State-of-the-art of 3D cultures (organs-on-a-chip) in safety testing and pathophysiology.

Integrated approaches using different in vitro methods in combination with bioinformatics can (i) increase the success rate and speed of drug development; (ii) improve the accuracy of toxicological risk assessment; and (iii) increase our understanding of disease. Three-dimensional (3D) cell culture models are important building blocks of this strategy which has emerged during the last years. The majority of these models are organotypic, i.e., they aim to reproduce major functions of an organ or organ system. This implies in many cases that more than one cell type forms the 3D structure, and often matrix elements play an important role. This review summarizes the state of the art concerning commonalities of the different models. For instance, the theory of mass transport/metabolite exchange in 3D systems and the special analytical requirements for test endpoints in organotypic cultures are discussed in detail. In the next part, 3D model systems for selected organs--liver, lung, skin, brain--are presented and characterized in dedicated chapters. Also, 3D approaches to the modeling of tumors are presented and discussed. All chapters give a historical background, illustrate the large variety of approaches, and highlight up- and downsides as well as specific requirements. Moreover, they refer to the application in disease modeling, drug discovery and safety assessment. Finally, consensus recommendations indicate a roadmap for the successful implementation of 3D models in routine screening. It is expected that the use of such models will accelerate progress by reducing error rates and wrong predictions from compound testing.

Early functional differentiation in the chick embryonic disc: interaction between mechanical activity and extracellular matrix

The mechanical behaviour of ectodermal cells in the area opaca and the supracellular organization of fibronectin in the adjacent extracellular matrix were studied in whole chick blastoderms developing in vitro. The pattern of spontaneous mechanical activity and its modification by immunoglobulins against fibronectin were determined using a real-time image-analysis system. The pattern of fibronectin was studied using immunocytochemical techniques. It was found that the ectodermal cells in the area opaca actively develop a radially oriented contraction, which leads to a distension of the area pellucida from which the embryo develops. Abnormally increased tension resulted in perturbations of gastrulation and neurulation. An optimized mechanical equilibrium within the blastoderm seems to be necessary for normal development. Anti-fibronectin antibodies applied to the basal side of the blastoderm led rapidly and reversibly to an increase of tension in the contracted cells. This observation indicates that modifications of the extracellular matrix can be transmitted to cytoskeletal elements within adjacent cells. The extracellular matrix of the area opaca contains fibronectin arranged in radially oriented fibrils. This orientation corresponds to the direction of migration of the mesodermal cells. Interestingly, the radial pattern of fibronectin is found in the regions where the ectodermal cells are contracted and develop radially oriented forces. This observation suggests that the supracellular assembly of the extracellular materials could be influenced by the mechanical activity of adjacent cells. Possible modulations of the supracellular organization of extracellular matrix by other factors, e.g. diffusible metabolites, is also discussed. The presence of characteristically organized extracellular matrix components, of spatially differentiated cell activities and of reciprocal interactions between them makes the young chick blastoderm an excellent system for physiological studies of the coordinated cellular activities that lead to changes in form, complexity and function.

Use of the chicken lysozyme 5' matrix attachment region to generate high producer CHO cell lines.

Scaffold or matrix attachment region (S/MAR) genetic elements have previously been proposed to insulate transgenes from repressive effects linked to their site of integration within the host cell genome. We have evaluated their use in various stable transfection settings to increase the production of recombinant proteins such as monoclonal antibodies from Chinese hamster ovary (CHO) cell lines. Using the green fluorescent protein coding sequence, we show that S/MAR elements mediate a dual effect on the population of transfected cells. First, S/MAR elements almost fully abolish the occurrence of cell clones that express little transgene that may result from transgene integration in an unfavorable chromosomal environment. Second, they increase the overall expression of the transgene over the whole range of expression levels, allowing the detection of cells with significantly higher levels of transgene expression. An optimal setting was identified as the addition of a S/MAR element both in cis (on the transgene expression vector) and in trans (co-transfected on a separate plasmid). When used to express immunoglobulins, the S/MAR element enabled cell clones with high and stable levels of expression to be isolated following the analysis of a few cell lines generated without transgene amplification procedures.

MAR elements as tools to increase protein production by CHO cells

One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection, analysis and characterization of the numerous clones required to identify one with the desired characteristics. Various boundary elements, matrix attachment regions, and locus control regions were screened for for their ability to augment the expression of heterologous genes in CHO and other cells. The 5'-matrix-attachment region (MAR) of the chicken lysozyme gene was found to significantly increase stable gene expression, in culture dishes and in bioreactors. These MAR elements can be easily combined with various existing expression systems, as they can be added in trans (i.e. on a separate plasmid) in co-transfections with previously constructed expression vectors. Using cell population analysis, we found that the use of the MAR increases the proportion of high-producing CHO cell clones, thus reducing the number of cell lines that need to be screened while increasing maximal productivity. Random cDNA cloning and sequencing indicated that over 12% of the ESTs correspond to the transgene. Thus, productivity is no longer limited by transcriptional events in such MAR-containing cell lines. The identification of small and more convenient active MAR portions will also be summarized. Finally, we will show examples of how MAR elements can be combined with short term expression to increase the simultaneous synthesis of many proteins in parallel by CHO cells. Overall, we conclude that the MAR sequence is a versatile tool to increase protein expression in short and long term production processes.

Combining MAR elements and transposable vectors for more reliable transgene integration and expression

Integration without cytotoxic effects and long-term expression of a transgene constitutes a major challenge in gene therapy and biotechnology applications. In this context, transposons represent an attractive system for gene transfer because of their ability to promote efficient integration of a transgene in a variety of cell lines. However, the transgene integration can lead to insertional mutagenesis and/or unstable transgene expression by epigenetic modifications. These unwanted events may be limited by the use of chromatin control elements called MARs (matrix attachment regions). Indeed, the insertion of these DNA elements next to the transgene usually results in higher and more stable expression by maintaining transgene chromatin in an active configuration and preventing gene silencing. In this study, we tested if the inclusion of the MAR 1-68 in the piggyBac transposon system may lead to efficient and safer transgene integration and ensure reliable stable and long-term expression of a transgene. The MAR-containing transposon construct was tested in CHO cells, for biotechnology applications, and in mesoangioblast cells that can differentiate into muscle cells and are important candidates for potential stem cell therapies of myopathies. We showed that the addition of the MAR 1 -68 in the piggyBac transposon did not interfere with transposition, thereby maintaining high frequency of transgene integrations in these cells. Moreover, the MAR allowed higher transgene expression from fewer transposon integration events. We also found that enriched transgene-expressing cell populations could be obtained without the need of selection pressure. Since antibiotic-enforced selection protocols often result in a higher integrated copy number and mosaic expression patterns, this strategy could benefit many applications in which a low copy number of integrated transgenes and antibiotic-free conditions are desired. In addition, the intramuscular transplantation of mouse tibialis anterior muscles with mesoangioblasts containing the transposon led to widespread and sustained myofiber transgene expression after differentiation of these cells in vivo. These findings indicated that piggyBac vectors may provide a viable approach to achieve stable gene transfer in the context of Duchenne muscular dystrophy therapy. - L'intégration sans effets cytotoxiques et l'expression à long terme d'un transgène constituent un défi majeur en thérapie génique et en biotechnologie. Dans ce contexte, les transposons représentent un système attrayant pour le transfert de gènes en raison de leur capacité à promouvoir l'intégration efficace d'un transgène dans une variété de lignées cellulaires. Toutefois, l'intégration d'un transgène peut conduire à une mutagénèse insertionnelle et/ou à une expression instable due au silençage du transgène suite à des modifications épigénétiques. Ces événements indésirables de silençage génique peuvent être diminués par l'utilisation d'éléments de contrôle de la chromatine appelés MAR (matrix attachment region). En effet, l'insertion de ces éléments d'ADN à proximité du transgène se traduit généralement par une expression plus élevée et plus stable de celui-ci, en permettant le maintien d'une chromatine dans une configuration active autour du transgène et en empêchant l'inactivation du gène. Dans cette étude, nous avons testé si l'inclusion du MAR 1-68 dans le système transposon piggyBac peut améliorer l'efficacité d'intégration de façon sécuritaire et l'expression à long terme d'un transgène. Le transposon contenant l'élément MAR a été testé dans les cellules CHO, couramment utilisées en biotechnologie, et dans des cellules progénitrices appelées mésoangioblastes, qui peuvent se différencier en cellules musculaires, et qui constituent ainsi des candidats prometteurs pour la thérapie à partir de cellules souches de patients souffrant de myopathie. Nous avons montré que l'addition du MAR 1-68 dans le transposon piggyBac n'interfère pas avec la transposition et permet de maintenir une fréquence élevée d'intégration du transgène dans ces deux types cellulaires. De plus, il semble que cette association mène à une meilleure expression du transgène à partir de peu d'événements d'intégration du transposon. En outre, ces populations enrichies en cellules exprimant de façon stable le transgène ont pu être obtenues sans avoir recours à une pression de sélection. Etant donné que les protocoles de sélection basée sur l'utilisation d'antibiotiques conduisent souvent à un nombre plus élevé de copies intégrées et à la variégation de l'expression du transgène et qu'ils impliquent une longue culture in vitro, cette stratégie pourrait profiter à des applications pour lesquelles on souhaite un faible nombre de copies intégrées et/ou l'utilisation d'antibiotiques n'est pas souhaitable. De plus, la transplantation intramusculaire de mésoangioblastes contenant le transposon dans le muscle tibial antérieur de souris a conduit, après la différentiation de ces cellules in vivo, à une expression constante et étendue du transgène dans les myofibres. Ces résultats indiquent que les vecteurs piggyBac pourraient fournir une approche viable pour assurer un transfert de gènes stables dans le contexte d'un traitement de la dystrophic musculaire de Duchenne.

Effects of epigenetic regulatory elements on telomeric gene expression

Transgene expression in eukaryotic cells strongly depends on the locus of integration in the host genome and often results in limited transcription level because of unfavorable chromatin structure at the integration site. Epigenetic regulators are DNA sequences which are believed to act on the chromatin structure and may protect transgenes from this so-called position effect. Despite being extensively used to increase transgene expression, the mechanism of action of many of these elements remains largely unknown. Here we evaluated different epigenetic regulatory DNA elements for their ability to protect transgene transcription at telomeres, a defined chromatin environment associated to low or inconsistent expression caused by the Telomere Position Effect (TPE). For the assessment of the effects of epigenetic regulators at telomeres, a novel dual reporter system had to be designed. Telomeric integration of the newly-developed dual reporter system carrying different epigenetic regulators showed that MARs (Matrix Attachment Regions), a UCOE (Ubiquitous Chromatin-Opening Element) or the chicken cHS4 insulator have strong barrier activity which prevented TPE from spreading toward the centromere, resulting in stable and in some cases increased expression of a telomeric-distal reporter gene. In addition, MARs and STAR element 40 resulted in an increase of cells expressing the telomeric-proximal reporter gene, suggesting also an anti-silencing effect. Chromatin immunoprecipitation assays revealed that at telomeres MARs actively promote the deposition of euchromatic histone marks, especially acetylation of both histone H3 and H4, which might be involved in MARs' barrier and transcriptional activator activities. Differently, the chromatin in proximity of the UCOE element was depleted of several repressive chromatin marks, such as trimethylated lysine 9 and lysine 27 on histone H3 and trimethylated lysine 20 of histone H4, possibly favoring the preservation of an open chromatin structure at the integration site. We conclude that epigenetic regulatory elements that may be used to enhance and sustain transgene expression have all a specific epigenetic signature which might be at the basis of their mechanism of action, and that a combination of different classes of epigenetic regulators might be advantageous when high levels of protein expression are required. - L'expression des transgènes dans les cellules eucaryotes est fortement influencée par leur site d'intégration dans le génome. En effet, une structure chromatinienne défavorable au niveau du site d'intégration peut fortement limiter le degré d'expression d'un transgène. Il existe toutefois des séquences d'ADN qui, en agissant sur la structure de la chromatine, permettent de limiter cet effet de position et, par conséquent, de promouvoir l'expression soutenue d'un transgène. Ces éléments génomiques, connus comme régulateurs épigénétiques, sont largement utilisés dans plusieurs domaines où une expression élevée et soutenue est requise, malgré un mode de fonctionnement parfois méconnu. Dans cette étude, j'ai évalué la capacité de différents régulateurs épigénétiques à protéger la transcription de transgènes au niveau des télomères, régions chromatiniennes bien définies qui ont été associées à un fort effet de silençage, connu comme «effet de position télomérique». Pour cela, un nouveau système à deux gènes rapporteurs a été développé. Lorsque des MARs (Matrix Attachment Regions, séquences d'ADN pouvant s'associer à la matrice nucléaire), un UCOE (Ubiquitous Chromatin-Opening Element, élément pouvant ouvrir la chromatine) ou l'isolateur génétique cHS4 (dérivé du locus de la β-globine de poulet) sont placés entre les deux gènes rapporteurs, une forte activité barrière bloquant la propagation de la chromatine répressive télomérique est observée, résultant en un plus grand nombre de cellules exprimant le gène télomérique-distal. D'autre part, une augmentation du nombre de cellules exprimant le gène télomérique-proximal, observée en présence des éléments MAR et STAR 40 (Stabilizing Anti-Repressor element 40, un élément pouvant prévenir la répression génique), suggère aussi un faible effet anti-silençage pour ces éléments. Des expériences d'immunoprécipitation de la chromatine démontrent qu'au télomère, les MARs favorisent l'assemblage de marqueurs de la chromatine active, surtout l'acétylation des histones H3 et H4, qui pourraient être à la base de l'activité barrière et de celle d'activateur transcriptionel. Différemment, la chromatine à proximité de l'élément UCOE est particulièrement pauvre en marqueurs de la chromatine silencieuse, comme la trimethylation des lysines 9 et 27 de l'histone H3, ainsi que la trimethylation de la lysine 20 de l'histone H4. Cela suggère que UCOE pourrait préserver une structure chromatinienne ouverte au site d'intégration, favorisant l'expression des gènes à sa proximité. En conclusion, les régulateurs épigénétiques analysés lors de cette étude ont tous montré une signature épigénétique spécifique qui pourrait être à la base de leurs mécanismes de fonctionnement, suggérant aussi qu'une utilisation d'éléments épigénétiques de classe différente dans un même vecteur d'expression pourrait être avantageuse lorsque de hauts et soutenus niveaux d'expression sont nécessaires.

MAR elements and transposons for improved transgene integration and expression.

Reliable and long-term expression of transgenes remain significant challenges for gene therapy and biotechnology applications, especially when antibiotic selection procedures are not applicable. In this context, transposons represent attractive gene transfer vectors because of their ability to promote efficient genomic integration in a variety of mammalian cell types. However, expression from genome-integrating vectors may be inhibited by variable gene transcription and/or silencing events. In this study, we assessed whether inclusion of two epigenetic control elements, the human Matrix Attachment Region (MAR) 1-68 and X-29, in a piggyBac transposon vector, may lead to more reliable and efficient expression in CHO cells. We found that addition of the MAR 1-68 at the center of the transposon did not interfere with transposition frequency, and transgene expressing cells could be readily detected from the total cell population without antibiotic selection. Inclusion of the MAR led to higher transgene expression per integrated copy, and reliable expression could be obtained from as few as 2-4 genomic copies of the MAR-containing transposon vector. The MAR X-29-containing transposons was found to mediate elevated expression of therapeutic proteins in polyclonal or monoclonal CHO cell populations using a transposable vector devoid of selection gene. Overall, we conclude that MAR and transposable vectors can be used to improve transgene expression from few genomic transposition events, which may be useful when expression from a low number of integrated transgene copies must be obtained and/or when antibiotic selection cannot be applied.

MAR elements regulate the probability of epigenetic switching between active and inactive gene expression.

Gene expression often cycles between active and inactive states in eukaryotes, yielding variable or noisy gene expression in the short-term, while slow epigenetic changes may lead to silencing or variegated expression. Understanding how cells control these effects will be of paramount importance to construct biological systems with predictable behaviours. Here we find that a human matrix attachment region (MAR) genetic element controls the stability and heritability of gene expression in cell populations. Mathematical modeling indicated that the MAR controls the probability of long-term transitions between active and inactive expression, thus reducing silencing effects and increasing the reactivation of silent genes. Single-cell short-terms assays revealed persistent expression and reduced expression noise in MAR-driven genes, while stochastic burst of expression occurred without this genetic element. The MAR thus confers a more deterministic behavior to an otherwise stochastic process, providing a means towards more reliable expression of engineered genetic systems.

Use of human MAR elements to improve retroviral vector production.

Retroviral vectors have many favorable properties for gene therapies, but their use remains limited by safety concerns and/or by relatively lower titers for some of the safer self-inactivating (SIN) derivatives. In this study, we evaluated whether increased production of SIN retroviral vectors can be achieved from the use of matrix attachment region (MAR) epigenetic regulators. Two MAR elements of human origin were found to increase and to stabilize the expression of the green fluorescent protein transgene in stably transfected HEK-293 packaging cells. Introduction of one of these MAR elements in retroviral vector-producing plasmids yielded higher expression of the viral vector RNA. Consistently, viral titers obtained from transient transfection of MAR-containing plasmids were increased up to sixfold as compared with the parental construct, when evaluated in different packaging cell systems and transfection conditions. Thus, use of MAR elements opens new perspectives for the efficient generation of gene therapy vectors.

Sustained transgene expression using MAR elements.

Matrix attachment regions (MARs) are DNA sequences that may be involved in anchoring DNA/chromatin to the nuclear matrix and they have been described in both mammalian and plant species. MARs possess a number of features that facilitate the opening and maintenance of euchromatin. When incorporated into viral or non-viral vectors MARs can increase transgene expression and limit position-effects. They have been used extensively to improve transgene expression and recombinant protein production and promising studies on the potential use of MAR elements for mammalian gene therapy have appeared. These illustrate how MARs may be used to mediate sustained or higher levels of expression of therapeutic genes and/or to reduce the viral vector multiplicity of infection required to achieve consistent expression. More recently, the discovery of potent MAR elements and the development of improved vectors for transgene delivery, notably non-viral episomal vectors, has strengthened interest in their use to mediate expression of therapeutic transgenes. This article will describe the progress made in this field, and it will discuss future directions and issues to be addressed.

Development of stable cell lines for production or regulated expression using matrix attachment regions.

One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection procedure. Given the variation in the expression levels of the same construct in individual clones, hundreds of clones must be isolated and tested to identify one or more with the desired characteristics. Various boundary elements (BEs), matrix attachment regions, and locus control regions (LCRs) were screened for their ability to augment the expression of heterologous genes in Chinese hamster ovary (CHO) cells. Of the chromatin elements assayed, the chicken lysozyme matrix-attachment region (MAR) was the only element to significantly increase stable reporter expression. We found that the use of the MAR increases the proportion of high-producing clones, thus reducing the number of clones that need to be screened. These benefits are observed both for constructs with MARs flanking the transgene expression cassette, as well as when constructs are co-transfected with the MAR on a separate plasmid. Moreover, the MAR was co-transfected with a multicomponent regulatable beta-galactosidase expression system in C2C12 cells and several clones exhibiting regulated expression were identified. Hence, MARs are useful in the development of stable cell lines for production or regulated expression.

Epigenetic regulatory elements associate with specific histone modifications to prevent silencing of telomeric genes.

In eukaryotic cells, transgene expression levels may be limited by an unfavourable chromatin structure at the integration site. Epigenetic regulators are DNA sequences which may protect transgenes from such position effect. We evaluated different epigenetic regulators for their ability to protect transgene expression at telomeres, which are commonly associated to low or inconsistent expression because of their repressive chromatin environment. Although to variable extents, matrix attachment regions (MARs), ubiquitous chromatin opening element (UCOE) and the chicken cHS4 insulator acted as barrier elements, protecting a telomeric-distal transgene from silencing. MARs also increased the probability of silent gene reactivation in time-course experiments. Additionally, all MARs improved the level of expression in non-silenced cells, unlike other elements. MARs were associated to histone marks usually linked to actively expressed genes, especially acetylation of histone H3 and H4, suggesting that they may prevent the spread of silencing chromatin by imposing acetylation marks on nearby nucleosomes. Alternatively, an UCOE was found to act by preventing deposition of repressive chromatin marks. We conclude that epigenetic DNA elements used to enhance and stabilize transgene expression all have specific epigenetic signature that might be at the basis of their mode of action.

The role of MAR elements in DNA recombination

The untargeted integration of foreign DNA into the mammalian cell genome, extensively used in gene therapy and biotechnology, remains an incompletely understood process. It is believed to be based on cellular DNA double strand break (DSB) repair machinery and to involve two major steps: i) the formation of long gene arrays (concatemers), and ii) recombination of the resulting concatemer with the genome. The main DSB repair pathways in eukaryotes include non-homologous end-joining (NHEJ), homologous recombination (HR), and microhomology-mediated end-joining (MMEJ). However, it is still not clear, which of these pathways are responsible for transgene integration. Here, we show that NHEJ is not the primary pathway used by mammalian cells in the transgene integration process, while the components of the HR pathway seem to be important for genomic integration but not concatemerization. Instead, concatemer formation appears to be mediated by a subset of the MMEJ pathway, termed synthesis-dependent MMEJ (SD-MMEJ). This mechanism also seems to be preferentially used for plasmid integration into the genome, as confirmed by the analysis of plasmid-to-genome junction sequences, which were found to display an SD-MMEJ pattern. Therefore, we propose the existence of two distinct SD-MMEJ subpathways, relying on different subsets of enzymes. One of these mechanisms appears to be responsible for concatemerization, while the other mechanism, partially dependent in HR enzymes, seems to mediate recombination with the genome. Previous studies performed by our group suggested that matrix attachment regions (MARs), which are epigenetic regulatory DNA elements that participate in the formation of chromatin boundaries and augment transcription, may mediate increased plasmid integration into the genome of CHO cells by stimulating DNA recombination. In the present work, we demonstrate that MAR-mediated plasmid integration results from the enhanced SD-MMEJ pathway. Analysis of transgene integration loci and junction DNA sequences validated the prevalent use of this pathway by the MAR elements to target plasmid DNA into gene-rich areas of the CHO genome. We propose that this finding should in the future help to engineer cells for improved recombinant protein production. In addition to investigating the process of transgene integration, we designed recombination assays to better characterize the components of the MMEJ and SD-MMEJ pathways. We also used CHO cells expressing cycle-sensitive reporter genes to demonstrate a potential role of HR proteins in the cell cycle regulation.

Epigenetic regulatory elements: Recent advances in understanding their mode of action and use for recombinant protein production in mammalian cells.

Successful generation of high producing cell lines requires the generation of cell clones expressing the recombinant protein at high levels and the characterization of the clones' ability to maintain stable expression levels. The use of cis-acting epigenetic regulatory elements that improve this otherwise long and uncertain process has revolutionized recombinant protein production. Here we review and discuss new insights into the molecular mode of action of the matrix attachment regions (MARs) and ubiquitously-acting chromatin opening elements (UCOEs), i.e. cis-acting elements, and how these elements are being used to improve recombinant protein production. These elements can help maintain the chromatin environment of the transgene genomic integration locus in a transcriptionally favorable state, which increases the numbers of positive clones and the transgene expression levels. Moreover, the high producing clones tend to be more stable in long-term cultures even in the absence of selection pressure. Therefore, by increasing the probability of isolating a high producing clone, as well as by increasing transcription efficiency and stability, these elements can significantly reduce the time and cost required for producing large quantities of recombinant proteins.