Characterization of insulators and barrier elements for gene therapy

Gene transfer that relies on integrating vectors often suffers from epigenetic or regulatory effects that influence the expression of the therapeutic gene and=or of cellular genes located near the vector integration site in the chromosome. Insulator elements act to block gene activation by enhancers, while chromatin domain boundary or barrier sequences prevent gene-silencing effects. At present, the modes of action of insulator and barriers are poorly understood, and their use in the context of gene therapies remains to be documented. Using combinations of reporter genes coding for indicator fluorescent proteins, we constructed assay systems that allow the quantification of the insulator or barrier activities of genetic elements in individual cells. This presentation will illustrate how these assay systems were used to identify short DNA elements that insulate nearby genes from activation by viral vector elements, and=or that block the propagation of a silent chromatin structure that leads to gene silencing. We will show that some barrier elements do not merely block repressive effects, but that they can act to stabilize and sustain transgene expression. We will illustrate that this may be beneficial when transgenes are introduced into stem or precursor cells using non-viral vectors, where later differentiation may lead to the silencing of the therapeutic gene. We will show that these elements can be used to maintain efficient transgene expression upon the differentiation of murine precursor cells towards myofibers, in a model of cell therapy for muscle dystrophies.

C/EBPbeta couples dopamine signalling to substance P precursor gene expression in striatal neurones.

Dopamine-induced changes in striatal gene expression are thought to play an important role in drug addiction and compulsive behaviour. In this study we report that dopamine induces the expression of the transcription factor CCAAT/Enhancer Binding Protein beta (C/EBP)-beta in primary cultures of striatal neurones. We identified the preprotachykinin-A (PPT-A) gene coding for substance P and neurokinin-A as a potential target gene of C/EBPbeta. We demonstrated that C/EBPbeta physically interacts with an element of the PPT-A promoter, thereby facilitating substance P precursor gene transcription. The regulation of PPT-A gene by C/EBPbeta could subserve many important physiological processes involving substance P, such as nociception, neurogenic inflammation and addiction. Given that substance P is known to increase dopamine signalling in the striatum and, in turn, dopamine increases substance P expression in medium spiny neurones, our results implicate C/EBPbeta in a positive feedback loop, changes of which might contribute to the development of drug addiction.

Transduction of CpG DNA-stimulated primary human B cells with bicistronic lentivectors.

Recently, using HIV-1-derived lentivectors, we obtained efficient transduction of primary human B lymphocytes cocultured with murine EL-4 B5 thymoma cells, but not of isolated B cells activated by CD40 ligation. Coculture with a cell line is problematic for gene therapy applications or study of gene functions. We have now found that transduction of B cells in a system using CpG DNA was comparable to that in the EL-4 B5 system. A monocistronic vector with a CMV promoter gave 32 +/- 4.7% green fluorescent protein (GFP)+ cells. A bicistronic vector, encoding IL-4 and GFP in the first and second cistrons, respectively, gave 14.2 +/- 2.1% GFP+ cells and IL-4 secretion of 1.3 +/- 0.2 ng/10(5) B cells/24 h. This was similar to results obtained in CD34+ cells using the elongation factor-1alpha promoter. Activated memory and naive B cells were transducible. After transduction with a bicistronic vector encoding a viral FLIP molecule, vFLIP was detectable by FACS or Western blot in GFP+, but not in GFP-, B cells, and 57% of sorted GFP+ B cells were protected against Fas ligand-induced cell death. This system should be useful for gene function research in primary B cells and development of gene therapies.

Myeloid differentiation factor-88/interleukin-1 signaling controls cardiac fibrosis and heart failure progression in inflammatory dilated cardiomyopathy.

RATIONALE: The myeloid differentiation factor (MyD)88/interleukin (IL)-1 axis activates self-antigen-presenting cells and promotes autoreactive CD4(+) T-cell expansion in experimental autoimmune myocarditis, a mouse model of inflammatory heart disease. OBJECTIVE: The aim of this study was to determine the role of MyD88 and IL-1 in the progression of acute myocarditis to an end-stage heart failure. METHODS AND RESULTS: Using alpha-myosin heavy chain peptide (MyHC-alpha)-loaded, activated dendritic cells, we induced myocarditis in wild-type and MyD88(-/-) mice with similar distributions of heart-infiltrating cell subsets and comparable CD4(+) T-cell responses. Injection of complete Freund's adjuvant (CFA) or MyHC-alpha/CFA into diseased mice promoted cardiac fibrosis, induced ventricular dilation, and impaired heart function in wild-type but not in MyD88(-/-) mice. Experiments with chimeric mice confirmed the bone marrow origin of the fibroblasts replacing inflammatory infiltrates and showed that MyD88 and IL-1 receptor type I signaling on bone marrow-derived cells was critical for development of cardiac fibrosis during progression to heart failure. CONCLUSIONS: Our findings indicate a critical role of MyD88/IL-1 signaling in the bone marrow compartment in postinflammatory cardiac fibrosis and heart failure and point to novel therapeutic strategies against inflammatory cardiomyopathy.

Dpp signaling activity requires Pentagone to scale with tissue size in the growing Drosophila wing imaginal disc.

The wing of the fruit fly, Drosophila melanogaster, with its simple, two-dimensional structure, is a model organ well suited for a systems biology approach. The wing arises from an epithelial sac referred to as the wing imaginal disc, which undergoes a phase of massive growth and concomitant patterning during larval stages. The Decapentaplegic (Dpp) morphogen plays a central role in wing formation with its ability to co-coordinately regulate patterning and growth. Here, we asked whether the Dpp signaling activity scales, i.e. expands proportionally, with the growing wing imaginal disc. Using new methods for spatial and temporal quantification of Dpp activity and its scaling properties, we found that the Dpp response scales with the size of the growing tissue. Notably, scaling is not perfect at all positions in the field and the scaling of target gene domains is ensured specifically where they define vein positions. We also found that the target gene domains are not defined at constant concentration thresholds of the downstream Dpp activity gradients P-Mad and Brinker. Most interestingly, Pentagone, an important secreted feedback regulator of the pathway, plays a central role in scaling and acts as an expander of the Dpp gradient during disc growth.

Using matrix attachment regions to improve recombinant protein production.

Chinese hamster ovary (CHO) cells are the system of choice for the production of complex molecules, such as monoclonal antibodies. Despite significant progress in improving the yield from these cells, the process to the selection, identification, and maintenance of high-producing cell lines remains cumbersome, time consuming, and often of uncertain outcome. Matrix attachment regions (MARs) are DNA sequences that help generate and maintain an open chromatin domain that is favourable to transcription and may also facilitate the integration of several copies of the transgene. By incorporating MARs into expression vectors, an increase in the proportion of high-producer cells as well as an increase in protein production are seen, thereby reducing the number of clones to be screened and time to production by as much as 9 months. In this chapter, we describe how MARs can be used to increase transgene expression and provide protocols for the transfection of CHO cells in suspension and detection of high-producing antibody cell clones.

Development of a microfluidics biosensor for agarose-bead immobilized Escherichia coli bioreporter cells for arsenite detection in aqueous samples.

Contamination with arsenic is a recurring problem in both industrialized and developing countries. Drinking water supplies for large populations can have concentrations much higher than the permissible levels (for most European countries and the United States, 10 μg As per L; elsewhere, 50 μg As per L). Arsenic analysis requires high-end instruments, which are largely unavailable in developing countries. Bioassays based on genetically engineered bacteria have been proposed as suitable alternatives but such tests would profit from better standardization and direct incorporation into sensing devices. The goal of this work was to develop and test microfluidic devices in which bacterial bioreporters could be embedded, exposed and reporter signals detected, as a further step towards a complete miniaturized bacterial biosensor. The signal element in the biosensor is a nonpathogenic laboratory strain of Escherichia coli, which produces a variant of the green fluorescent protein after contact to arsenite and arsenate. E. coli bioreporter cells were encapsulated in agarose beads and incorporated into a microfluidic device where they were captured in 500 × 500 μm(2) cages and exposed to aqueous samples containing arsenic. Cell-beads frozen at -20 °C in the microfluidic chip retained inducibility for up to a month and arsenic samples with 10 or 50 μg L(-1) could be reproducibly discriminated from the blank. In the 0-50 μg L(-1) range and with an exposure time of 200 minutes, the rate of signal increase was linearly proportional to the arsenic concentration. The time needed to reliably and reproducibly detect a concentration of 50 μg L(-1) was 75-120 minutes, and 120-180 minutes for a concentration of 10 μg L(-1).

Testing mouse mammary tumor virus superantigen as adjuvant in cytotoxic T-lymphocyte responses against a melanoma tumor antigen.

Cytotoxic T cells represent a powerful strategy for antitumor treatment. Depending on the route of injection, an important role for CD4 T cell-mediated help was observed in the induction of this response. For this reason, we investigated whether induction of a CTL response to the HLA-A2-restricted immunodominant peptide melanoma antigen Melan-A was improved by using rVVs expressing the CTL-defined epitope alone or in combination with an SAg. In the latter case, the few infected dendritic cells simultaneously presented an SAg and an antigen, i.e., peptide. Here, we show that the anti-Melan-A response was efficiently induced but not significantly improved by coexpression of the SAg.

Study of the regulation of antifungal activity in plant-associated pseudomas fluorescens

RésuméEn agriculture d'énormes pertes sont causées par des champignons telluriques pathogènes tels que Thielaviopsis, Fusarium, Gaeumannomyces et Rhizoctonia ou encore l'oomycète Pythium. Certaines bactéries dites bénéfiques, comme Pseudomonas fluorescens, ont la capacité de protéger les plantes de ces pathogènes par la colonisation de leur racines, par la production de métabolites secondaires possédants des propriétés antifongiques et par l'induction des mécanismes de défenses de la plante colonisée. P. fluorescens CHAO, une bactérie biocontrôle isolée d'un champ de tabac à Payerne, a la faculté de produire un large spectre de métabolites antifongiques, en particulier le 2,4- diacétylphloroglucinol (DAPG), la pyolutéorine (PLT), le cyanure d'hydrogène (HCN), la pyrrolnitrine (PRN) ainsi que des chélateurs de fer.La plante, par sécrétion racinaire, produit des rhizodéposites, source de carbone et d'azote, qui profitent aux populations bactériennes vivant dans la rhizosphere. De plus, certains stresses biotiques et abiotiques modifient cette sécrétion racinaire, en terme quantitatif et qualitatif. De leur côté, les bactéries bénéfiques, améliorent, de façon direct et/ou indirect, la croissance de la plante hôte. De nombreux facteurs biotiques et abiotiques sont connus pour réguler la production de métabolites secondaires chez les bactéries. Des études récentes ont démontré l'importance de la communication entre la plante et les bactéries bénéfiques afin que s'établisse une interaction profitant à chacun des deux partis. Il est ainsi vraisemblable que les populations bactériennes associées aux racines soient capables d'intégrer ces signaux et d'adapter spécifiquement leur comportement en conséquence.La première partie de ce travail de thèse a été la mise au point d'outils basés sur la cytométrie permettant de mesurer l'activité antifongique de cellules bactériennes individuelles dans un environnent naturel, les racines des plantes. Nous avons démontré, grâce à un double marquage aux protéines autofluorescentes GFP et mCherry, que les niveaux d'expression des gènes impliqués dans la biosynthèse des substances antifongiques DAPG, PLT, PRN et HCN ne sont pas les mêmes dans des milieux de cultures liquides que sur les racines de céréales. Par exemple, l'expression de pltA (impliqué dans la biosynthèse du PLT) est quasiment abolie sur les racines de blé mais atteint un niveau relativement haut in vitro. De plus cette étude a mis en avant l'influence du génotype céréalien sur l'expression du gène phlA qui est impliqué dans la biosynthèse du DAPG.Une seconde étude a révélé la communication existant entre une céréale (orge) infectée par le pathogène tellurique Pythium ultimum et P. fluorescens CHAO. Un système de partage des racines nous a permis de séparer physiquement le pathogène et la bactérie bénéfique sur la plante. Cette méthode a donné la possibilité d'évaluer l'effet systémique, causé par l'attaque du pathogène, de la plante sur la bactérie biocontrôle. En effet, l'infection par le phytopathogène modifie la concentration de certains composés phénoliques dans les exsudats racinaires stimulant ainsi l'expression de phi A chez P.fluorescens CHAO.Une troisième partie de ce travail focalise sur l'effet des amibes qui sont des micro-prédateurs présents dans la rhizosphere. Leur présence diminue l'expression des gènes impliqués dans la biosynthèse du DAPG, PLT, PRN et HCN chez P.fluorescens CHAO, ceci en culture liquide et sur des racines d'orge. De plus, des molécules provenant du surnageant d'amibes, influencent l'expression des gènes requis pour la biosynthèse de ces antifongiques. Ces résultats illustrent que les amibes et les bactéries de la rhizosphere ont développé des stratégies pour se reconnaître et adapter leur comportement.La dernière section de ce travail est consacrée à l'acide indole-acétique (LA.A), une phytohormone connue pour son effet stimulateur sur phlA. Une étude moléculaire détaillée nous a démontré que cet effet de l'IAA est notamment modulé par une pompe à efflux (FusPl) et de son régulateur transcriptionnel (MarRl). De plus, les gènes fusPl et marRl sont régulés par d'autres composés phénoliques tels que le salicylate (un signal végétal) et l'acide fusarique (une phytotoxine du pathogène Fusarium).En résumé, ce travail de thèse illustre la complexité des interactions entre les eucaryotes et procaryotes de la rhizosphère. La reconnaissance mutuelle et l'instauration d'un dialogue moléculaire entre une plante hôte et ses bactéries bénéfiques associées? sont indispensables à la survie des deux protagonistes et semblent être hautement spécifiques.SummaryIn agriculture important crop losses result from the attack of soil-borne phytopathogenic fungi, including Thielaviopsis, Fusarium, Gaeumannomyces and Rhizoctonia, as well as from the oomycete Pythium. Certain beneficial microorganisms of the rhizosphere, in particular Pseudomonas fluorescens, have the ability to protect plants against phytopathogens by the intense colonisation of roots, by the production of antifungal exoproducts, and by induction of plant host defences. P. fluorescens strain CHAO, isolated from a tobacco field near Payerne, produces a large array of antifungal exoproducts, including 2,4-diacetylphloroglucinol (DAPG), pyoluteorin (PLT), hydrogen cyanide (HCN), pyrrolnitrin (PRN) and iron chelators. Plants produce rhizodeposites via root secretion and these represent a relevant source of carbon and nitrogen for rhizosphere microorganisms. Various biotic and abiotic stresses influence the quantity and the quality of released exudates. One the other hand, beneficial bacteria directly or indirectly promote plant growth. Biotic and abiotic factors regulate exoproduct production in biocontrol microorganisms. Recent studies have highlighted the importance of communication in establishing a fine-tuned mutualist interaction between plants and their associated beneficial bacteria. Bacteria may be able to integrate rhizosphere signals and adapt subsequently their behaviour.In a first part of the thesis, we developed a new method to monitor directly antifungal activity of individual bacterial cells in a natural environment, i.e. on roots of crop plants. We were able to demonstrate, via a dual-labelling system involving green and red fluorescent proteins (GFP, mCherry) and FACS-based flow cytometry, that expression levels of biosynthetic genes for the antifungal compounds DAPG, PLT, PRN, and HCN are highly different in liquid culture and on roots of cereals. For instance, expression of pltA (involved in PLT biosynthesis) was nearly abolished on wheat roots whereas it attained a relatively high level under in vitro conditions. In addition, we established the importance of the cereal genotype in the expression of phi A (involved in DAPG biosynthesis) in P. fluorescens CHAO.A second part of this work highlighted the systemic communication that exists between biocontrol pseudomonads and plants following attack by a root pathogen. A split-root system, allowing physical separation between the soil-borne oomycete pathogen Phytium ultimum and P. fluorescens CHAO on barley roots, was set up. Root infection by the pathogen triggered a modification of the concentration of certain phenolic root exudates in the healthy root part, resulting in an induction ofphlA expression in P. fluorescens CHAO.Amoebas are micro-predators of the rhizosphere that feed notably on bacteria. In the third part of the thesis, co-habitation of Acanthamoeba castellanii with P. fluorescens CHAO in culture media and on barley roots was found to significantly reduce bacterial expression of genes involved in the biosynthesis of DAPG, PLT, HCN and PRN. Interestingly, molecular cues present in supernatant of A. castelanii induced the expression of these antifungal genes. These findings illustrate the strategies of mutual recognition developed by amoeba and rhizosphere bacteria triggering responses that allow specific adaptations of their behaviour.The last section of the work focuses on indole-3-acetic acid (IAA), a phytohormone that stimulates the expression of phi A. A detailed molecular study revealed that the IAA-mediated effect on phi A is notably modulated by an efflux pump (FusPl) and its transcriptional regulator (MarRl). Remarkably, transcription of fusPl and marRl was strongly upregulated in presence of other phenolic compounds such as salicylate (a plant signal) and fusaric acid (a phytotoxin of the pathogenic fungus Fusarium).To sum up, this work illustrates the great complexity of interactions between eukaryotes and prokaryotes taking place in the rhizosphere niche. The mutual recognition and the establishment of a molecular cross-talk between the host plant and its associated beneficial bacteria are essential for the survival of the two partners and these interactions appear to be highly specific.

HDLs protect pancreatic β-cells against ER stress by restoring protein folding and trafficking.

Endoplasmic reticulum (ER) homeostasis alteration contributes to pancreatic β-cell dysfunction and death and favors the development of diabetes. In this study, we demonstrate that HDLs protect β-cells against ER stress induced by thapsigargin, cyclopiazonic acid, palmitate, insulin overexpression, and high glucose concentrations. ER stress marker induction and ER morphology disruption mediated by these stimuli were inhibited by HDLs. Using a temperature-sensitive viral glycoprotein folding mutant, we show that HDLs correct impaired protein trafficking and folding induced by thapsigargin and palmitate. The ability of HDLs to protect β-cells against ER stress was inhibited by brefeldin A, an ER to Golgi trafficking blocker. These results indicate that HDLs restore ER homeostasis in response to ER stress, which is required for their ability to promote β-cell survival. This study identifies a cellular mechanism mediating the beneficial effect of HDLs on β-cells against ER stress-inducing factors.

Role of glast-positive glial cells during photoreceptor development

Purpose: Taking advantage of two transgenic lines, glast.DsRed and crx.gfp, that express fluorescent proteins in glial and photoreceptor cells respectively, we investigate the role of glast-positive glial cells (GPCs) in the survival/differentiation/proliferation of age-matched photoreceptor cells. Methods: Primary retinal cells were isolated from newborn transgenic mouse retina (glast.dsRed::crx.gfp) at postnatal day (P0/P1) and propagated in defined medium containing epidermal growth factor (EGF) and fibroblast growth factor 2 (bFGF). By flow-sorting another population of pure GPCs was isolated. Both populations were expanded and analyzed for the presence of specific retinal cell markers. Notably, the primary cell culture collected from the transgenic line glast.dsRed::crx.gfp showed a conspicuous presence of immature photoreceptors growing on top of GPCs. In order to reveal the role of such cells in the survival/differentiation/proliferation of photoreceptors we set up in vitro cultures of retina-derived cells that allowed long-term time-lapse recordings charting every cell division, death and differentiation event. To assess the regenerative potential of GPCs we challenged them with compounds mimicking retinal degeneration (NMU, NMDA, Zaprinast). Mass spectrometry (MS), immunostainings and other molecular approaches were performed to reveal adhesion molecules involved in the relationship between glial cells and photoreceptors. Results: Both primary cell lines were highly homogenous, with an elongated morphology and the majority expressed Müller glia markers (MG) such as glast, blbp, glt-1, vimentin, glutamine synthetase (GS), GFAP, cd44, mash1 and markers of reactive Müller glia such as nestin, pax6. Conversely, none of them were found positive for retinal neuron markers like tuj1, otx2, recoverin. Primary cultures of GPCs show the incapability of glial cells to give rise to photoreceptors in both wild type or degenerative environment. Furthermore, primary cultures of pure GPCs challenged with different compounds did not highlight the production of new glial cell-derived photoreceptors. Adhesion molecules involved in the contact between photoreceptors and glial cells are still under investigation. Conclusions: Primary glia cells do not give rise to photoreceptor cells in wt and degenerative conditions at least in vitro. The roles of glial cells seem to be more linked to the maintenance/proliferation of photoreceptor cells.

Identification of a potent MAR element from the mouse genome and assessment of its activity in stable and transient transfections.

Matrix attachment regions are DNA sequences found throughout eukaryotic genomes that are believed to define boundaries interfacing heterochromatin and euchromatin domains, thereby acting as epigenetic regulators. When included in expression vectors, MARs can improve and sustain transgene expression, and a search for more potent novel elements is therefore actively pursued to further improve recombinant protein production. Here we describe the isolation of new MARs from the mouse genome using a modified in silico analysis. One of these MARs was found to be a powerful activator of transgene expression in stable transfections. Interestingly, this MAR also increased GFP and/or immunoglobulin expression from some but not all expression vectors in transient transfections. This effect was attributed to the presence or absence of elements on the vector backbone, providing an explanation for earlier discrepancies as to the ability of this class of elements to affect transgene expression under such conditions.

Avoiding epigenetic variations in transgene expression with genetic insulators

Gene transfer that relies on integrating vectors often suffers from epigenetic or regulatory effects that influence the expression of the therapeutic gene and=or of cellular genes located near the vector integration site in the chromosome. Insulator elements act to block gene activation by enhancers, while chromatin domain boundary or barrier sequences prevent gene-silencing effects. At present, the modes of action of insulator and barriers are poorly understood, and their use in the context of gene therapies remains to be documented. Using combinations of reporter genes coding for indicator fluorescent proteins, we constructed assay systems that allow the quantification of the insulator or of the barrier activities of genetic elements in individual cells. This presentation will illustrate how these assay systems were used to identify short DNA elements that can insulate nearby genes from activation by viral vector enhancer elements, and=or that can block the propagation of a silent chromatin structure that leads to gene silencing. We will show that small elements of the order of 100-400 nucleotides can be designed to achieve both insulator and boundary function, as needed for safer integrating viral vectors.

An effector-reduced anti-β-amyloid (Aβ) antibody with unique aβ binding properties promotes neuroprotection and glial engulfment of Aβ.

Passive immunization against β-amyloid (Aβ) has become an increasingly desirable strategy as a therapeutic treatment for Alzheimer's disease (AD). However, traditional passive immunization approaches carry the risk of Fcγ receptor-mediated overactivation of microglial cells, which may contribute to an inappropriate proinflammatory response leading to vasogenic edema and cerebral microhemorrhage. Here, we describe the generation of a humanized anti-Aβ monoclonal antibody of an IgG4 isotype, known as MABT5102A (MABT). An IgG4 subclass was selected to reduce the risk of Fcγ receptor-mediated overactivation of microglia. MABT bound with high affinity to multiple forms of Aβ, protected against Aβ1-42 oligomer-induced cytotoxicity, and increased uptake of neurotoxic Aβ oligomers by microglia. Furthermore, MABT-mediated amyloid plaque removal was demonstrated using in vivo live imaging in hAPP((V717I))/PS1 transgenic mice. When compared with a human IgG1 wild-type subclass, containing the same antigen-binding variable domains and with equal binding to Aβ, MABT showed reduced activation of stress-activated p38MAPK (p38 mitogen-activated protein kinase) in microglia and induced less release of the proinflammatory cytokine TNFα. We propose that a humanized IgG4 anti-Aβ antibody that takes advantage of a unique Aβ binding profile, while also possessing reduced effector function, may provide a safer therapeutic alternative for passive immunotherapy for AD. Data from a phase I clinical trial testing MABT is consistent with this hypothesis, showing no signs of vasogenic edema, even in ApoE4 carriers.