TY - JOUR A1 - Patil, Sandeep S. A1 - Gentschev, Ivaylo A1 - Adelfinger, Marion A1 - Donat, Ulrike A1 - Hess, Michael A1 - Weibel, Stephanie A1 - Nolte, Ingo A1 - Frentzen, Alexa A1 - Szalay, Aladar A. T1 - Virotherapy of Canine Tumors with Oncolytic Vaccinia Virus GLV-1h109 Expressing an Anti-VEGF Single-Chain Antibody JF - PLoS One N2 - Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for cancer therapy. We have previously reported that oncolytic vaccinia virus strains expressing an anti-VEGF (Vascular Endothelial Growth Factor) single-chain antibody (scAb) GLAF-1 exhibited significant therapeutic efficacy for treatment of human tumor xenografts. Here, we describe the use of oncolytic vaccinia virus GLV-1h109 encoding GLAF-1 for canine cancer therapy. In this study we analyzed the virus-mediated delivery and production of scAb GLAF-1 and the oncolytic and immunological effects of the GLV-1h109 vaccinia virus strain against canine soft tissue sarcoma and canine prostate carcinoma in xenograft models. Cell culture data demonstrated that the GLV-1h109 virus efficiently infect, replicate in and destroy both tested canine cancer cell lines. In addition, successful expression of GLAF-1 was demonstrated in virus-infected canine cancer cells and the antibody specifically recognized canine VEGF. In two different xenograft models, the systemic administration of the GLV-1h109 virus was found to be safe and led to anti-tumor and immunological effects resulting in the significant reduction of tumor growth in comparison to untreated control mice. Furthermore, tumor-specific virus infection led to a continued production of functional scAb GLAF-1, resulting in inhibition of angiogenesis. Overall, the GLV-1h109-mediated cancer therapy and production of immunotherapeutic anti-VEGF scAb may open the way for combination therapy concept i.e. vaccinia virus mediated oncolysis and intratumoral production of therapeutic drugs in canine cancer patients. KW - angiogenesis KW - microenvironment KW - model KW - cancer KW - therapy KW - pet dogs KW - nude-mice KW - breast-tumors KW - microvascular density KW - endothelial growth-factor Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130039 VL - 7 IS - 10 ER - TY - JOUR A1 - Schäfer, Simon A1 - Weibel, Stephanie A1 - Donat, Ulrike A1 - Zhang, Quian A1 - Aguilar, Richard J. A1 - Chen, Nanhai G. A1 - Szalay, Aladar A. T1 - Vaccinia virus-mediated intra-tumoral expression of matrix metalloproteinase 9 enhances oncolysis of PC-3 xenograft tumors JF - BMC Cancer N2 - Background Oncolytic viruses, including vaccinia virus (VACV), are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, we enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9)-mediated degradation of proteins of the tumoral extracellular matrix (ECM), leading to increased viral distribution within the tumors. Methods For this study, the oncolytic vaccinia virus GLV-1h255, containing the mmp-9 gene, was constructed and used to treat PC-3 tumor-bearing mice, achieving an intra-tumoral over-expression of MMP-9. The intra-tumoral MMP-9 content was quantified by immunohistochemistry in tumor sections. Therapeutic efficacy of GLV-1h255 was evaluated by monitoring tumor growth kinetics and intra-tumoral virus titers. Microenvironmental changes mediated by the intra-tumoral MMP-9 over-expression were investigated by microscopic quantification of the collagen IV content, the blood vessel density (BVD) and the analysis of lymph node metastasis formation. Results GLV-1h255-treatment of PC-3 tumors led to a significant over-expression of intra-tumoral MMP-9, accompanied by a marked decrease in collagen IV content in infected tumor areas, when compared to GLV-1h68-infected tumor areas. This led to considerably elevated virus titers in GLV-1h255 infected tumors, and to enhanced tumor regression. The analysis of the BVD, as well as the lumbar and renal lymph node volumes, revealed lower BVD and significantly smaller lymph nodes in both GLV-1h68- and GLV-1h255- injected mice compared to those injected with PBS, indicating that MMP-9 over-expression does not alter the metastasis-reducing effect of oncolytic VACV. Conclusions Taken together, these results indicate that a GLV-1h255-mediated intra-tumoral over-expression of MMP-9 leads to a degradation of collagen IV, facilitating intra-tumoral viral dissemination, and resulting in accelerated tumor regression. We propose that approaches which enhance the oncolytic effect by increasing the intra-tumoral viral load, may be an effective way to improve therapeutic outcome. KW - microenvironment KW - angiogenesis KW - therapy KW - cancer KW - breast-tumors KW - matrix metalloproteinases KW - adenovirus KW - carcinoma KW - prostate KW - mice Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-140800 VL - 12 IS - 366 ER - TY - JOUR A1 - Schäfer, Simon A1 - Weibel, Stephanie A1 - Donat, Ulrike A1 - Zhang, Qian A1 - Aguilar, Richard J. A1 - Chen, Nanhai G. A1 - Szalay, Aladar A. T1 - Vaccinia virus-mediated intra-tumoral expression of matrix metalloproteinase 9 enhances oncolysis of PC-3 xenograft tumors N2 - Background: Oncolytic viruses, including vaccinia virus (VACV), are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, we enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9)-mediated degradation of proteins of the tumoral extracellular matrix (ECM), leading to increased viral distribution within the tumors. Methods: For this study, the oncolytic vaccinia virus GLV-1h255, containing the mmp-9 gene, was constructed and used to treat PC-3 tumor-bearing mice, achieving an intra-tumoral over-expression of MMP-9. The intra-tumoral MMP-9 content was quantified by immunohistochemistry in tumor sections. Therapeutic efficacy of GLV-1h255 was evaluated by monitoring tumor growth kinetics and intra-tumoral virus titers. Microenvironmental changes mediated by the intra-tumoral MMP-9 over-expression were investigated by microscopic quantification of the collagen IV content, the blood vessel density (BVD) and the analysis of lymph node metastasis formation. Results: GLV-1h255-treatment of PC-3 tumors led to a significant over-expression of intra-tumoral MMP-9, accompanied by a marked decrease in collagen IV content in infected tumor areas, when compared to GLV-1h68-infected tumor areas. This led to considerably elevated virus titers in GLV-1h255 infected tumors, and to enhanced tumor regression. The analysis of the BVD, as well as the lumbar and renal lymph node volumes, revealed lower BVD and significantly smaller lymph nodes in both GLV-1h68- and GLV-1h255- injected mice compared to those injected with PBS, indicating that MMP-9 over-expression does not alter the metastasis-reducing effect of oncolytic VACV. Conclusions: Taken together, these results indicate that a GLV-1h255-mediated intra-tumoral over-expression of MMP-9 leads to a degradation of collagen IV, facilitating intra-tumoral viral dissemination, and resulting in accelerated tumor regression. We propose that approaches which enhance the oncolytic effect by increasing the intra-tumoral viral load, may be an effective way to improve therapeutic outcome. KW - Biochemie Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-78220 ER - TY - JOUR A1 - Spivey, Tara L. A1 - De Giorgi, Valeria A1 - Zhao, Yingdong A1 - Bedognetti, Davide A1 - Pos, Zoltan A1 - Liu, Qiuzhen A1 - Tomei, Sara A1 - Ascierto, Maria Libera A1 - Uccellini, Lorenzo A1 - Reinboth, Jennifer A1 - Chouchane, Lotfi A1 - Stroncek, David F. A1 - Wang, Ena A1 - Marincola, Francesco M. T1 - The stable traits of melanoma genetics: an alternate approach to target discovery JF - BMC Genomics N2 - Background: The weight that gene copy number plays in transcription remains controversial; although in specific cases gene expression correlates with copy number, the relationship cannot be inferred at the global level. We hypothesized that genes steadily expressed by 15 melanoma cell lines (CMs) and their parental tissues (TMs) should be critical for oncogenesis and their expression most frequently influenced by their respective copy number. Results: Functional interpretation of 3,030 transcripts concordantly expressed (Pearson's correlation coefficient p-value < 0.05) by CMs and TMs confirmed an enrichment of functions crucial to oncogenesis. Among them, 968 were expressed according to the transcriptional efficiency predicted by copy number analysis (Pearson's correlation coefficient p-value < 0.05). We named these genes, "genomic delegates" as they represent at the transcriptional level the genetic footprint of individual cancers. We then tested whether the genes could categorize 112 melanoma metastases. Two divergent phenotypes were observed: one with prevalent expression of cancer testis antigens, enhanced cyclin activity, WNT signaling, and a Th17 immune phenotype (Class A). This phenotype expressed, therefore, transcripts previously associated to more aggressive cancer. The second class (B) prevalently expressed genes associated with melanoma signaling including MITF, melanoma differentiation antigens, and displayed a Th1 immune phenotype associated with better prognosis and likelihood to respond to immunotherapy. An intermediate third class (C) was further identified. The three phenotypes were confirmed by unsupervised principal component analysis. Conclusions: This study suggests that clinically relevant phenotypes of melanoma can be retraced to stable oncogenic properties of cancer cells linked to their genetic back bone, and offers a roadmap for uncovering novel targets for tailored anti-cancer therapy. KW - tumors KW - comparative genomic hybridization KW - coloteral cancer KW - prognostic relevance KW - aquired resistance KW - malignant melanoma KW - antigen expression KW - tissue microarray KW - cell carcinoma KW - T cells Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131992 VL - 13 IS - 156 ER - TY - JOUR A1 - Krehan, Mario A1 - Heubeck, Christian A1 - Menzel, Nicolas A1 - Seibel, Peter A1 - Schön, Astrid T1 - RNase MRP RNA and RNase P activity in plants are associated with a Pop1p containing complex JF - Nucleic Acids Research N2 - RNase P processes the 5'-end of tRNAs. An essential catalytic RNA has been demonstrated in Bacteria, Archaea and the nuclei of most eukaryotes; an organism-specific number of proteins complement the holoenzyme. Nuclear RNase P from yeast and humans is well understood and contains an RNA, similar to the sister enzyme RNase MRP. In contrast, no protein subunits have yet been identified in the plant enzymes, and the presence of a nucleic acid in RNase P is still enigmatic. We have thus set out to identify and characterize the subunits of these enzymes in two plant model systems. Expression of the two known Arabidopsis MRP RNA genes in vivo was verified. The first wheat MRP RNA sequences are presented, leading to improved structure models for plant MRP RNAs. A novel mRNA encoding the central RNase P/MRP protein Pop1p was identified in Arabidopsis, suggesting the expression of distinct protein variants from this gene in vivo. Pop1p-specific antibodies precipitate RNase P activity and MRP RNAs from wheat extracts. Our results provide evidence that in plants, Pop1p is associated with MRP RNAs and with the catalytic subunit of RNase P, either separately or in a single large complex. KW - enzyme KW - binding KW - sequence KW - cyanelle KW - in vitro KW - partial purification KW - protein subunit KW - ribonuclease-P KW - genes KW - identification Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130648 VL - 40 IS - 16 ER - TY - JOUR A1 - Gentschev, Ivaylo A1 - Adelfinger, Marion A1 - Josupeit, Rafael A1 - Rudolph, Stephan A1 - Ehrig, Klaas A1 - Donat, Ulrike A1 - Weibel, Stephanie A1 - Chen, Nanhai G. A1 - Yu, Yong A. A1 - Zhang, Qian A1 - Heisig, Martin A1 - Thamm, Douglas A1 - Stritzker, Jochen A1 - MacNeill, Amy A1 - Szalay, Aladar A. T1 - Preclinical Evaluation of Oncolytic Vaccinia Virus for Therapy of Canine Soft Tissue Sarcoma JF - PLoS One N2 - Virotherapy using oncolytic vaccinia virus (VACV) strains is one promising new strategy for canine cancer therapy. In this study we describe the establishment of an in vivo model of canine soft tissue sarcoma (CSTS) using the new isolated cell line STSA-1 and the analysis of the virus-mediated oncolytic and immunological effects of two different Lister VACV LIVP1.1.1 and GLV-1h68 strains against CSTS. Cell culture data demonstrated that both tested VACV strains efficiently infected and destroyed cells of the canine soft tissue sarcoma line STSA-1. In addition, in our new canine sarcoma tumor xenograft mouse model, systemic administration of LIVP1.1.1 or GLV-1h68 viruses led to significant inhibition of tumor growth compared to control mice. Furthermore, LIVP1.1.1 mediated therapy resulted in almost complete tumor regression and resulted in long-term survival of sarcoma-bearing mice. The replication of the tested VACV strains in tumor tissues led to strong oncolytic effects accompanied by an intense intratumoral infiltration of host immune cells, mainly neutrophils. These findings suggest that the direct viral oncolysis of tumor cells and the virus-dependent activation of tumor-associated host immune cells could be crucial parts of anti-tumor mechanism in STSA-1 xenografts. In summary, the data showed that both tested vaccinia virus strains and especially LIVP1.1.1 have great potential for effective treatment of CSTS. KW - breast-tumors KW - animal-model KW - nude-mice KW - cell-line KW - in-vitro KW - glv-1h68 KW - cancer KW - virotherapy KW - dogs KW - neutrophils Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-129998 VL - 7 IS - 5 ER - TY - JOUR A1 - Wang, Huiqiang A1 - Chen, Nanhai G. A1 - Minev, Boris R. A1 - Szalay, Aladar A. T1 - Oncolytic vaccinia virus GLV-1h68 strain shows enhanced replication in human breast cancer stem-like cells in comparison to breast cancer cells JF - Journal of Translational Medicine N2 - Background: Recent data suggest that cancer stem cells (CSCs) play an important role in cancer, as these cells possess enhanced tumor-forming capabilities and are responsible for relapses after apparently curative therapies have been undertaken. Hence, novel cancer therapies will be needed to test for both tumor regression and CSC targeting. The use of oncolytic vaccinia virus (VACV) represents an attractive anti-tumor approach and is currently under evaluation in clinical trials. The purpose of this study was to demonstrate whether VACV does kill CSCs that are resistant to irradiation and chemotherapy. Methods: Cancer stem-like cells were identified and separated from the human breast cancer cell line GI-101A by virtue of increased aldehyde dehydrogenase 1 (ALDH1) activity as assessed by the ALDEFLUOR assay and cancer stem cell-like features such as chemo-resistance, irradiation-resistance and tumor-initiating were confirmed in cell culture and in animal models. VACV treatments were applied to both ALDEFLUOR-positive cells in cell culture and in xenograft tumors derived from these cells. Moreover, we identified and isolated CD44\(^+\)CD24\(^+\)ESA\(^+\) cells from GI-101A upon an epithelial-mesenchymal transition (EMT). These cells were similarly characterized both in cell culture and in animal models. Results: We demonstrated for the first time that the oncolytic VACV GLV-1h68 strain replicated more efficiently in cells with higher ALDH1 activity that possessed stem cell-like features than in cells with lower ALDH1 activity. GLV-1h68 selectively colonized and eventually eradicated xenograft tumors originating from cells with higher ALDH1 activity. Furthermore, GLV-1h68 also showed preferential replication in CD44\(^+\)CD24\(^+\)ESA\(^+\) cells derived from GI-101A upon an EMT induction as well as in xenograft tumors originating from these cells that were more tumorigenic than CD44\(^+\)CD24\(^-\)ESA\(^+\) cells. Conclusions: Taken together, our findings indicate that GLV-1h68 efficiently replicates and kills cancer stem-like cells. Thus, GLV-1h68 may become a promising agent for eradicating both primary and metastatic tumors, especially tumors harboring cancer stem-like cells that are resistant to chemo and/or radiotherapy and may be responsible for recurrence of tumors. KW - tumors KW - therapy KW - metastasis KW - identification KW - lines KW - gene expression KW - in-vitro propagation KW - acute myeloid leukemia KW - epithelial-mesenchymal transition KW - subpopulation Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130019 VL - 10 IS - 167 ER - TY - JOUR A1 - Haddad, Dana A1 - Chen, Chun-Hao A1 - Carlin, Sean A1 - Silberhumer, Gerd A1 - Chen, Nanhai G. A1 - Zhang, Qian A1 - Longo, Valerie A1 - Carpenter, Susanne G. A1 - Mittra, Arjun A1 - Carson, Joshua A1 - Au, Joyce A1 - Gonen, Mithat A1 - Zanzonico, Pat B. A1 - Szalay, Aladar A. A1 - Fong, Yuman T1 - Imaging Characteristics, Tissue Distribution, and Spread of a Novel Oncolytic Vaccinia Virus Carrying the Human Sodium Iodide Symporter JF - PLoS One N2 - Introduction: Oncolytic viruses show promise for treating cancer. However, to assess therapy and potential toxicity, a noninvasive imaging modality is needed. This study aims to determine the in vivo biodistribution, and imaging and timing characteristics of a vaccinia virus, GLV-1h153, encoding the human sodium iodide symporter (hNIS. Methods: GLV-1h153 was modified from GLV-1h68 to encode the hNIS gene. Timing of cellular uptake of radioiodide \(^{131}\)I in human pancreatic carcinoma cells PANC-1 was assessed using radiouptake assays. Viral biodistribution was determined in nude mice bearing PANC-1 xenografts, and infection in tumors confirmed histologically and optically via Green Fluorescent Protein (GFP) and bioluminescence. Timing characteristics of enhanced radiouptake in xenografts were assessed via \(^{124}\)I-positron emission tomography (PET). Detection of systemic administration of virus was investigated with both \(^{124}\)I-PET and 99m-technecium gamma-scintigraphy. Results: GLV-1h153 successfully facilitated time-dependent intracellular uptake of \(^{131}\)I in PANC-1 cells with a maximum uptake at 24 hours postinfection (P < 0.05). In vivo, biodistribution profiles revealed persistence of virus in tumors 5 weeks postinjection at 10\(^9\) plaque-forming unit (PFU)/gm tissue, with the virus mainly cleared from all other major organs. Tumor infection by GLV-1h153 was confirmed via optical imaging and histology. GLV-1h153 facilitated imaging virus replication in tumors via PET even at 8 hours post radiotracer injection, with a mean % ID/gm of 3.82 \(\pm\) 60.46 (P < 0.05) 2 days after intratumoral administration of virus, confirmed via tissue radiouptake assays. One week post systemic administration, GLV1h153-infected tumors were detected via \(^{124}\)I-PET and 99m-technecium-scintigraphy. Conclusion: GLV-1h153 is a promising oncolytic agent against pancreatic cancer with a promising biosafety profile. GLV-1h153 facilitated time-dependent hNIS-specific radiouptake in pancreatic cancer cells, facilitating detection by PET with both intratumoral and systemic administration. Therefore, GLV-1h153 is a promising candidate for the noninvasive imaging of virotherapy and warrants further study into longterm monitoring of virotherapy and potential radiocombination therapies with this treatment and imaging modality. KW - nude mice KW - pancreatic cancer KW - engineered measles-virus KW - positron-emission-tomography KW - malignant pleural mesothelioma KW - reporter gene KW - replicating adenovirus KW - NA/I symporter KW - breast cancer KW - viral therapy Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130041 VL - 7 IS - 8 ER - TY - THES A1 - Dill, Holger T1 - Functional characterization of the microRNA-26 family in zebrafish neurogenesis T1 - Funktionelle Charakterisierung der microRNA-26 Familie während der Zebrafisch Neurogenese N2 - Formation oft the central nervous system (CNS) from multipotent neuronal stem cells (NSCs) requires a tightly controlled, step-wise activation of the neuronal gene expression program. Expression of neuronal genes at the transition from neural stem cell to mature neuron (i. e. neuronal cell differentiation) is controlled by the Repressor element 1 (RE1) silencing transcription factor (REST) complex. As a master transcriptional regulator, the REST-complex specifically inhibits expression of neuronal genes in non-neuronal tissues and neuronal progenitor cells. Differentiation of NSCs to mature neurons requires the activation of genes controlled by the REST-complex, but how abrogation of REST-complex mediated repression is achieved during neurogenesis is only poorly understood. MicroRNAs (miRNAs) are a class of small regulatory RNAs that posttranscriptionally control target gene expression. Binding of miRNAs to target sequences in the 3’UTR of mRNAs, leads either to degradation or translational inhibition of the mRNA. Distinct neuronal miRNAs (e.g. miR-124) were shown to modulate REST-complex activity by silencing expression of REST-complex components. Interestingly, these miRNAs are also under transcriptional control of the REST-complex and inactivation of the REST-complex precedes their expression. Hence, additional factors are required for derepression of neuronal genes at the onset of neurogenesis. In this study function of the miR-26 family during neurogenesis of the zebrafish (Danio rerio) was analyzed. Computational target prediction revealed a number of REST-complex components as putative miR-26 targets. One of these predicted target genes, the C-terminal domain small phosphatase 2 (Ctdsp2) was validated as an in vivo target for miR-26b. Ctdsps are important cofactors of REST and suppress neuronal gene expression by dephosphorylating the C-terminal domain (CTD) of RNA polymerase II (Pol II). Interestingly, miR-26b is encoded in an intron of the ctdsp2 primary transcript and is cotranscribed together with its host gene. Hence, miR-26b modulates expression of its host gene ctdsp2 in an intrinsic negative autoregulatory loop. This negative autoregulatory loop is inactive in NSCs because miR-26b biogenesis is inhibited at the precursor level. Generation of mature miR-26b is activated during neurogenesis, where it suppresses Ctdsp2 protein expression and is required for neuronal cell differentiation in vivo. Strikingly, miR-26b is expressed prior to miR-124 during neuronal cell differentiation. Thus, it is reasonable to speculate about a function of miR-26b in early events of neurogenesis. In line with this assumption, knockdown of miR-26b in zebrafish embryos results in downregulation of REST-complex controlled neuronal genes and a block in neuronal cell differentiation, most likely due to aberrant regulation of Ctdsp2 expression. This is evident by reduced numbers of secondary motor neurons compared to control siblings. In contrast, motor neuron progenitor cells and glia cells were not affected by depletion of miR-26b.This study identifies the ctdsp2/miR-26b autoregulatory loop as the first experimentally validated interaction between an intronic miRNA and its host gene transcript. Silencing of ctdsp2 by miR-26b in neurons is possible because biogenesis of the ctdsp2 mRNA and mature mir-26b is uncoupled at the posttranscriptional level. Furthermore the obtained data indicate a cell type specific role for miR-26b in vertebrate neurogenesis and CNS development. N2 - Die Entwicklung des Zentralen Nervensystems (ZNS) aus multipotenten neuronalen Stammzellen erfordert eine stufenweise und genau regulierte Aktivierung der neuronalen Genexpression. Bei der Differenzierung neuronaler Stammzellen zu Neuronen wird die Expression neuronaler Gene durch den sogenannten „Repressor element 1 (RE1) silencing transcription factor (REST)”-Komplex gesteuert. Der REST-Komplex unterdrückt spezifisch in proliferierenden neuronalen Vorläuferzellen die Expression neuronaler Gene. Während der neuronalen Zelldifferenzierung wird die Expression dieser Gene jedoch benötigt. Wie die Inaktivierung neuronaler Gene durch den REST-Komplex während des Prozesses der Neurogenese aufgehoben wird ist bislang nicht genau bekannt. MicroRNAs (miRNAs) sind kleine regulatorische RNAs, die die Expression ihrer Zielgene auf posttranskriptioneller Ebene regulieren. Dazu binden miRNAs an Zielsequenzen in 3’UTRs von mRNAs, was zu einer Inhibition der Translation oder Abbau der mRNA führt. Auch Komponenten des REST-Komplexes stehen unter Kontrolle bestimmter neuronaler miRNAs (z.B. miR-124). Erstaunlicherweise stehen diese miRNAs selber wiederum unter der transkriptionellen Inhibition des REST-Komplexes und können daher nicht für die Inaktivierung des REST-Komplexes zu Beginn der Neurogenese verantwortlich sein. Übereinstimmend damit konnte beobachtet werden, dass der REST-Komplex aus differenzierenden Zellen entfernt wird, bevor die genannten neuronalen miRNAs exprimiert werden. Diese Umstände legen die Existenz weiterer, bis jetzt unbekannter Faktoren nahe, die die Expression des REST-Komplexes selber inhibieren und so die Neurogenese erlauben Im Rahmen dieser Dissertation wurde die Funktion der miR-26 Familie während der Neurogenese des Zebrafisches (Danio rerio) untersucht. Eine bioinformatische Zielgenvorhersage für die miR-26 Familie ergab, dass unter anderem zahlreiche bekannte Komponenten des REST-Komplexes unter den Kandidatengenen sind. Für eines dieser vorhergesagten Zielgene, die sogenannte „C-terminal domain small phosphatase 2 (Ctdsp2)” wurde daraufhin gezeigt, dass seine Expression in der Tat durch die miR-26b inhibiert wird. Ctdsps sind wichtige Kofaktoren des REST-Komplexes und unterdrücken die Expression neuronaler Gene, indem die die C-terminale Domäne (CTD) der RNA Polymerase II dephosphorylieren und diese dadurch inaktivieren. In diesem Zusammenhang von besonderer Bedeutung ist die Tatsache, dass die miR-26b in einem Intron des ctdsp2 Gens kodiert ist und mit ctdsp2 zusammen transkribiert wird. Folglich beeinflusst die miR-26b die Expression ihres eigenen „Host genes“ in einer Art autoregulativer Rückkopplungsschleife. Die beschriebene negative Regulation ist in neuronalen Stammzellen nicht aktiv, da dort die Biogenese der miR-26b auf Vorläuferebene angehalten wird. Reife miR-26b wird erst während der Neurogenese produziert, wo sie daraufhin die Expression von Ctdsp2 Protein verhindert. Während der neuronalen Zelldifferenzierung wird die miR-26b deutlich früher exprimiert als zum Beispiel die miR-124. Daher liegt es nahe eine Funktion der miR-26b während früher Prozesse in der Neurogenese anzunehmen. In Übereinstimmung mit dieser Annahme führt ein „Knockdown“ der miR-26b zu einer schwächeren Expression von neuronalen Genen, die unter der Kontrolle des REST-Komplex stehen. Weiterhin führt ein reduziertes Maß an miR-26b zu fehlerhafter oder gänzlich ausbleibender neuronaler Zelldifferenzierung. Dies konnte anhand einer verringerten Anzahl differenzierter spinaler Motorneuronen aufgezeigt werden. Die Vorläufer dieser Motorneuronen und Gliazellen waren hingegen vom miR-26b-„Knockdown“ nicht beeinflusst. Die hier präsentierte Studie zeigt erstmals in experimenteller Weise das Vorhandensein einer direkten Interaktion zwischen einer intronischen miRNA und ihrem eigenen Primärtranskript. Die negative Regulation der Ctdsp2 Expression in Neuronen wird erst dadurch möglich, dass die Biogenese der ctdsp2 mRNA und der reifen miR-26b durch einen posttranskriptionellen Mechanismus voneinander getrennt werden. Weiterhin legen die Daten aus dieser Studie nahe, dass die miR-26b in der Tat eine spezifische Funktion in der Entwicklung des ZNS von Vertebraten hat. KW - Zebrabärbling KW - Neurogenese KW - miRNS KW - Zelldifferenzierung KW - miR-26b KW - ctdsp2 KW - REST Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-70757 ER - TY - THES A1 - Buckel, Lisa T1 - Evaluating the combination of oncolytic vaccinia virus and ionizing radiation in therapy of preclinical glioma models T1 - Evaluierung der Kombination von onkolytischem Vaccinia Virus und ionisierender Strahlung in vorklinischen Gliomamodellen N2 - Glioblastoma multiforme (GBM) represents the most aggressive form of malignant brain tumors and remains a therapeutically challenge. Intense research in the field has lead to the testing of oncolytic viruses to improve tumor control. Currently, a variety of different oncolytic viruses are being evaluated for their ability to be used in anti-cancer therapy and a few have entered clinical trials. Vaccinia virus, is one of the viruses being studied. GLV-1h68, an oncolytic vaccinia virus engineered by Genelux Corporation, was constructed by insertion of three gene cassettes, RUC-GFP fusion, β-galactosidase and β- glucuronidase into the genome of the LIVP strain. Since focal tumor radiotherapy is a mainstay for cancer treatment, including glioma therapy, it is of clinical relevance to assess how systemically administered oncolytic vaccinia virus could be combined with targeted ionizing radiation for therapeutic gain. In this work we show how focal ionizing radiation (IR) can be combined with multiple systemically delivered oncolytic vaccinia virus strains in murine models of human U-87 glioma. After initial experiments which confirmed that ionizing radiation does not damage viral DNA or alter viral tropism, animal studies were carried out to analyze the interaction of vaccinia virus and ionizing radiation in the in vivo setting. We found that irradiation of the tumor target, prior to systemic administration of oncolytic vaccinia virus GLV-1h68, increased viral replication within the U-87 xenografts as measured by viral reporter gene expression and viral titers. Importantly, while GLV-1h68 alone had minimal effect on U-87 tumor growth delay, IR enhanced GLV-1h68 replication, which translated to increased tumor growth delay and mouse survival in subcutaneous and orthotopic U-87 glioma murine models compared to monotherapy with IR or GLV-1h68. The ability of IR to enhance vaccinia replication was not restricted to the multi-mutated GLV-1h68, but was also seen with the less attenuated oncolytic vaccinia, LIVP 1.1.1. We have demonstrated that in animals treated with combination of ionizing radiation and LIVP 1.1.1 a strong pro-inflammatory tissue response was induced. When IR was given in a more clinically relevant fractionated scheme, we found oncolytic vaccinia virus replication also increased. This indicates that vaccinia virus could be incorporated into either larger hypo-fraction or more conventionally fractionated radiotherapy schemes. The ability of focal IR to mediate selective replication of systemically injected oncolytic vaccinia was demonstrated in a bilateral glioma model. In mice with bilateral U-87 tumors in both hindlimbs, systemically administered oncolytic vaccinia replicated preferentially in the focally irradiated tumor compared to the shielded non- irradiated tumor in the same mouse We demonstrated that tumor control could be further improved when fractionated focal ionizing radiation was combined with a vaccinia virus caring an anti-angiogenic payload targeting vascular endothelial growth factor (VEGF). Our studies showed that following ionizing radiation expression of VEGF is upregulated in U-87 glioma cells in culture. We further showed a concentration dependent increase in radioresistance of human endothelial cells in presence of VEGF. Interestingly, we found effects of vascular endothelial growth factor on endothelial cells were reversible by adding purified GLAF-1 to the cells. GLAF-1 is a single- chain antibody targeting human and murine VEGF and is expressed by oncolytic vaccinia virus GLV-109. In U-87 glioma xenograft murine models the combination of fractionated ionizing radiation with GLV-1h164, a vaccinia virus also targeting VEGF, resulted in the best volumetric tumor response and a drastic decrease in vascular endothelial growth factor. Histological analysis of embedded tumor sections 14 days after viral administration confirmed that blocking VEGF translated into a decrease in vessel number to 30% of vessel number found in control tumors in animals treated with GLV-164 and fractionated IR which was lower than for all other treatment groups. Our experiments with GLV-1h164 and fractionated radiotherapy have shown that in addition to ionizing radiation and viral induced tumor cell destruction we were able to effectively target the tumor vasculature. This was achieved by enhanced viral replication translating in increased levels of GLAF-2 disrupting tumor vessels as well as the radiosensitization of tumor vasculature to IR by blocking VEGF. Our preclinical results have important clinical implications of how focal radiotherapy can be combined with systemic oncolytic viral administration for highly aggressive, locally advanced tumors with the potential, by using a vaccinia virus targeting human vascular endothelial growth factor, to further increase tumor radiation sensitivity by engaging the vascular component in addition to cancer cells. N2 - Glioblastoma multiforme (GBM) verkörpert die aggressivste Form von bösartigen Gehirntumoren und seine Therapie gestaltet sich schwierig. Weitläufige Forschung hat dazu geführt, dass onkolytische Viren zur Verbesserung der Tumorbehandlung untersucht wurden. Gegenwärtig wird eine Vielzahl an verschiedenen onkolytischen Viren untersucht und einige wenige befinden sich bereits in klinischen Studien. Eines der Viren die untersucht werden, ist das Vaccinia-Virus. GLV-1h68, ein onkolytisches Vaccinia- Virus, wurde durch die Einfügung von drei Genkasseten, RUC-GFP Fusion, β- Galaktosidase und β- Glucuronidase in das Genom des LIVP Stammes hergestellt. Da fokale Bestrahlungstherapie aus der Behandlung von Krebs, nicht nur im Falle von Glioblastomen, nicht wegzudenken ist, ist es klinisch relevant, zu untersuchen, wie ein systemisch verabreichtes Vaccinia-Virus mit gezielter ionisierender Strahlung (IR) kombiniert werden könnte, um Therapiechancen zu verbesseren. In dieser Arbeit konnte gezeigt werden, wie gezielte IR mit verschiedenen sytemisch injizierten Vaccinia-Virus Stämmen in einem Mausmodell für humane U-87-Glioma kombiniert wurde. Nachdem einleitende Versuche bestätigten, dass IR die virale Erbinformation nicht beschädigt und auch nicht den viralen Tropismus verändert, wurden Tierstudien durchgeführt, die die Interaktion des Vaccinia-Virus mit Bestrahlungtherapie in vivo untersuchten. Wir konnten zeigen, dass eine vorherige Bestrahlung des Tumors, bevor das GLV-1h68-Virus systemisch injiziert wurde, eine erhöhte viraler Replikation im Tumor zur Folge hatte, wie wir durch gesteigerte virale Titer und Markergenexpression belegen konnten. Von wesentlicher Bedeutung ist, dass eine Verabreichung von ausschliesslich GLV-1h68 einen minimalen Einfluss auf das U-87 Tumorwachstum hatte, während die durch die Bestrahlung ausgelöste erhöhte Vermehrung von Virus im Tumor eine Verzögerung des Tumorwachstums sowie ein verlängertes Überleben von Mäusen mit U-87-Xenografts zur Folge hatte. Die Fähigkeit von IR virale Vermehrung zu erhöhen, wurde auch für das weniger attenuierte LIVP 1.1.1-Virus gezeigt. Wenn die Bestrahlung in einem klinisch relevanten fraktionierten Bestrahlungsschema verabreicht wurde, war virale Replikation ebenfalls erhöht. Dies verdeutlicht, dass das Vaccinia-Virus klinisch entweder in eine Bestrahlung mit einer einzelnen Dosis oder in eine konventionelle fraktionierte Bestrahlung integriert werden kann. Die Fähigkeit von fokaler IR, eine selektive Vermehrung von systemisch injizierten onkolytischen Vaccinia-Viren zu ermöglichen, wurde in einem bilateralen Gliomamausmodell bestätigt. In Mausen mit Tumoren an beiden Hinterbeinen, vermehrte sich das systemisch gespritzte Vaccinia-Virus bevorzugt im bestrahlten Tumor. Wir konnten zeigen, wie die Tumorkontrolle darüber hinaus weiter verbessert werden kann, wenn fraktionierte fokale Bestrahlung mit einem Vaccinia-Virus kombiniert wird, das eine anti-angiogenetische Ladung, die den vaskulaeren endothelialen Wachstumsfaktor (VEGF) inhibiert, exprimiert. Unsere Studien konnten zeigen, dass durch die Bestrahlung von U-87 Gliomazellen eine Hochregulation von VEGF-Expression ausgelöst wurde, die Radioresistenz von Endothelzellen konzentrationsabhängig induzierte. Wir konnten zeigen, dass die durch VEGF verursache Radioresistenz umkehrbar ist, wenn zusätzlich aufgereinigtes GLAF-1, einen Vaccinia Virus exprimierten Antikörper, zu den Zellen gegeben wurde. In einem Mausmodell zeigte die Kombination aus fraktionierter Bestrahlung und GLV-1h164, ein Vaccinia-Virus, das ebenfalls einen VEGF Antikörper mit Ähnlichkeit zu GLAF-1 exprimiert, resultierte in der stärksten volumetrischen Tumorantwort. Es wurde ebenfalls eine drastische Abnahme an VEGF im Tumor bereits 3 Tagen nach Virus- Injektion nachgewiesen. Histologische Analyse bestätigte, dass die Blockade von VEGF eine Erniedrigung der Anzahl von Tumorblutgefäßen, zu 30% von Kontrolltumoren, zur Folge hatte. Dieser Wert war niedriger als in allen anderen Behandlungsgruppen. Unsere Versuche mit fraktionierter Bestrahlung und GLV-1h164 konnten zeigen, dass zusätzlich zu der durch Virus und Bestrahlung ausgelösten Tumorzellzerstörung, eine effiziente Degeneration der Tumorblutgefäße möglich war. Dies wurde durch eine erhöhte Virus-Vermehrung als Folge der Bestrahlung, sowie durch Sensitiveren der tumoralen Endothelzellen durch Blockierung von VEGF-A erreicht. Die Ergebnisse, die in dieser Arbeit zeigen, wie fokale Bestrahlungstherapie mit systemisch verabreichten onkolytische Vaccinia-Viren für aggressive, fortgeschrittene Tumore kombiniert werden kann. Es ist denkbar, dass die Tumortherapie weiter verbessert werden kann, wenn ein Vaccinia-Virus benutzt wird, das sich zusätzlich gegen VEGF richtet, so werden zu den Krebszellen zusätzlich Tumorblutgefäße in die Therapie miteinbezogen, um die Sensitivität von Endothelzellen gegen Bestrahlung weiter zu erhöhen. KW - Gliom KW - Vaccinia-Virus KW - Strahlentherapie KW - Kombinationstherapie KW - onkolytische Virotherapie KW - Glioma KW - vaccinia virus KW - ionizing radiation KW - combination therapy KW - oncolytic virotherapy Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-85309 ER -