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Adenovirus-based vectors are playing an important role as efficacious genetic vaccines to fight the current COVID-19 pandemic. Furthermore, they have an enormous potential as oncolytic vectors for virotherapy and as vectors for classic gene therapy. However, numerous vector–host interactions on a cellular and noncellular level, including specific components of the immune system, must be modulated in order to generate safe and efficacious vectors for virotherapy or classic gene therapy. Importantly, the current widespread use of Ad vectors as vaccines against COVID-19 will induce antivector immunity in many humans. This requires the development of strategies and techniques to enable Ad-based vectors to evade pre-existing immunity. In this review article, we discuss the current status of genetic and chemical capsid modifications as means to modulate the vector–host interactions of Ad-based vectors.
Denice Weklak; Daniel Pembaur; Georgia Koukou; Franziska Jönsson; Claudia Hagedorn; Florian Kreppel. Genetic and Chemical Capsid Modifications of Adenovirus Vectors to Modulate Vector–Host Interactions. Viruses 2021, 13, 1300 .
AMA StyleDenice Weklak, Daniel Pembaur, Georgia Koukou, Franziska Jönsson, Claudia Hagedorn, Florian Kreppel. Genetic and Chemical Capsid Modifications of Adenovirus Vectors to Modulate Vector–Host Interactions. Viruses. 2021; 13 (7):1300.
Chicago/Turabian StyleDenice Weklak; Daniel Pembaur; Georgia Koukou; Franziska Jönsson; Claudia Hagedorn; Florian Kreppel. 2021. "Genetic and Chemical Capsid Modifications of Adenovirus Vectors to Modulate Vector–Host Interactions." Viruses 13, no. 7: 1300.
Adenovirus-based gene transfer vectors are the most frequently used vector type in gene therapy clinical trials to date, and they play an important role as genetic vaccine candidates during the ongoing SARS-CoV-2 pandemic. Immediately upon delivery, adenovirus-based vectors exhibit multiple complex vector-host interactions and induce innate and adaptive immune responses. This can severely limit their safety and efficacy, particularly after delivery through the blood stream. In this review article we summarize two strategies to modulate Ad vector-induced immune responses: extensive genomic and chemical capsid modifications. Both strategies have shown beneficial effects in a number of preclinical studies while potential synergistic effects warrant further investigations.
Florian Kreppel; Claudia Hagedorn. Capsid and Genome Modification Strategies to Reduce the Immunogenicity of Adenoviral Vectors. International Journal of Molecular Sciences 2021, 22, 2417 .
AMA StyleFlorian Kreppel, Claudia Hagedorn. Capsid and Genome Modification Strategies to Reduce the Immunogenicity of Adenoviral Vectors. International Journal of Molecular Sciences. 2021; 22 (5):2417.
Chicago/Turabian StyleFlorian Kreppel; Claudia Hagedorn. 2021. "Capsid and Genome Modification Strategies to Reduce the Immunogenicity of Adenoviral Vectors." International Journal of Molecular Sciences 22, no. 5: 2417.
Adenovirus vectors are potent tools for genetic vaccination and oncolytic virotherapy. However, they are prone to multiple undesired vector-host interactions, especially after in vivo delivery. It is a consensus that the limitations imposed by undesired vector-host interactions can only be overcome if defined modifications of the vector surface are performed. These modifications include shielding of the particles from unwanted interactions and targeting by the introduction of new ligands. The goal of the protocol presented here is to enable the reader to generate shielded and, if desired, retargeted human adenovirus gene transfer vectors or oncolytic viruses. The protocol will enable researchers to modify the surface of adenovirus vector capsids by specific chemical attachment of synthetic polymers, carbohydrates, lipids, or other biological or chemical moieties. It describes the cutting-edge technology of combined genetic and chemical capsid modifications, which have been shown to facilitate the understanding and overcoming of barriers for in vivo delivery of adenovirus vectors. A detailed and commented description of the crucial steps for performing specific chemical reactions with biologically active viruses or virus-derived vectors is provided. The technology described in the protocol is based on the genetic introduction of (naturally absent) cysteine residues into solvent-exposed loops of adenovirus-derived vectors. These cysteine residues provide a specific chemical reactivity that can, after production of the vectors to high titers, be exploited for highly specific and efficient covalent chemical coupling of molecules from a wide variety of substance classes to the vector particles. Importantly, this protocol can easily be adapted to perform a broad variety of different (non-thiol-based) chemical modifications of adenovirus vector capsids. Finally, it is likely that non-enveloped virus-based gene transfer vectors other than adenovirus can be modified from the basis of this protocol.
Franziska Jönsson; Claudia Hagedorn; Florian Kreppel. Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting. Journal of Visualized Experiments 2018, e58480 -e58480.
AMA StyleFranziska Jönsson, Claudia Hagedorn, Florian Kreppel. Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting. Journal of Visualized Experiments. 2018; (140):e58480-e58480.
Chicago/Turabian StyleFranziska Jönsson; Claudia Hagedorn; Florian Kreppel. 2018. "Combined Genetic and Chemical Capsid Modifications of Adenovirus-Based Gene Transfer Vectors for Shielding and Targeting." Journal of Visualized Experiments , no. 140: e58480-e58480.
Adenovirus-based vectors comprise the most frequently used vector type in clinical studies to date. Both intense lab research and insights from the clinical trials reveal the importance of a comprehensive understanding of vector–host interactions. Especially for systemic intravenous adenovirus vector delivery, it is paramount to develop safe and efficacious vectors. Very early vector–host interactions that take place in blood long before the first cell is being transduced are phenomena triggered by the surface, shape, and size of the adenovirus vector particles. Not surprisingly, a multitude of different technologies ranging from genetics to chemistry has been developed to alter the adenovirus vector surface. In this review, we discuss the most important technologies and evaluate them for their suitability to overcome hurdles imposed by early vector–host interactions.
Claudia Hagedorn; Florian Kreppel. Capsid Engineering of Adenovirus Vectors: Overcoming Early Vector–Host Interactions for Therapy. Human Gene Therapy 2017, 28, 820 -832.
AMA StyleClaudia Hagedorn, Florian Kreppel. Capsid Engineering of Adenovirus Vectors: Overcoming Early Vector–Host Interactions for Therapy. Human Gene Therapy. 2017; 28 (10):820-832.
Chicago/Turabian StyleClaudia Hagedorn; Florian Kreppel. 2017. "Capsid Engineering of Adenovirus Vectors: Overcoming Early Vector–Host Interactions for Therapy." Human Gene Therapy 28, no. 10: 820-832.
CRISPR/Cas9 RNA-guided nucleases refashioned in vivo gene editing approaches for specific gene disruption, gene correction, or gene addition. Moreover, chimeric Cas9 proteins can be applied to direct fused cis-acting effector protein domains, enzymes, or fluorescent markers to DNA to target sequences to regulate gene expression, to introduce epigenetic changes, or to fluorescently label DNA sequences of interest. Here we show how to design guide RNAs for specific DNA targeting. We provide a protocol to customize the CRISPR/Cas9 machinery encoded on commercially available plasmids and present how to test the targeting efficiency of Cas9 with a target-specific gRNA by testing mutation induction efficiency. To exemplify related applications we provide a guideline of how to apply the CRISPR/Cas9 technology for gene labeling.
Eric Ehrke-Schulz; Maren Schiwon; Claudia Hagedorn; Anja Ehrhardt. Establishment of the CRISPR/Cas9 System for Targeted Gene Disruption and Gene Tagging. Advanced Structural Safety Studies 2017, 1654, 165 -176.
AMA StyleEric Ehrke-Schulz, Maren Schiwon, Claudia Hagedorn, Anja Ehrhardt. Establishment of the CRISPR/Cas9 System for Targeted Gene Disruption and Gene Tagging. Advanced Structural Safety Studies. 2017; 1654 ():165-176.
Chicago/Turabian StyleEric Ehrke-Schulz; Maren Schiwon; Claudia Hagedorn; Anja Ehrhardt. 2017. "Establishment of the CRISPR/Cas9 System for Targeted Gene Disruption and Gene Tagging." Advanced Structural Safety Studies 1654, no. : 165-176.
Autonomously replicating vectors represent a simple and versatile model system for genetic modifications, but their localization in the nucleus and effect on endogenous gene expression is largely unknown. Using circular chromosome conformation capture we mapped genomic contact sites of S/MAR-based replicons in HeLa cells. The influence of cis-active sequences on genomic localization was assessed using replicons containing either an insulator sequence or an intron. While the original and the insulator-containing replicons displayed distinct contact sites, the intron-containing replicon showed a rather broad genomic contact pattern. Our results indicate a preference for certain chromatin structures and a rather non-dynamic behaviour during mitosis. Independent of inserted cis-active elements established vector molecules reside preferentially within actively transcribed regions, especially within promoter sequences and transcription start sites. However, transcriptome analyses revealed that established S/MAR-based replicons do not alter gene expression profiles of host genome. Knowledge of preferred contact sites of exogenous DNA, e.g. viral or non-viral episomes, contribute to our understanding of episome behaviour in the nucleus and can be used for vector improvement and guiding of DNA sequences to specific subnuclear sites.
Claudia Hagedorn; Andreas Gogol-Döring; Sabrina Schreiber; Jörg T. Epplen; Hans J. Lipps. Genome-wide profiling of S/MAR-based replicon contact sites. Nucleic Acids Research 2017, 45, 7841 -7854.
AMA StyleClaudia Hagedorn, Andreas Gogol-Döring, Sabrina Schreiber, Jörg T. Epplen, Hans J. Lipps. Genome-wide profiling of S/MAR-based replicon contact sites. Nucleic Acids Research. 2017; 45 (13):7841-7854.
Chicago/Turabian StyleClaudia Hagedorn; Andreas Gogol-Döring; Sabrina Schreiber; Jörg T. Epplen; Hans J. Lipps. 2017. "Genome-wide profiling of S/MAR-based replicon contact sites." Nucleic Acids Research 45, no. 13: 7841-7854.
Gene therapeutic approaches offer great opportunities to treat genetic diseases which require long-term effects after a single administration of a customized vector. For these specific approaches the optimal vector system should combine the following features: (1) it should efficiently transport the genetic cargo into target cells in vitro or in vivo, (2) it should lead to sufficient long-term expression of the therapeutic transgene, (3) it should not interfere with the expression profile or the composition of the host genome, and (4) it should not result in unwanted side effects such as immune responses or other toxic effects. Predominantly used vectors for maintenance of therapeutic DNA and long-term transgene expression in preclinical and clinical studies are based on integrase-, recombinase-, transposase- or designer nuclease-mediated somatic integration into the host genome. However, for these systems the risk of insertional mutagenesis represents a potential unwanted adverse event. Therefore, autonomously replicating genetic elements were developed and there is accumulating evidence that these episomal vectors which are maintained extrachromosomally are suitable for therapeutic applications in dividing cells. In this review we provide a state-of-the-art overview of used viral hybrid-vectors which efficiently deliver autonomous DNA (plasmid replicon pEPI and Epstein-Barr Virus-based replicons) and RNA replicons (Semliki Forest Virus replicons) into target cells. To date adenoviruses, herpesviruses and baculovirus were explored for efficient delivery of autonomous replicons into various cell types and tissues. Applications and advantages and limitations of these hybrid-vectors are discussed in this review. We believe that with further optimization autonomous replicons may play an increasingly important role in gene therapeutic applications.
Wenli Zhang; Claudia Hagedorn; Eric Schulz; Hans-Joachim Lipps; Anja Ehrhardt. Viral hybrid-vectors for delivery of autonomous replicons. Current Gene Therapy 2014, 14, 10 -23.
AMA StyleWenli Zhang, Claudia Hagedorn, Eric Schulz, Hans-Joachim Lipps, Anja Ehrhardt. Viral hybrid-vectors for delivery of autonomous replicons. Current Gene Therapy. 2014; 14 (1):10-23.
Chicago/Turabian StyleWenli Zhang; Claudia Hagedorn; Eric Schulz; Hans-Joachim Lipps; Anja Ehrhardt. 2014. "Viral hybrid-vectors for delivery of autonomous replicons." Current Gene Therapy 14, no. 1: 10-23.
The vector pEPI was the first nonviral and episomally replicating vector. Its functional element is an expression unit linked to a chromosomal scaffold/matrix attached region (S/MAR). The vector replicates autonomously with low copy number in various cell lines, is mitotically stable in the absence of selection over hundreds of generations, and was successfully used for the efficient generation of genetically modified pigs. Since it is assumed that establishment of the vector is a stochastic event and strongly depends on the nuclear compartment it reaches after transfection, it is of great interest to identify genomic sequences that guide DNA sequences into certain nuclear compartments. Here we inserted genomic cis-acting sequences into pEPI and examined their impact on transgene expression, long-term stability, and vector establishment. We demonstrated that a ubiquitous chromatin-opening element (UCOE) mediated enhanced transgene expression, while an insulator sequence (cHS4) increased establishment efficiency, presumably via an additional interaction with the nuclear matrix. Thus, besides being a promising alternative to currently used viral vectors in gene therapeutic approaches, pEPI may also serve as a tool to study nuclear compartmentalization; identification of genomic cis-acting sequences that are involved in nuclear organization will contribute to our understanding of the interplay between transgene expression, plasmid establishment, and nuclear architecture.
Claudia Hagedorn; Michael N Antoniou; Hans J Lipps. Genomic cis-acting Sequences Improve Expression and Establishment of a Nonviral Vector. Molecular Therapy - Nucleic Acids 2013, 2, e118 .
AMA StyleClaudia Hagedorn, Michael N Antoniou, Hans J Lipps. Genomic cis-acting Sequences Improve Expression and Establishment of a Nonviral Vector. Molecular Therapy - Nucleic Acids. 2013; 2 (8):e118.
Chicago/Turabian StyleClaudia Hagedorn; Michael N Antoniou; Hans J Lipps. 2013. "Genomic cis-acting Sequences Improve Expression and Establishment of a Nonviral Vector." Molecular Therapy - Nucleic Acids 2, no. 8: e118.
Claudia Hagedorn; Hans J. Lipps. S/MAR VECTORS — ALTERNATIVE EXPRESSION SYSTEMS FOR GENE THERAPY? Gene Therapy and Regulation 2012, 7, 1 .
AMA StyleClaudia Hagedorn, Hans J. Lipps. S/MAR VECTORS — ALTERNATIVE EXPRESSION SYSTEMS FOR GENE THERAPY? Gene Therapy and Regulation. 2012; 7 (1):1.
Chicago/Turabian StyleClaudia Hagedorn; Hans J. Lipps. 2012. "S/MAR VECTORS — ALTERNATIVE EXPRESSION SYSTEMS FOR GENE THERAPY?" Gene Therapy and Regulation 7, no. 1: 1.
Nonviral episomal vectors represent attractive alternatives to currently used virus-based expression systems. In the late 1990s, it was shown that a plasmid containing an expression cassette linked to a scaffold/matrix attached region (S/MAR) replicates as a low copy number episome in all cell lines tested, as well as primary cells, and can be used for the genetic modification of higher animals. Once established in the cell, the S/MAR vector replicates early during S-phase and, in the absence of selection, is stably retained in the cells for an unlimited period of time. This vector can therefore be regarded as a minimal model system for studying the epigenetic regulation of replication and functional nuclear architecture. In theory, this construct represents an almost “ideal” expression system for gene therapy. In practice, S/MAR-based vectors stably modify mammalian cells with efficiencies far below those of virus-based constructs. Consequently, they have not yet found application in gene therapy trials. Furthermore, S/MAR vector systems are not trivial to handle and several critical technical issues have to be considered when modifying these vectors for various applications.
Claudia Hagedorn; Armin Baiker; Jan Postberg; Anja Ehrhardt; Hans J. Lipps. Handling S/MAR Vectors. Cold Spring Harbor Protocols 2012, 2012, 1 .
AMA StyleClaudia Hagedorn, Armin Baiker, Jan Postberg, Anja Ehrhardt, Hans J. Lipps. Handling S/MAR Vectors. Cold Spring Harbor Protocols. 2012; 2012 (6):1.
Chicago/Turabian StyleClaudia Hagedorn; Armin Baiker; Jan Postberg; Anja Ehrhardt; Hans J. Lipps. 2012. "Handling S/MAR Vectors." Cold Spring Harbor Protocols 2012, no. 6: 1.
The episomal status of S/MAR (scaffold/matrix attached region)-based vectors can be confirmed by several methods including Southern blots, fluorescence in situ hybridization (FISH) analysis, or plasmid rescue experiments. In rescue experiments, genomic DNA (gDNA) or DNA from Hirt extracts is isolated from cell clones or mixed populations in which S/MAR plasmids are stably established. Bacteria are transformed with this DNA and if episomal plasmid DNA (pDNA) is present, resistant bacterial colonies will form.
Claudia Hagedorn; Armin Baiker; Jan Postberg; Anja Ehrhardt; Hans J. Lipps. Rescue of S/MAR-Containing Nonviral Episomal Expression Vectors. Cold Spring Harbor Protocols 2012, 2012, 1 .
AMA StyleClaudia Hagedorn, Armin Baiker, Jan Postberg, Anja Ehrhardt, Hans J. Lipps. Rescue of S/MAR-Containing Nonviral Episomal Expression Vectors. Cold Spring Harbor Protocols. 2012; 2012 (6):1.
Chicago/Turabian StyleClaudia Hagedorn; Armin Baiker; Jan Postberg; Anja Ehrhardt; Hans J. Lipps. 2012. "Rescue of S/MAR-Containing Nonviral Episomal Expression Vectors." Cold Spring Harbor Protocols 2012, no. 6: 1.
As with all eukaryotic replicons, the stable establishment of S/MAR (scaffold/matrix attached region) vectors is a stochastic event that depends on poorly understood epigenetic factors such as chromatin structure and nuclear localization. Establishment efficiency describes the percentage of cells in which a particular S/MAR vector is stably retained as an episome after an initial selection period. Expected establishment efficiency for S/MAR vectors is 1–5%. This article describes a colony-forming assay that may be used either to determine establishment efficiency or to generate single cell clones.
Claudia Hagedorn; Armin Baiker; Jan Postberg; Anja Ehrhardt; Hans J. Lipps. A Colony-Forming Assay for Determining the Establishment Efficiency of S/MAR-Containing Nonviral Episomal Expression Vectors. Cold Spring Harbor Protocols 2012, 2012, 1 .
AMA StyleClaudia Hagedorn, Armin Baiker, Jan Postberg, Anja Ehrhardt, Hans J. Lipps. A Colony-Forming Assay for Determining the Establishment Efficiency of S/MAR-Containing Nonviral Episomal Expression Vectors. Cold Spring Harbor Protocols. 2012; 2012 (6):1.
Chicago/Turabian StyleClaudia Hagedorn; Armin Baiker; Jan Postberg; Anja Ehrhardt; Hans J. Lipps. 2012. "A Colony-Forming Assay for Determining the Establishment Efficiency of S/MAR-Containing Nonviral Episomal Expression Vectors." Cold Spring Harbor Protocols 2012, no. 6: 1.
Because of their high efficiency, virus-based vectors are currently used in most gene therapy trials. Because such vectors bear some potential safety risks, nonviral expression systems could be an attractive alternative. Ideally, these vectors should be completely based on chromosomal elements and replicate as an autonomous unit in the recipient cell, thus avoiding the risk of insertional mutagenesis or immunological reactions of the recipient organism. Our limited knowledge of the epigenetic regulation of replication in mammalian cells does not yet allow the rational design of such constructs. But in the late 1990s it was shown that scaffold/matrix attached region (S/MAR)-based vectors can promote episomal replication and maintenance in mammalian cells. These vectors have found broad application in basic research but are now improved for their use in the safe and reproducible genetic modification of cells and organisms and in gene therapy.
Claudia Hagedorn; Suet-Ping Wong; Richard Harbottle; Hans J. Lipps. Scaffold/Matrix Attached Region-Based Nonviral Episomal Vectors. Human Gene Therapy 2011, 22, 915 -923.
AMA StyleClaudia Hagedorn, Suet-Ping Wong, Richard Harbottle, Hans J. Lipps. Scaffold/Matrix Attached Region-Based Nonviral Episomal Vectors. Human Gene Therapy. 2011; 22 (8):915-923.
Chicago/Turabian StyleClaudia Hagedorn; Suet-Ping Wong; Richard Harbottle; Hans J. Lipps. 2011. "Scaffold/Matrix Attached Region-Based Nonviral Episomal Vectors." Human Gene Therapy 22, no. 8: 915-923.
Neuropsychopharmacology, the official publication of the American College of Neuropsychopharmacology, publishing the highest quality original research and advancing our understanding of the brain and behavior.
H Wersching; K Guske; S Hasenkamp; Claudia Hagedorn; S Schiwek; S Jansen; V Witte; J Wellmann; H Lohmann; K Duning; J Kremerskothen; Stefan Knecht; E Brand; A Flöel. Impact of Common KIBRA Allele on Human Cognitive Functions. Neuropsychopharmacology 2011, 36, 1296 -1304.
AMA StyleH Wersching, K Guske, S Hasenkamp, Claudia Hagedorn, S Schiwek, S Jansen, V Witte, J Wellmann, H Lohmann, K Duning, J Kremerskothen, Stefan Knecht, E Brand, A Flöel. Impact of Common KIBRA Allele on Human Cognitive Functions. Neuropsychopharmacology. 2011; 36 (6):1296-1304.
Chicago/Turabian StyleH Wersching; K Guske; S Hasenkamp; Claudia Hagedorn; S Schiwek; S Jansen; V Witte; J Wellmann; H Lohmann; K Duning; J Kremerskothen; Stefan Knecht; E Brand; A Flöel. 2011. "Impact of Common KIBRA Allele on Human Cognitive Functions." Neuropsychopharmacology 36, no. 6: 1296-1304.
An ideal vector to be used in gene therapy should allow long-term and regulated expression of the therapeutic sequence, but in many cases, it would be most desirable to remove all ectopic vector sequences from the cell once expression is no longer required. The vector pEPI is the first nonviral autonomous replicon that was constructed for mammalian cells. It represents a minimal model system to study the epigenetic regulation of replication and transcription but is also regarded as a promising alternative to currently used viral vector systems in gene therapy. Its function relies on a transcription unit linked to an S/MAR sequence. We constructed an inducible pEPI vector system based on the Tet ON system in which transcription is switched on in the presence of doxycycline. We show that for vector replication and long-term maintenance an ongoing transcription running into the S/MAR element is required. Once established, the vector is lost from the cell upon switching off transcription from the gene linked to the S/MAR. This feature provides not only controlled transgene expression but also the possibility to remove all vector molecules from the cells upon demand. This inducible episomal nonviral vector system will find broad application in gene therapy but also in reprogramming of somatic cells or modification of stem cells.
Sina Rupprecht; Claudia Hagedorn; Davide Seruggia; Terese Magnusson; Ernst Wagner; Manfred Ogris; Hans J. Lipps. Controlled removal of a nonviral episomal vector from transfected cells. Gene 2010, 466, 36 -42.
AMA StyleSina Rupprecht, Claudia Hagedorn, Davide Seruggia, Terese Magnusson, Ernst Wagner, Manfred Ogris, Hans J. Lipps. Controlled removal of a nonviral episomal vector from transfected cells. Gene. 2010; 466 (1-2):36-42.
Chicago/Turabian StyleSina Rupprecht; Claudia Hagedorn; Davide Seruggia; Terese Magnusson; Ernst Wagner; Manfred Ogris; Hans J. Lipps. 2010. "Controlled removal of a nonviral episomal vector from transfected cells." Gene 466, no. 1-2: 36-42.
The discovery of autonomous replicating sequences (ARSs) inSaccharomyces cerevisiaein 1979 was considered a milestone in unraveling the regulation of replication in eukaryotic cells. However, shortly afterwards it became obvious that inSaccharomyces pombeand all other higher organisms ARSs were not sufficient to initiate independent replication. Understanding the mechanisms of replication is a major challenge in modern cell biology and is also a prerequisite to developing application-oriented autonomous replicons for gene therapeutic treatments. This review will focus on the development of non-viral episomal vectors, their use in gene therapeutic applications and our current knowledge about their epigenetic regulation.
Claudia Hagedorn; Hans J. Lipps; Sina Rupprecht. The epigenetic regulation of autonomous replicons. Biomolecular Concepts 2010, 1, 17 -30.
AMA StyleClaudia Hagedorn, Hans J. Lipps, Sina Rupprecht. The epigenetic regulation of autonomous replicons. Biomolecular Concepts. 2010; 1 (1):17-30.
Chicago/Turabian StyleClaudia Hagedorn; Hans J. Lipps; Sina Rupprecht. 2010. "The epigenetic regulation of autonomous replicons." Biomolecular Concepts 1, no. 1: 17-30.
Objective— Osteoprotegerin (OPG) has been reported to be involved in the development of atherosclerotic disease, and OPG gene variation has been associated with plasma OPG levels and different cardiovascular disease phenotypes. However, the genetic architecture of the OPG promoter and its transcriptional regulation are poorly characterized.Methods and Results— We identified 1008 bp of the OPG 5′-flanking region to be sufficiently transcriptionally active in osteosarcoma cell lines and generated serial promoter deletion constructs. Individual subcloning revealed the existence of 3 molecular haplotypes (MolHaps): [T−960-A−946-G−900-T−864; MolHap1, wild type], [T−960-G−946-G−900-T−864; MolHap2], [C−960-G−946-A−900-G−864; MolHap4]. Compared to MolHap1, transcriptional activities of MolHaps 2 and 4 were significantly reduced (P=0.0018). Whereas introduction of the −159C allele reduced transcriptional activities of the full-length constructs (P=0.0014), it significantly increased activities of the deletion constructs (P=0.0005). Electrophoretic mobility shift, competition, and chromatin immunoprecipitation assays revealed specific DNA:protein interactions for the MolHaps with Sp1 and NF-1, and identified Egr1 interacting exclusively with the −159T allele.Conclusions— We propose new structural and transcriptional features within the OPG promoter region and identified MolHaps being differentially transcriptionally active and allele-dependently interacting with a proximal polymorphic site. Using osteosarcoma cell lines, we (1) assessed transcriptionally active portions of the osteoprotegerin gene promoter, (2) assessed differential DNA-protein interactions involving Sp1, NF-1, and Egr1 depending on molecular haplotypes, promoter truncation, and interaction with proximal gene sites, and (3) evidenced differential transcription start site usage.
Claudia Hagedorn; Ralph Telgmann; Corinna Dördelmann; Boris Schmitz; Sandra Hasenkamp; François Cambien; Martin Paul; Eva Brand; Stefan-Martin Brand-Herrmann. Identification and Functional Analyses of Molecular Haplotypes of the Human Osteoprotegerin Gene Promoter. Arteriosclerosis, Thrombosis, and Vascular Biology 2009, 29, 1638 -1643.
AMA StyleClaudia Hagedorn, Ralph Telgmann, Corinna Dördelmann, Boris Schmitz, Sandra Hasenkamp, François Cambien, Martin Paul, Eva Brand, Stefan-Martin Brand-Herrmann. Identification and Functional Analyses of Molecular Haplotypes of the Human Osteoprotegerin Gene Promoter. Arteriosclerosis, Thrombosis, and Vascular Biology. 2009; 29 (10):1638-1643.
Chicago/Turabian StyleClaudia Hagedorn; Ralph Telgmann; Corinna Dördelmann; Boris Schmitz; Sandra Hasenkamp; François Cambien; Martin Paul; Eva Brand; Stefan-Martin Brand-Herrmann. 2009. "Identification and Functional Analyses of Molecular Haplotypes of the Human Osteoprotegerin Gene Promoter." Arteriosclerosis, Thrombosis, and Vascular Biology 29, no. 10: 1638-1643.
We aimed at associating common osteopontin (OPN) gene variants with cardiovascular disease phenotypes.We scanned the OPN gene in 190 chromosomes from myocardial infarction (MI) patients and identified five variants in the promoter, three synonymous and one non-synonymous variant. All variants were investigated in case–control studies for MI (ECTIM: 990 cases, 900 controls) and brain infarction (BI) (GÉNIC: 466 cases, 444 controls). Promoter variants were functionally analyzed by bandshift assays, the coding D147D [T/C] by Western blot. Allele D147D C was independently and significantly associated with lower apoB levels (P = 0.044 [ECTIM] P = 0.03 [GENIC]), its allele frequency was significantly lower in patients with BI compared to controls (OR [95% CI] 0.39 [0.20–0.74], P = 0.004), and C allele carriers had a significantly lower frequency of presence of carotid plaques (P = 0.02). Bandshifts with HepG2 and Ea.hy926 nuclear proteins did not reveal any functionality of promoter variants, whereas the OPN-441C-containing construct resulted in reduced OPN protein expression in Western blots, complying with its potential protective effect on the phenotypes studied.We here provide evidence that a portion of the OPN locus is likely to associate with cardiovascular disease-related phenotypes. However, further experiments are warranted to clarify the functional role of OPN variants.
Klaus Schmidt-Petersen; Eva Brand; Ralph Telgmann; Viviane Nicaud; Claudia Hagedorn; Julien Labreuche; Corinna Dördelmann; Alexis Elbaz; Marion Gautier-Bertrand; Jens W. Fischer; Alun Evans; Caroline Morrison; Minique Arveiler; Monika Stoll; Pierre Amarenco; Francois Cambien; Martin Paul; Stefan-Martin Brand-Herrmann. Osteopontin gene variation and cardio/cerebrovascular disease phenotypes. Atherosclerosis 2009, 206, 209 -215.
AMA StyleKlaus Schmidt-Petersen, Eva Brand, Ralph Telgmann, Viviane Nicaud, Claudia Hagedorn, Julien Labreuche, Corinna Dördelmann, Alexis Elbaz, Marion Gautier-Bertrand, Jens W. Fischer, Alun Evans, Caroline Morrison, Minique Arveiler, Monika Stoll, Pierre Amarenco, Francois Cambien, Martin Paul, Stefan-Martin Brand-Herrmann. Osteopontin gene variation and cardio/cerebrovascular disease phenotypes. Atherosclerosis. 2009; 206 (1):209-215.
Chicago/Turabian StyleKlaus Schmidt-Petersen; Eva Brand; Ralph Telgmann; Viviane Nicaud; Claudia Hagedorn; Julien Labreuche; Corinna Dördelmann; Alexis Elbaz; Marion Gautier-Bertrand; Jens W. Fischer; Alun Evans; Caroline Morrison; Minique Arveiler; Monika Stoll; Pierre Amarenco; Francois Cambien; Martin Paul; Stefan-Martin Brand-Herrmann. 2009. "Osteopontin gene variation and cardio/cerebrovascular disease phenotypes." Atherosclerosis 206, no. 1: 209-215.
Nebivolol (NEB) is a β1-receptor blocker with nitric oxide-dependent vasodilating properties. NEB-induced nitric oxide release is mediated through the estrogen receptor. Here, we tested the hypothesis that NEB decreases endothelial cell stiffness and that these effects can be abolished by both endothelial nitric oxide synthase and estrogen receptor blockade. Human endothelial cells (EAHy-926) were incubated with vehicle, NEB 0.7 nmol/l, metoprolol 200 nmol/l, 17β-estradiol (E2) 15 nmol/l, the estrogen receptor antagonists tamoxifen 100 nmol/l and ICI 182780 (ICI) 100 nmol/l, the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester 1 mmol/l and combinations of NEB and E2 with either tamoxifen, ICI or Nω-nitro-L-arginine methyl ester as well as metoprolol and ICI. Atomic force microscopy was performed to measure cellular stiffness, cell volume and apical surface. Presence of estrogen receptor protein in EAHy-926 was confirmed by western blot analysis; quantification of ERα and ERβ total RNA was performed by semiquantitative PCR. Both NEB as well as E2 decreased cellular stiffness to a similar extent (NEB: 0.83 ± 0.03 pN/nm, E2: 0.87 ± 0.03 pN/nm, vehicle: 2.19 ± 0.07 pN/nm), whereas metoprolol had no effect on endothelial stiffness (2.07 ± 0.04 pN/nm, all n = 60, P N NEB decreases cellular stiffness and causes endothelial cell growth. These effects are nitric oxide-dependent and mediated through nongenomic ERβ pathways. The morphological and functional alterations observed in endothelial cells may explain improved endothelial function with NEB treatment.
Uta Hillebrand; Detlef Lang; Ralph G Telgmann; Claudia Hagedorn; Stefan Reuter; Katrin Kliche; Christian M Stock; Hans Oberleithner; Hermann Pavenstädt; Eckhart Büssemaker; Martin Hausberg. Nebivolol decreases endothelial cell stiffness via the estrogen receptor beta: a nano-imaging study. Journal of Hypertension 2009, 27, 517 -526.
AMA StyleUta Hillebrand, Detlef Lang, Ralph G Telgmann, Claudia Hagedorn, Stefan Reuter, Katrin Kliche, Christian M Stock, Hans Oberleithner, Hermann Pavenstädt, Eckhart Büssemaker, Martin Hausberg. Nebivolol decreases endothelial cell stiffness via the estrogen receptor beta: a nano-imaging study. Journal of Hypertension. 2009; 27 (3):517-526.
Chicago/Turabian StyleUta Hillebrand; Detlef Lang; Ralph G Telgmann; Claudia Hagedorn; Stefan Reuter; Katrin Kliche; Christian M Stock; Hans Oberleithner; Hermann Pavenstädt; Eckhart Büssemaker; Martin Hausberg. 2009. "Nebivolol decreases endothelial cell stiffness via the estrogen receptor beta: a nano-imaging study." Journal of Hypertension 27, no. 3: 517-526.
Insulin-like growth factor 1 (IGF1) exerts important endocrine and paracrine functions in the cardiovascular system. We identified the common variant −1411C>T in the IGF1 upstream promoter P1, located within several overlapping transcription factor binding sites. Using transient transfection assays, we identified this site as a functional enhancer. The T allele-carrying enhancer, compared with the C allelic portion, exerts significantly reduced or even abrogated activity, respectively, in SaOs-2 and HepG2 (all PT is also associated with other IGF1-related disease phenotypes should be evaluated further in population studies.—Telgmann, R., Dördelmann, C., Brand, E., Nicaud, V., Hagedorn, C., Pavenstädt, H., Cambien, F., Tiret, L., Paul, M., Brand-Herrmann, S.-M. Molecular genetic analysis of a human insulin growth factor 1 promoter P1 variation.
Ralph Telgmann; Corinna Dördelmann; Eva Brand; Viviane Nicaud; Claudia Hagedorn; Hermann Pavenstädt; Franşois Cambien; Laurence Tiret; Martin Paul; Stefan‐Martin Brand‐Herrmann. Molecular genetic analysis of a human insulin‐like growth factor 1 promoter P1 variation. The FASEB Journal 2008, 23, 1303 -1313.
AMA StyleRalph Telgmann, Corinna Dördelmann, Eva Brand, Viviane Nicaud, Claudia Hagedorn, Hermann Pavenstädt, Franşois Cambien, Laurence Tiret, Martin Paul, Stefan‐Martin Brand‐Herrmann. Molecular genetic analysis of a human insulin‐like growth factor 1 promoter P1 variation. The FASEB Journal. 2008; 23 (5):1303-1313.
Chicago/Turabian StyleRalph Telgmann; Corinna Dördelmann; Eva Brand; Viviane Nicaud; Claudia Hagedorn; Hermann Pavenstädt; Franşois Cambien; Laurence Tiret; Martin Paul; Stefan‐Martin Brand‐Herrmann. 2008. "Molecular genetic analysis of a human insulin‐like growth factor 1 promoter P1 variation." The FASEB Journal 23, no. 5: 1303-1313.