related deaths in men in the Western world. Advanced PCa is initially managed by anti-androgen therapy however, resistance frequently develops resulting in progressive metastatic disease. The current standard of care for hormone-insensitive PCa includes the cytotoxic drugs docetaxel and mitoxantrone although resistance rapidly develops to all available therapies. We demonstrated that the replication-selective oncolytic adenoviral mutant AdΔΔ enhanced drug-induced cell killing in several preclinical cancer models. AdΔΔ is deleted in the viral E1ACR2 and E1B19K, to prevent pRb-binding and enhance drug-mediated apoptotic cell killing, respectively. In drug-insensitive PCa tumour-xenografts, in vivo administration of AdΔΔ greatly enhanced drug-mediated tumour regression. The aim of my thesis project was to investigate the role of apoptosis and pro-survival pathways, including drug-induced autophagy, in AdΔΔ-mediated drug-sensitisation. I have demonstrated that autophagy was activated in a dose-dependent manner in response to mitoxantrone in the human PCa cell lines PC3, PC3M and 22Rv1. Low doses of mitoxantrone (< EC50-values) caused initiation of autophagy, determined as increased conversion of LC3I to LC3II and increased number of acidic vesicles, indicating autophagosome formation. At higher doses degradation of p62 was also observed, suggesting autophagosome fusion with the lysosome. AdΔΔ attenuated the drug-induced activation of autophagy by restoring basal LC3II/I ratios, and increasing apoptosis, determined as increased PARP-cleavage and mitochondrial depolarization. The autophagyinducer rapamycin prevented AdΔΔ-mediated sensitization in PC3 cells increasing mitoxantrone EC50-values 3-fold and attenuating apoptosis induction. In contrast, the autophagy-inhibitor chloroquine further sensitized PC3 and 22Rv1 cells to the combinationtreatment, decreasing mitoxantrone EC50-values by 40% and increasing apoptotic cell death. Atg7 is a key-factor in the autophagy pathway and siRNA-mediated knockdown prevented increases in LC3II/I ratios. In siAtg7-transfected PC3 cells mitochondrial depolarization was further promoted in combination-treated cells, similar to the results with chloroquine. The cellular Bcl-2-protein has important roles as a mediator of both anti-apoptotic and antiautophagic functions. In cells transfected with siBcl-2 the LC3II/I ratios increased and AdΔΔ- mediated sensitization to mitoxantrone was prevented, indicating initiation of autophagy. In addition, mitoxantrone-induced degradation of Bcl-2 was attenuated by AdΔΔ infection, suggesting stabilization of the protein. The mechanisms for the AdΔΔ-mediated increases in cell killing were also demonstrated in 3-dimensional co-culture models of PC3 or 22Rv1 in combination with prostate stromal cells and extracellular matrix proteins using confocal microscopy. These data demonstrate that AdΔΔ attenuates drug-induced cell survival/rescue and promotes elimination of cancer cells through apoptosis and viral lysis, and that Bcl-2 was essential for the sensitisation.
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Rojas, Expósito Luis Alfonso. "Blood barriers for oncolytic adenovirus efficacy: study of binding to erythrocytes via CAR and albumin‐mediated evasion of neutralizing antibodies." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/404054.
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Cancer virotherapy with oncolytic adenoviruses represents a promising therapeutic approach due to the capacity of these viruses to infect and selectively kill tumor cells without damaging normal tissues. Although the intravenous is the preferred route of administration in order to reach disseminated metastasis, several interactions with blood components cause the neutralization of the virus. Thus, improving the delivery of such adenoviruses to tumors by systemic injection is crucial for the success of the therapy. In this work we have studied the interaction of the adenovirus serotype 5 (Ad5) with human erythrocytes through the receptor CAR, which was described to sequester and inactivate the virus. Although erythrocyte binding was observed, it did not reduce viral transduction of tumor cells in vitro. Since mouse erythrocytes do not express CAR, human erythrocytes were transferred into nude mice to analyze the impact of erythrocyte binding after systemic administration. However, adenovirus extravasation and transduction of liver and tumors was not reduced, suggesting that this binding is reversible and does not neutralize the virus.
On the other hand, the high prevalence of anti-Ad5 neutralizing antibodies (NAbs) is a major obstacle for the intravenous administration of adenoviruses. To protect adenovirus against NAbs we inserted an albumin-binding domain (ABD) in the main adenovirus capsid protein, the hexon. This domain binds serum albumin to shield the virus upon systemic administration. The ABD-modified adenoviruses bind human and mouse albumin, which allow them to maintain the infectivity and replication capacity in presence of NAbs. Non-modified adenoviruses are completely neutralized after systemic administration in pre-immune mice, whereas ABD-modified viruses preserve the ability to transduce target organs and induce oncolysis. The data presented in this thesis supports the use of this strategy to treat patients systemically with oncolytic adenoviruses.
In summary, this thesis focused on improving the intravenous delivery of oncolytic adenoviruses, which is one of the main limitations of this therapy. The results presented in this work demonstrate that while erythrocyte binding via CAR does not inactivate the virus, NAbs represent a major obstacle for efficacy. In this regard, albumin coating of the virus capsid represents an effective approach to evade pre-existing NAbs. This strategy has translational relevance in the use of adenovirus by systemic injection not only for cancer virotherapy, but also for gene therapy and vaccination.Els adenovirus oncolítics són agents terapèutics prometedors, degut a la seva capacitat d’infectar i eliminar selectivament les cèl·lules tumorals, sense afectar les cèl·lules normals. Tot i que la ruta preferida d’administració és la intravenosa per tal d’arribar a totes les metàstasis, la interacció del virus amb diversos components de la sang provoca la seva neutralització. Per tant, millorar l’arribada dels virus als tumors per via sistèmica és un aspecte clau per a l’èxit d’aquesta teràpia. En aquest treball s’ha estudiat la interacció de l’adenovirus serotip 5 amb els eritròcits humans a través del receptor CAR, la qual es va descriure que provocava el segrest i la inactivació del virus. Malgrat es va observar unió als eritròcits, aquesta no va reduir la transducció de cèl·lules tumorals in vitro. Degut a que els eritròcits murins no expressen CAR, es van transferir eritròcits humans a ratolins immunodeprimits per tal d’analitzar l’efecte de la interacció després de la injecció sistèmica. Tot i així, aquesta unió als eritròcits no va alterar la extravasació ni la transducció del fetge per part del virus, suggerint que la interacció és reversible i no neutralitzant. Per altra banda, l’alta prevalença d’anticossos neutralitzants contra l’adenovirus 5 en la població humana representa un obstacle molt important per la injecció intravenosa d’aquest. Per protegir l’adenovirus contra els anticossos neutralitzants s’ha inserit un domini d’unió a albúmina (ABD) a la proteïna principal de la càpside viral, la proteïna hexó. Aquest domini s’uneix a l’albúmina sèrica, recobrint el virus amb aquesta després de l’administració sistèmica. Els virus modificats amb ABD són capaços d’unir-se tant a l’albúmina humana com a la murina, fet que els permet mantenir la infectivitat i la capacitat replicativa en presència d’anticossos neutralitzants. Els adenovirus no modificats són completament neutralitzats després de la administració sistèmica en ratolins pre-immunes, mentre que els virus modificats amb ABD mantenen la capacitat de transduïr els òrgans i controlar el creixement tumoral. Els resultats presentats en aquesta tesi recolzen l’ús d’aquesta estratègia per a tractar pacients amb adenovirus oncolítics per via sistèmica.
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Rodrigues, Margret S. Tong Alex W. "Growth inhibition of human multiple myeloma cells by a conditional-replicative, oncolytic adenovirus armed with the CD154 (CD40-ligand) transgene." Waco, Tex. : Baylor University, 2006. http://hdl.handle.net/2104/5016.
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Raimondi, Giulia. "Broadening Adenoviral Oncolysis in PDAC: Interrogation of Patient-Derived Organoids for personalized virotherapy and modulation of miRNA content to boost adenoviral potency." Doctoral thesis, Universitat de Barcelona, 2020. http://hdl.handle.net/10803/671205.
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The general goal of this thesis has been to progress oncolytic adenovirus therapy for PDAC, by the incorporation of novel preclinical models to test for patient-specific responses and the generation of oncolytic adenoviruses with enhanced therapeutic index.
The two main objectives have been the following:
i) Evaluate patients-derived organoids (PDOs) technology as a platform to screen for personalized virotherapy in vitro
1) Establishment of a battery of PDOs from PDAC and normal pancreatic tissues, and evaluation of their applicability in the study of adenoviral infection;
2) Screening of a battery of PDOs to identify individual sensitivities to virotherapies, and the effects derived from the combination with chemotherapy;
3) Study virotherapy-responses in metastasis originated from
PDOs xenografted in mice;
(ii) Improve oncolytic adenovirus potency by modulation of miRNAs deregulated in PDAC
4) Screening of aberrantly expressed miRNAs sensitizing viral oncolysis in PDAC via CRISPR/Cas9 system;
5) Generation of a miRNA sponge-adenovirus and evaluation of its oncolytic effects in vitro and in vivo;
6) Modulation of miRNA levels with the THZ1 transcriptional inhibitor, and assessment of the effects of its combination
with oncolytic adenoviruses.
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Gomes, Erica Manuela Tong Alex W. "Anti-tumor properties of CD40 ligand when delivered as a transgene by the conditional replicative oncolytic adenovirus AdEH to breast cancer cells." Waco, Tex. : Baylor University, 2006. http://hdl.handle.net/2104/4901.
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Rodríguez, García Alba. "Enhancing the Antitumor Activity of Oncolytic Adenoviruses by Combining Tumor Targeting with Hyaluronidase Expression or by Increasing the Immunogenicity of Exogenous Epitopes." Doctoral thesis, Universitat de Barcelona, 2015. http://hdl.handle.net/10803/290068.
Full textAbstract:
Oncolytic adenoviruses represent an appealing therapeutic approach to treat cancer regarding its capability to infect and kill selectively tumor cells without damaging normal tissues. Tumor targeting upon intravenous administration and subsequent intratumoral virus dissemination are key features to improve oncolytic adenovirus therapy. To address these hurdles, in this work we have combined two different genetic modifications previously described by our group in an oncolytic adenovirus backbone with selective replication conditional to pRB pathway deregulation. First, the replacement of the heparan sulfate glycosaminoglycan-binding site KKTK of the fiber shaft with an integrin-binding motif RGDK for tumor-targeting that has shown to prolong blood persistence and significantly enhance the therapeutic index compared with a nonRGDK-modified virus. Second, the expression of hyaluronidase to degrade the extracellular matrix and improve the intratumoral spread of the virus. Preclinical toxicology and biodistribution studies conducted in non-permissive mouse and semi-permissive Syrian hamster models supported the selectivity and safety of this novel virus, ICOVIR-17K. Antitumor efficacy was also demonstrated in different tumor models in immunodeficient mice and immunocompetent hamsters upon different routs of administration. Moreover, the combination of ICOVIR-17K with the chemotherapeutic drug gemcitabine further increased the antitumor activity of the virus. The data presented in this thesis strongly supports ICOVIR-17K as a promising clinical candidate which is currently being tested in phase I clinical trials.
Besides direct killing of cancer cells, oncolytic viruses may induce antitumor immune responses. Local inflammation of tumor tissue during an infection by an oncolytic virus provides suitable conditions to trigger antitumor immune responses against the tumor-associated antigens that are released in the immunogenic cell death process caused by these agents. Oncolytic adenoviruses can be used to promote immune responses against tumors by expressing and/or displaying tumor-associated antigens. However, a key limitation of this immunotherapeutic approach is the bias of the response towards the immunodominant viral antigens instead of the less immunogenic tumor antigens. In addition, defects in MHC class I antigen presentation pathway such as the downregulation of the transporter associated with antigen processing (TAP) are frequently associated with immune evasion of tumor cells and further impair the generation of specific immune responses against tumor antigens carried by oncolytic viruses. In this work we present a novel strategy that benefit from the TAP deficiency on tumor cells to enhance the response against those tumor epitopes, which had been attached to the viral protein E3-19K. This protein has a signal sequence that targets it to the endoplasmic reticulum, bypassing the necessity of TAP to transport the epitopes to that compartment. Compared to the display of the epitopes at the adenoviral capsid, this strategy promoted the immunogenicity of the epitopes and resulted in more potent antitumor immune responses, tackling a crucial aspect of virotherapy that is often overlooked.
In summary, the two different projects involved in this thesis addressed the main limitations of oncolytic adenoviruses from distinct points of view that may eventually be combined in order to maximize the opportunities of success of oncolytic adenoviruses in the clinics.La viroteràpia del càncer amb adenovirus oncolítics es basa en l’habilitat d’aquests agents en replicar selectivament en cèl·lules tumorals, produint la seva mort sense afectar cèl·lules normals. Les principals limitacions d’aquesta teràpia són la dificultat dels adenovirus per arribar als tumors després de ser administrats sistèmicament i també la seva incapacitat per dispersar-se de manera homogènia dins dels tumors. En aquest treball s’ha generat un adenovirus oncolític que combina dues mutacions descrites amb anterioritat pel nostre grup. Per una banda, la substitució del motiu d’unió a heparan-sulfats glicosaminoglicans situat al domini shaft de la fibra pel motiu d’unió a integrines RGD (mutació RGDK) per tal de millorar la ratio de transducció tumor/fetge i d’augmentar la persistència en sang de l’adenovirus. Per altra banda, l’expressió de hialuronidasa amb l’objectiu de degradar l’àcid hialurònic de la matriu extracel·lular del tumor i millorar la dispersió intratumoral de l’adenovirus. Aquest nou virus, l’ICOVIR-17K, va mostrar una potent eficàcia antitumoral en models de ratolí i hàmster que va ser fins i tot incrementada mitjançant la combinació amb gemcitabina, tot mantenint el perfil de toxicitat dels adenovirus oncolítics parentals. Per altra banda, a més de matar directament les cèl·lules tumorals, els adenovirus oncolítics poden contribuir a la generació de respostes immunes contra el tumor. El tipus de mort cel·lular que generen és altament immunogènic i ajuda al reclutament de cèl·lules del sistema immune que generen respostes contra els antígens tumorals alliberats en aquest procés. Una de les principals limitacions de la immunoteràpia amb virus oncolítics és la resposta esbiaixada cap als antígens virals, que són immunodominants, en lloc de cap als antígens tumorals, que són poc immunogènics. Per tal d’afavorir la generació de respostes immunes antitumorals, en aquest treball s’han incorporat epítops tumorals en la proteïna E3-19K de l’adenovirus, que conté una seqüència senyal que la dirigeix directament al reticle endoplasmàtic, de manera que evadeix els passos previs de processament antigènic per la via del MHC de classe I, comunament afectada en cèl·lules tumorals. Aquesta estratègia va permetre la generació de respostes immunes antitumorals més potents que quan els mateixos epítops eren incorporats a la càpside de l’adenovirus, i a més, van ser traduïdes en una millor eficàcia antitumoral en un model murí de melanoma. En resum, en aquest treball s’han abordat les principals limitacions dels adenovirus oncolítics des de diferents punts de vista que, eventualment, poden ser combinats per tal d’aconseguir un millor candidat per ser testat exitosament a la clínica.
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Brüggemann, Sabrina [Verfasser], Dagmar [Akademischer Betreuer] Knebel-Mörsdorf, and Hildegard [Akademischer Betreuer] Büning. "Generation of an oncolytic adenovirus vector combining three cancer targeting strategies and characterization of a new preclinical model for breast cancer virotherapy / Sabrina Brüggemann. Gutachter: Dagmar Knebel-Mörsdorf ; Hildegard Büning." Köln : Universitäts- und Stadtbibliothek Köln, 2012. http://d-nb.info/1038234689/34.
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Bressy, Christian. "Potentialisation de la virothérapie anti-tumorale basée sur des adénovirus oncolytiques dans le traitement des cancers côliques et rénaux." Phd thesis, Université Paris Sud - Paris XI, 2013. http://tel.archives-ouvertes.fr/tel-00921952.
Full textAbstract:
Nous avons mis en place au cours de ce travail de thèse différentes stratégies permettant d'améliorer l'efficacité thérapeutique des adénovirus (Ad) oncolytiques contre différents types de tumeurs. Une première stratégie a été de combiner un inhibiteur d'histone-désacétylase, l'acide valproique (VPA) avec un Ad oncolytique à capside sauvage E1Δ24 (CRAd) dans le traitement de carcinomes côliques. Nous avons dans un premier temps démontré que la combinaison du CRAd et du VPA permettait une diminution plus importante de la survie des cellules cancéreuses côliques comparé au simple traitement basé sur le CRAd ou le VPA in vitro mais aussi in vivo.De plus, nous avons observé que cet effet n'était pas lié à une meilleure réplication du CRAd par le VPA. En effet, le VPA provoquait un ralentissement de la réplication virale à des temps précoces mais ne modifiait pas la production virale. Nous avons également découvert que le co-traitement CRAd+VPA conduisait à une forte inhibition de la croissance cellulaire mais aussi à une mort cellulaire non apoptotique. Par ailleurs, nous avons mis en évidence que les cellules co-traitées par le CRAd et le VPA affichaient une forte polyploïdie accompagnée d'une augmentation de la phosphorylation de l'histone H2AX, un marqueur de dommages à l'ADN. Une deuxième stratégie a été de fournir aux Ad oncolytiques de nouvelles voies d'entrée afin d'infecter et de détruire plus efficacement des cellules de carcinomes rénaux réfractaires à l'infection adénovirale. Nous avons démontré que les CRAd à hexon modifié porteurs d'un ligand CKS-17 (Ad-HCKS-17-E1Δ24) ou à fibre modifiée de sérotype 3 (AdF3-E1Δ24) étaient capables d'infecter et de tuer plus efficacement ces cellules qu'un CRAd à capside sauvage in vitro. Malheureusement in vivo, les modifications de capside n'ont permis ni d'améliorer l'entrée des CRAd dans les tumeurs rénales, ni d'améliorer leur efficacité anti-tumorale. Cependant, nous avons observé qu'après administration intra-tumorale, les Ad à capside modifiée présentaient un plus faible tropisme hépatique comparé à un Ad à capside sauvage.
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Rifai, Bassel. "Cavitation-enhanced delivery of therapeutics to solid tumors." Thesis, University of Oxford, 2011. http://ora.ox.ac.uk/objects/uuid:374b2ee1-0711-4994-8434-bf90358d9e47.
Full textAbstract:
Poor drug penetration through tumor tissue has emerged as a fundamental obstacle to cancer therapy. The solid tumor microenvironment presents several physiological abnormalities which reduce the uptake of intravenously administered therapeutics, including leaky, irregularly spaced blood vessels, and a pressure gradient which resists transport of therapeutics from the bloodstream into the tumor. Because of these factors, a systemically administered anti-cancer agent is unlikely to reach 100% of cancer cells at therapeutic dosages, which is the efficacy required for curative treatment. The goal of this project is to use high-intensity focused ultrasound (HIFU) to enhance drug delivery via phenomena associated with acoustic cavitation. ‘Cavitation’ is the formation, oscillation, and collapse of bubbles in a sound field, and can be broadly divided into two types: ‘inertial’ and ‘stable’. Inertial cavitation involves violent bubble collapse and is associated with phenomena such as heating, fluid jetting, and broadband noise emission. Stable cavitation occurs at lower pressure amplitudes, and can generate liquid microstreaming in the bubble vicinity. It is the combination of fluid jetting and microstreaming which it is attempted to explore, control, and apply to the drug delivery problem in solid tumors. First, the potential for cavitation to enhance the convective transport of a model therapeutic into obstructed vasculature in a cell-free in vitro tumor model is evaluated. Transport is quantified using post-treatment image analysis of the distribution of a dye-labeled macromolecule, while cavitation activity is quantified by analyzing passively recorded acoustic emissions. The introduction of exogenous cavitation nuclei into the acoustic field is found to dramatically enhance both cavitation activity and convective transport. The strong correlation between inertial cavitation activity and drug delivery in this study suggested both a mechanism of action and the clinical potential for non-invasive treatment monitoring. Next, a flexible and efficient method to simulate numerically the microstreaming fields instigated by cavitating microbubbles is developed. The technique is applied to the problem of quantifying convective transport of a scalar quantity in the vicinity of acoustically cavitating microbubbles of various initial radii subject to a range of sonication parameters, yielding insight regarding treatment parameter choice. Finally, in vitro and in vivo models are used to explore the effect of HIFU on delivery and expression of a biologically active adenovirus. The role of cavitation in improving the distribution of adenovirus in porous media is established, as well as the critical role of certain sonication parameters in sustaining cavitation activity in vivo. It is shown that following intratumoral or intravenous co-injection of ultrasound contrast agents and adenovirus, both the distribution and expression of viral transgenes are enhanced in the presence of inertial cavitation. This ultrasound-based drug delivery system has the potential to be applied in conjunction with a broad range of macromolecular therapeutics to augment their bioavailability for cancer treatment. In order to reach this objective, further developmental work is recommended, directed towards improving therapeutic transducer design, using transducer arrays for treatment monitoring and mapping, and continuing the development of functionalized monodisperse cavitation nuclei.
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Wang, Wei-Shiuan, and 王偉璇. "Oncolytic adenovirus driven by hypoxia-inducible hTERT promoter for cancer therapy." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/47378699877191665993.
Full textAbstract:
碩士國立成功大學
生物化學研究所
93
Hypoxia, a condition that oxygen density is low at local areas, plays a critical role in tumor malignancy and is associated with resistance of cancer cells to conventional chemotherapy and radiotherapy. Hypoxia-inducible factor-1 (HIF-1) is stabilized and accumulated when tissues are exposed to hypoxia. HIF-1 is a heterodimeric transcription factor that regulates the physiologic reaction to hypoxia by binding to hypoxia response element (HRE) of target genes. Human telomerase reverse transcriptase (hTERT), the catalytic subunit of the telomerase is transcriptionally upregulated in about 90% of cancers. Therefore, overexpression of hTERT is considered as a tumorigenesis marker. It has been suggested that hypoxia activates telomerase via transcriptional activation of hTERT, and that HIF-1 plays an important role as a transcription factor. Novel therapeutic strategies to target tumor cells in hypoxia regions with high TERT promoter activity to overcome their resistance to chemotherapy and radiotherapy are urgently needed. Therefore, we have exploited 6 copies of HRE ligated to hTERT promoter to modify the transcription activity of hTERT and constructed AdWiSh (Ad5-6xHRE-hTERT), an oncolytic adenovirus driven by this modified promoter. The transcription activity of the 6xHRE-hTERT promoter has been proved higher than that of the original promoter in hypoxia conditions. Similarly the oncolytic efficacy of AdWiSh under hypoxia is better than under normoxia. Intratumoral injection of AdWiSh resulted in suppressing of tumor growth and prolonging survival in mice bearing subcutaneous Lewis lung carcinoma. Cisplatin combined with hypoxia stimulated HIF-1α upregulation and enhanced 6xHRE-hTERT promoter activity, and Ad.WiSh could have better cytolytic efficacy in this condition. Combination of hypoxia-inducible adenovirus and chemotherapeutic drug cisplatin exhibited higher antitumor efficacy compared with either treatment alone. Taken together, these results suggest that AdWiSh, a 6xHRE-hTERT-driven oncolytic adenovirus may have therapeutic potential for solid tumors.
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Wu, Hong-Yi, and 吳泓毅. "MSC delivery system of oncolytic adenovirus in PDAC xenograft animal models." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/n5x4hs.
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Wu, I.-Hui, and 吳怡蕙. "Cotargeting Prostate Cancer and Supporting Bone Stroma Using Dual Controlled Oncolytic Adenovirus." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/95613304539966060268.
Full textAbstract:
碩士中國醫藥大學
癌症生物學研究所碩士班
99
Our laboratory previously developed a conditional replicating adenoviral vector (CRAd), Ad-hOC-E1 to co-target bone metastatic prostate cancer cells (PCa) and bone stroma. Owing to the importance of tumor microenvironment in supporting cancer growth and metastasis, Ad-hOC-E1 is superior to currently clinically used signal-targeting CRAds for the treatment of bone metastasis PCa. It has been known that microRNAs inhibit gene expression at post-transcriptional level and many of them are dyregulated during cancer progression. We hypothesized that incorporating microRNA regulation into Ad-hOC-E1 could restrict toxicity of microRNAs to cancer-associated stroma but not normal tissues. To select the appropriate microRNAs, we performed microRNA microarray using normal and PCa-associated bone stromal cell lines. By RT-qPCR, we confirmed that miR-195 and miR-199a were down-regulated in both PCa and cancer-associated stroma but over-expressed in normal stroma. To test whether the miR-195 target sequence (miR-195T) or miR-199a target sequence (mir-199aT) is able to suppress the expression of transgene in normal stroma, we constructed microRNA-regulated luciferase reporter by insertion of synthetic miR-195T, miR-199aT and miR-scrambleT into the 3’-UTR of pMIR. Our results demonstrated that miR-199aT significantly suppressed luciferase expression in normal stroma but not in Pca and Pca-associated stroma in comparison to mir-scrambleT. Similarly, incorporating miR-199aT into luciferase expression cassette driven by tumor-specific hOC-promoter also decreased luciferase expression in normal cells. Further, inserting miR-199aT into oncolytic adenovirus driven by bidirectional hOC-promoter could also de-target virus replication in normal cells. These results suggested that miR-199aT-regulated oncolytic adenovirus dual control by tumor-specific promoter and microRNA regulation could provide a safe tumor-targeting approach.
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Liu, Chin-Cheng, and 劉錦誠. "Construction of an oncolytic adenovirus for KRAS activating mutant colorectal cancer cells." Thesis, 2010. http://ndltd.ncl.edu.tw/handle/75160038999894140520.
Full textAbstract:
博士國立陽明大學
生化暨分子生物研究所
98
Abstract E1B-55kD-deleted adenoviruses (ΔE1B-55kD Ads) have been used as conditionally replicative adenoviruses (CRAds) for therapeutic purpose in tumors with loss-of-function p53 mutation. To target cancer cells harboring activating mutant KRAS (KRASaMut) but spare p53wild normal cells, we constructed and examined by reporter assays a KRASaMut but not p53-responsive promoter, the ?愎53REP2 promoter. The ?愎53REP2 promoter, derived from human double minute 2 (hdm2) P2 promoter with its p53 response elements being deleted, was used to regulate the expression of hdm2 transgene in a novel ΔE1B-55kD CRAd, the Ad-KRhdm2. The Ad-KRhdm2 selectively replicated in and exerted cytopathic effects on KRASaMut colorectal cancer cell lines (HCT116, LoVo, LS174T, LS123, and SW620), regardless of their p53 gene statuses, by forming plaques and exhibiting cytopathic effect in cultured cells. Ad-KRhdm2, like other ΔE1B-55kD Ads, also exerted selective cytopathic effects on tumor cells with loss-of-function p53 mutant. The multiplicities of infection (MOIs) of Ad-KRhdm2 required to decrease 50% viability of KRASaMut tumor cells cultured for 7 days were 440 to 3400-time less than those of MRC5 normal fibroblasts and KRASwild/p53wild RKO tumor cells. Intratumoral injection of Ad-KRhdm2 vectors exhibited specific lytic activities in nude mouse xenografts of KRASaMut cell lines (LoVo, SW620, and LS174T) but not in xenografts of RKO cells. Transduction of KRASaMut /p53wild HCT116, LoVo, and LS174T cells by Ad-KRhdm2 significantly increased Hdm2 expression, decreased p53 level, and abolished the p53-transactivating p21Cip1 promoter activity. The Ad-KRhdm2 has demonstrated its therapeutic potential in KRASaMut cancer cells and warrants further clinical trials.
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Meng, Ching-Ting, and 孟慶庭. "MSC delivery system of oncolytic adenovirus harboring inducible promoter for pancreatic cancer microenvironments." Thesis, 2018. http://ndltd.ncl.edu.tw/handle/a4m83q.
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Wang, Hao-Tien, and 王皓恬. "Oncolytic adenovirus driven by hypoxia- inducible Met promoter for the treatment of hepatocellular carcinoma." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/27806092724080838195.
Full textAbstract:
碩士國立成功大學
生物化學研究所
93
Hepatocellular carcinoma (HCC) is one of the most human malignant tumors in Taiwan. As this disease has a poor prognosis, an effective therapeutic modality is urgently needed. Replication-selective adenovirus has been reported to kill tumor cells and therefore can be one of the promising therapeutic approaches for cancer. Since hypoxia is a common characteristic of human tumors, which adversely affects the prognosis of cancer patients, targeting hypoxic regions may increase the effectiveness of cancer treatment. It is the reason why many researches have exploited hypoxia response element (HRE) to control gene expression for tumor-targeted gene therapy. Met, the receptor for hepatocyte growth factor (HGF), is overexpressed in many human cancers, and may contribute to their progression and metastasis. In this study, we constructed oncolytic adenovirus driven by hypoxia-inducible Met promoter (Ad/mickey), or by Met promoter (Ad/WHaT), and examined their cytolytic effects on mouse and human HCC cell lines. We found that ML-1 and Hep3B HCC cells expressed high levels of Met protein, whereas LL/2 and PC14PE6 lung cancer cells expressed low levels of Met protein. Moreover, the Met promoter activity was positively correlated with Met protein expression. Based on the difference in promoter activity, both viruses caused more severe cytolytic effects on ML-1 and Hep3B cells than on LL/2 and PC14PE6 cells. We also used CoCl2 to mimic hypoxic condition and found hypoxia-inducible Met promoter could be up-regulated under hypoxic condition compared with Met promoter. Meanwhile, Ad/mickey exhibited better oncolytic effects than Ad/WHaT, but its cytolytic effect was relatively attenuated under normoxia. We also found that rapamycin, an immunosuppressive drug, could enhance Met promoter activity and Ad/WHaT protein expression. The in vivo antitumor effects of Ad/mickey and Ad/WHaT were evaluated in terms of tumor growth and survival in BALB/c mice bearing syngeneic ML-1 tumors. Combination of Ad/WHaT and chemotherapy exhibited higher antitumor efficacy compared with either treatment alone. These results suggest that oncolytic adenovirus driven by the Met promoter had therapeutic potential for the treatment of HCC overexpressing Met.
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Chen, Jie-Yi, and 陳傑宜. "Treating pancreatic ductal adenocarcinoma with oncolytic adenovirus and analyzing the immune properties in tumor microenvironment." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/92re8n.
Full textAbstract:
碩士國立東華大學
生命科學系
107
Pancreatic ductal adenocarcinoma (PDAC) with an extremely low 5-year survival rate is one of the most disastrous diseases. Some advanced therapies have been developed to meet the unmet medical need. However, owing to the immunosuppressive microenvironment and chemoresistant fibrous barriers, the efficacies of treatments are limited. Because oncolytic viruses (OVs) can specifically lyse the tumor cells and trigger anti tumor immune responses, they may provide alternative strategy as the cancer-targeted and immune therapy. In this study, oncolytic adenoviruses serotype C5 and F41 (OAdV-C5 and -F41), which are specific, replicative, relatively safe and easily manipulated, were used to treat PDAC in vitro and in vivo. OAdV-C5 and -F41 were isolated and characterized using RT-qPCR and immunofluorescence assay. As infected by OAdV-C5 or -F41, the tested cancer cells, especially in pancreatic malignancy, were more prone to be killed than normal cells. Therefore, the orthotopic PDAC nude mouse model was established and used to evaluate the efficacy of intratumor injection of OAdV-C5 or -F41. It was found that the tumor size was reduced in a dose dependence, and overall survival rates of the mice were accordingly prolonged 1.5-fold. Moreover, the infiltration of monocytes, NK cells and B cells in the PDAC microenvironment was increased in a time-dependent manner by analyzing the gene expression of Mcp1 (monocytes), Ncr1 (NK cells) and Cxcl13 (B cells). It was found that the expression of PD-1 was down-regulated at 2-wk post OAdV treatment. Afterwards PD-1 level increased at 1-3 months post treatment, and it could lead to the immunosuppression and attenuate the tumor-eliminating capability of immune cells. Taken together, the OAdV has the therapeutic potential, and its combination with immune checkpoint inhibitors may be promising for PDAC treatment.
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Huang, Ying-Hui, and 黃穎蕙. "Improvement of the selectivity of an oncolytic adenovirus carrying Oct-3/4 response elements in metastatic bladder cancer." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/29039096474756634610.
Full textAbstract:
碩士國立成功大學
生物化學研究所
94
Current treatment options for metastatic tumors lack efficacy and metastases targeting remains a major challenge for curing cancer. The POU homeodomain protein Oct-3/4 (where Oct stands for octamer binding protein) is an embryonic transcription factor expressed in germ cells and embryonic stem but not expressed in normal somatic tissues. It has been shown recently that embryonic genes are re-expressed in many cancer cells. Generally, cancer cells are immortal, undifferentiated and invasive. Therefore, it might be expected that cancer cells express genes in common with early embryonic cells. Bladder cancer is a common human malignancy and is the second most common genitourinary malignancy. Patients with metastatic disease have poor long-term survival rates despite systemic multiagent chemotherapy. Thus, the re-expression of the embryonic gene Oct-3/4 in cancer cells may have a high potential for cancer therapy. In our study, Oct-3/4 expression was found to be higher in metastatic bladder cancer cells than in non-metastatic bladder cancer cell lines. Therefore, we have exploited 9 copies of Oct-3/4 response elements ligated to CMVmini (CMVm) promoter to investigate its transcription activity in metastatic bladder cancer. The transcription activity of the Oct4RE-CMVmini promoter was higher in metastatic than in non-metastatic bladder cancer cells. We also determined whether oncolytic adenovirus carrying 9 copies of Oct-3/4 response elements (Ad-9OC) replicated more selectively in metastatic bladder cancer. Our results showed that Ad-9OC exhibited higher oncolytic activity in metastatic bladder cancer than non-metastatic bladder cancer. Furethermore, Ad.9OC had better antitumor effects on tumor growth and survival in metastatic bladder cancer than in non-metastatic bladder cancer animal model. Taken together, these results suggest that Ad.9OC, a 9x Oct4RE-CMVm-driven oncolytic adenovirus, may have therapeutic potential for Oct-3/4 re-expressed bladder metastatic bladder cancer.
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Tsai, Jeng-Liang, and 蔡政良. "Synergistic antitumor effect of c-Met-dependent oncolytic adenovirus combined with rapamycin in non-small cell lung cancer." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/09451060211457183819.
Full textAbstract:
碩士國立成功大學
微生物及免疫學研究所
96
Lung cancer is a deadly disease with high mortality and morbidity. Approximately 85% of these cases are non-small cell lung cancer (NSCLC) with the rest being small cell lung cancer (SCLC). Like normal cells, lung cancer cells express receptor tyrosine kinases. The difference is that these receptors may be overexpressed or mutated leading to increased activation. c-Met is a receptor tyrosine kinase whose activation by hepatocyte growth factor can lead to transformation and tumorigenicity. It is also implicated in growth, invasion, and metastasis of various tumors, including lung cancer. Here, we used an E1B 55KD-deleted replication-selective oncolytic adenovirus (Ad.What) driven by the c-Met promoter for the treatment of lung cancer. Ad.What replicated and hence lysed lung cancer cells with c-Met overexpression, whereas it did not induce noticeable cytopathic effects in normal cells. Previous studies showed that combination of oncolytic adenovirus with chemotherapeutic drugs could augment the antitumor efficacy. Rapamycin, a highly selective inhibitor of mammalian target of rapamycin (mTOR) serine/threonine kinase, has shown promise in clinical studies for treating different types of cancer. Accordingly, we combined rapamycin with Ad.What and found that they synergized in inducing cytopathic effects in lung cancer cells. Rapamycin enhanced coxsackievirus and adenovirus receptor (CAR) and αV integrin expression on cancer cells. Ad.What reduced total p70S6K and phosphorylated p70S6K, the downstream effector of mTOR, and induced autophagy. We concluded that the combination of c-Met promoter-driven oncolytic adenovirus with rapamycin has the potential to be an effective strategy for lung cancer treatment.
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Singleton, Dean Craig. "Antitumour efficacy of the nitroreductase-armed oncolytic adenovirus ONYX-411NTR in combination with dinitrobenzamide mustard prodrugs in preclinical models." 2009. http://hdl.handle.net/2292/4956.
Full textAbstract:
Oncolytic viruses that selectively replicate in and lyse cancer cells are a promising approach for the treatment of tumours that are resistant to conventional therapies. Clinical experience has shown that oncolytic viruses are safe and well tolerated but possess modest single agent activity. One approach to improve the efficacy of oncolytic viruses is to utilise their tumour tropism to deliver genes encoding enzymes able to activate prodrugs. ONYX-411 is an oncolytic adenovirus that replicates in cells that carry dysfunctions in the retinoblastoma (pRb) pathway, a common hallmark of cancer. ONYX-411 was ‘armed’ by inserting the Escherichia coli nfsB nitroreductase (NTR) gene into the E3B region of the viral genome under the control of the endogenous E3 viral transcriptional machinery. NTR is an oxygen-insensitive nitroreductase that is capable of activating dinitrobenzamide mustard (DNBM) prodrugs to cytotoxic metabolites. The main objective of this thesis was to determine the extent and mechanism of the therapeutic interaction between ONYX-411NTR and DNBM prodrugs. A fluorogenic probe was developed to monitor NTR activity non-invasively and revealed robust, replication dependent NTR activity in ONYX-411NTR-infected neoplastic but not primary human cell lines. In vitro exposure of ONYX-411NTR-infected cells to therapeutically relevant concentrations of the DNBM prodrugs (SN 27686 or PR-104A) did not inhibit virus replication. Tumour growth delay studies of systemic ONYX-411NTR followed by prodrug demonstrated different outcomes in three models (H1299, C33A, 22Rv1). To establish predictable viral infection of tumours a pre-infection model was developed using HCT 116 xenografts. This methodology demonstrated that prodrug administration (SN 28343 or PR-104) provided significant inhibition of tumour growth without suppression of ONYX-411NTR replication. Follow-on studies using intravenous virus administration confirmed titre amplification with time (24-fold between day 3 and 13 post administration; P < 0.001) and a marked survival gain for the virus/prodrug combinations. Neither the prodrugs nor ONYX-411NTR were active as single agents. The improvement in efficacy for the combination of ONYX-411NTR and prodrug was conditional on NTR-dependent prodrug activation resulting in improved virus distribution within the tumour. PR-104 is currently in clinical development making the combination of ONYX-411NTR with PR-104 a promising strategy for cancer selective therapy.Whole document restricted, but available by request, use the feedback form to request access.
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Schache, Peter [Verfasser]. "Adenovirus and VSV : investigations on virus-host-interactions to improve safety and efficacy of oncolytic viruses / von Peter Schache." 2009. http://d-nb.info/99494103X/34.
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Wang, Wei-Shyang, and 王瑋祥. "Evaluation of the therapeutic effects of interleukin- 8-dependent oncolytic adenovirus on orthotopic lung adenocarcinoma by in vivo non-invasive imaging." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/30604435409488956479.
Full textAbstract:
碩士國立成功大學
微生物及免疫學研究所
95
Non-small cell lung cancer (NSCLC), which comprises 80% of all lung cancer cases, is one of the most common human malignancies. The majority of NSCLC patients have incurable advanced disease with very poor therapeutic option. Therefore, new treatment approaches are urgently needed. Oncolytic adenoviruses have emerged as novel therapeutic agents for a variety of cancers. Interleukin-8/CXCL8 (IL-8) has been shown to be an angiogenic and growth factor, either autocrine or paracrine, in several cancers, including NSCLC. Moreover, the expression level of IL-8 in lung cancer is associated with angiogenesis, tumor progression, metastasis, and poor survival. Infiltrating macrophages may up-regulate tumor IL-8 expression through the NF-κB pathway and modulation by tumor necrosis factor-α (TNF-α) and interleukin-1. In the NSCLC microenvironment, IL-8 can be secreted from tumor cells, macrophages, and stromal cells. It is, therefore, feasible to exploit IL-8 promoter to control the replication of oncolytic adenovirus aiming to achieve more tumor-targeted oncolysis for treating NSCLC. Characterization of IL-8 promoter indicated that the minimal region essential for IL-8 expression contains the binding sites of AP-1, NF-κB, and C/EBP-β (NF-IL-6). Based on these observations, in this study we constructed an E1B 55kD-deleted oncolytic adenovirus driven by the human IL-8 promoter (-1481~+44), designated Ad.WSW, and tested its oncolytic activity for the treatment of NSCLC. Ad.WSW caused cytolytic effects in murine and human lung cancer cells with high IL-8 promoter activity, which could be further enhanced by TNF-α treatment. Ad.WSW also selectively replicated in murine and human lung cancer cell lines but not in normal cells. Moreover, we used A549 and Lewis lung carcinoma cells stably expressing firefly luciferase (A549-luc and LL-luc) to establish orthotopic lung adenocarcinoma models in NOD/SCID and C57BL/6 mice, respectively, which were useful for monitoring intrapleural lung tumors by in vivo bioluminescence imaging. Using in vivo non-invasive imaging, we found Ad.WSW alone reduced tumor burdens and prolonged survival in the orthotopic A549 lung tumor model. We concluded that IL-8 promoter-driven oncolytic virus has the potential to be an effective strategy for lung cancer treatment.
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Dorer, Dominik [Verfasser]. "Developing novel strategies for oncolytic adenovirus therapy by host cell gene expression profiling and arming with therapeutic antibodies / presented by Dominik Dorer." 2011. http://d-nb.info/1011486555/34.
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Lin, Chia-yi, and 林佳儀. "The use of oncolytic adenovirus carrying hypoxia-inducible OCT3/4 response element to improve the efficiency and selectivity for the treatment of bladder cancer." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/97800569626282183443.
Full textAbstract:
碩士國立成功大學
生物化學研究所
96
The POU homeodomain protein octamer binding protein 4 (Oct-4), also known as Oct3/4, Oct-3, and POU5F1) is an embryonic transcription factor expressed in germ and embryonic stem cells, but not in normal somatic tissues. It has been shown recently that the expression of Oct-4 is higher in bladder cancer than in normal genitourinary tissues, and that Oct-4 may be exploited as a therapeutic target for bladder cancer therapy. It has also been demonstrated that hypoxia-inducible factor -2α, but not HIF-1α, binds to the Oct-4 promoter and induces Oct-4 expression under hypoxic conditions. Our previous study has shown that 9 copies of Oct-4 response elements (ORE)-driven oncolytic adenovirus selectively killed Oct-4-overexpressing MBT-2 bladder cancer, but not normal cells. In this study, we generated a novel oncolytic adenovirus, designated Ad.LCY, under the control of 9 copies of ORE combined with 6 copies of hypoxia response element (HRE) and studied its antitumor activity. First, we showed that the protein levels of HIF-1α, HIF-2α and Oct-4 were upregulated in both murine MBT-2 and human TCCSUP bladder cancer cells under hypoxic conditions. In a reporter assay, we also found that the 9xORE-HRE promoter exerted higher hypoxia-inducible transcriptional activity than ORE or HRE alone in bladder cancer. The cytolytic effect of Ad.LCY on bladder cancer was more evident under hypoxic than under normoxic conditions. Furthermore, Ad.LCY had antitumor effects on tumor growth and survival in NOD/SCID and C3H/HeN mice bearing TCCSUP and MBT-2 tumors, respectively. CD133-positive cells were detected in TCCSUP bladder cancer cells and clinical tumor tissues, but not in the normal bladder tissues by immunohistochemical staining, suggesting that cancer stem cells exist in bladder cancer. Taken together, our results suggest that Ad.LCY, a 9xORE-6xHRE-driven oncolytic adenovirus, may have therapeutic potential for the treatment of bladder cancer.