Targeted epigenetic glyco-engineering in CHO cells

Extensive knowledge has been gathered by applying and generating –omics techniques and data towards a holistic understanding of the Chinese Hamster Ovary (CHO) cell’s regulatory network. However, these data are far from universally explanatory. The epigenome, i.e. the genetic signature that controls modulation of gene expression, has not yet been fully explored. To enable direct control of epigenetic regulation of individual genes, we constructed CRISPR-based epigenetic editing tools that induce site-specific DNA methylation or demethylation rather than double strand breaks at specific endogenous promoters. The current design targets the promoter of the silenced α (2,6)-sialyltransferase (ST6GAL1) gene, which is actively transcribed in human and there part of the protein glycosylation machinery. It is present in the CHO genome, but silenced. We aimed to induce its expression in CHO by targeted demethylation of the ST6GAL1 promoter. Flow cytometric analysis (based on glycosylation specific lectin staining) showed upregulation of ST6GAL1 in up to 67% of cells in transfected cell pools. ST6GAL1 expression was also confirmed by RT-qPCR and MS glycan analysis. Stable upregulation of ST6GAL1 was monitored over a period of more than 80 days, showing the applicability in industrial cell development pipelines for long-term changes in cell behavior. The effect could be readily reversed by subsequent targeted re-methylation of the ST6GAL1 promoter by our epigenetic editing tool set. In conclusion, this epigenetic tool does not only allow to build a new layer of cell control that complements existing techniques (e.g. genome engineering), but also enables a more sensitive investigation of gene function by induction and repression of genes without altering the DNA sequence. Finally, other than gene knockout or overexpression studies, the modulation is readily reversible, thus opening up a multitude of possibilities for fast, unbiased and stable testing of gene function and multiplex engineering approaches

A CRISPR/Cas9 based engineering tool to activate expression of multiple genes individually or in any specific combination

Engineering of cells by overexpression or knock-down/out of individual genes has demonstrated that in most cases the manipulation of single genes is not sufficient to alter a cellular phenotype. Rather, multiple genes involved in a pathway need to be manipulated. Especially in mammalian cells such as CHO, where clonal variation is large, it has been difficult to unequivocally assess whether the observed change in phenotype is due to such clonal variation or the engineered gene. This can in part be overcome by testing multiple subclones, however, once it comes to engineering multiple genes and combinations thereof, the required workload quickly becomes prohibitive. We here present a simple technology for successive and/or specific activation of multiple genes integrated into a single genomic locus, which presents a potential solution to this problem. The technology consists of a vector containing multiple genes to be engineered or copies of the same gene. The promoters of these genes/gene copies are separated from the translation start site by repressor elements, flanked by individual guide RNA (gRNA) target sites. After integration of the construct into the genome and clone selection, these repressor elements can be removed by transfection with Cas9 and the corresponding pair of gRNAs that target the repressor of the gene(s) to be activated. Efficiency of target gene activation was in the range of 20-30% of the population for individual genes. Using 4 different fluorescent genes, the success of the technology was shown by activation of different combinations of these genes, followed by sorting of cells with the correct combination of required target genes activated. For pathway engineering studies, the selected genes can be expressed linked to these fluorescent genes e.g. via an IRES or a 2A self-cleaving peptide and cells with the desired co-expression pattern sorted, thus obviating the necessity to subclone for subsequent phenotypic characterization of the engineered cells. The technology provides a rapid procedure to assess the effect of gene combination on cellular behavior. Please click Additional Files below to see the full abstract

Analysis of microRNA transcription and post-transcriptional processing by Dicer in the context of CHO cell proliferation

AbstractCHO cells are the mammalian cell line of choice for recombinant production of therapeutic proteins. However, their low rate of proliferation limits obtainable space-time yields due to inefficient biomass accumulation. We set out to correlate microRNA transcription to cell-specific growth-rate by microarray analysis of 5 CHO suspension cell lines with low to high specific growth rates. Global microRNA expression analysis and Pearson correlation studies showed that mature microRNA transcript levels are predominately up-regulated in a state of fast proliferation (46 positively correlated, 17 negatively correlated). To further validate this observation, the expression of three genes that are central to microRNA biogenesis (Dicer, Drosha and Dgcr8) was analyzed. The expression of Dicer, which mediates the final step in microRNA maturation, was found to be strongly correlated to growth rate. Accordingly, knockdown of Dicer impaired cell growth by reducing growth-correlating microRNA transcripts. Moderate ectopic overexpression of Dicer positively affected cell growth, while strong overexpression impaired growth, presumably due to the concomitant increase of microRNAs that inhibit cell growth. Our data therefore suggest that Dicer dependent microRNAs regulate CHO cell proliferation and that Dicer could serve as a potential surrogate marker for cellular proliferation

Epigenetic regulation of gene expression in response to a changing environment in CHO cell batch culture

Chinese Hamster Ovary (CHO) cells have been the workhorse for industrial production of recombinant therapeutic proteins since 1987. Variations in cellular environment and phenotypes that occur throughout the bioprocess can bring about significant changes in productivity and quality of recombinant proteins. This can potentially lead to rejection of the production lot. Hence, there is interest in an in-depth understanding of cell-line behavior and control to achieve more predictable and reliable process performance. Biological systems undergo dynamic changes over time, where individual genes are turned “on”, “off” or “paused” as and when required. So far, there is very little information available for CHO cell lines, that elucidates the effect of dynamic epigenetic regulation on temporal expression of genes in response to altered substrate availability and culture conditions. While DNA methylation levels around TSS induce either expression or silencing of genes, transcriptional regulation is primarily considered to be an interplay of transcription factors and chromatin modifiers. On top of these, there is a rapid increase in indications that connects phase-specific long non-coding RNAs (lncRNAs) in transcriptional and post-transcriptional gene regulation. Unfortunately, the mechanism of interaction of these lncRNAs with coding genes have not been studied extensively. In this study, the gene transcription dynamics throughout a batch culture of CHO cells was examined by analyzing expression profiles and histone modifications in regular 12-24 hour intervals. Chromatin states and differential methylation profiles were used to understand the role of epigenetic modifiers in the regulation of gene expression. A good correlation between expression level and absence of DNA-methylation in the promoter regions was observed. Genes having all essential active chromatin marks - specific for promoter activity, genic enhancer and active transcription, also showed significantly high positive correlation between the changes in expression levels and histone marks. Both transcription and chromatin modifications during different growth phases were found to be highly dynamic. Clusters of genes showing similar trends of expression depict gradual and continuous adaptation to the changing substrate concentrations. Less narrowly spaced temporal analyses would have prevented detection of critical regulators involved in transient changes during the batch culture. Here, we also report a plausible mode of interaction of lncRNAs with the coding genes mediated by RNA-DNA-DNA triplex formations. Based on the identified interactions, we could predict possible gene targets and the target sites for the expressed lncRNAs and show high level of correlation of expression levels between interacting pairs. To the best of our knowledge this is the first and most comprehensive report of genome wide transcriptional regulation by epigenetic modifiers for CHO. Please click Additional Files below to see the full abstract

What\u27s in a Phenotype?

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Providing a sound basis for microRNA engineering in CHO cells

Chinesische Hamster Ovarien (CHO) Zellen sind die Zellfabrik der Wahl für die Produktion von komplexen, menschenähnlichen therapeutischen Proteinen. Viel Aufwand wurde aufgebracht, um diese Zellen zu optimieren, insbesondere durch gentechnische Veränderungen. Eine neue Methode ist die Verwendung von microRNAs (miRNAs), um Zellen in Richtung gewünschter Phänotypen zu lenken. Seitdem die erste genomische Sequenz von CHO Zellen im Jahr 2011 veröffentlicht wurde, wurden Bemühungen unternommen, um miRNAs im CHO-Genom zu identifizieren und zu annotieren. Wie es bei anderen Spezies gezeigt worden ist, sind miRNAs oft in chromosomaler Nähe zueinander (miRNA Cluster) angeordnet, und werden gemeinsam transkribiert und reguliert. Der Schwerpunkt dieser Arbeit war es, die neu gewonnenen Sequenzinformationen zu verwenden, um endogene miRNA Cluster zu vervielfältigen und zu überexprimieren, und ihre Wirksamkeit zu chimären zu vergleichen, welche in CHO-Zellen in der prä-genomischen Sequenz Ära Stand der Technik waren. Außerdem, um miRNAs, welche mit dem Zellwachstum verbunden sind, zu identifizieren, wurden die miRNA Expressionsprofile von mehreren CHO-Zelllinien in verschiedenen Kultiviergefäßen und Wachstumsphasen analysiert und es zeigte sich, dass mehrere miRNAs eine konstante Korrelation zur Wachstumsrate aufweisen.Chinese Hamster Ovary (CHO) cells are the cell factory of choice when producing complex human-like therapeutic proteins. Lots of effort has been taken to optimize these cells, especially by using genetic engineering. A novel method is the use of microRNAs (miRNAs) to drive cells towards desired phenotypes. When the first genomic sequence of CHO cells became publicly available in 2011, effort has been taken to identify and annotate miRNAs in the CHO genome. As it has been shown in other species, miRNAs are often located in close chromosomal proximity to each other (miRNA cluster), and are transcribed and regulated jointly. The focus of this thesis was to use the newly gained sequence information to amplify and overexpress endogenous miRNA cluster, and compare their effectivity to chimeric ones, which were state-of the art in CHO cells in the pre-genomic sequence era. In addition, to identify miRNAs connected to cell growth, miRNA expression profiles of several CHO cell lines in different cultivation vessels and growth phases were analyzed and indicated several miRNAs constantly correlating to the growth rate.eingereicht von: Dipl.-Ing. Gerald KlanertZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersUniversität für Bodenkultur Wien, Dissertation, 2016OeBB(VLID)193035

Improved Bioavailability and Bioaccessibility of Lutein and Isoflavones in Cultured Cells In Vitro through Interaction with Ginger, Curcuma and Black Pepper Extracts

Intestinal absorption is intrinsically low for lipophilic micronutrients and phytochemicals. Plant extracts acting as bioavailability enhancers can complement for this deficiency by modulation of both, physicochemical and biochemical parameters, in the absorption process. However, these interactions often are limited to specific conditions and the mechanisms and potential synergisms are poorly understood. In this work, we used a human intestinal cell line to characterize the impact of extracts from C. longa (curcuma), Z. officinale (ginger) and P.nigrum (black pepper) on uptake and transport rates of the xanthophylls lutein and zeaxanthin as well as soy isoflavones measured by HPLC-DAD. We found a significant increase in the uptake of lutein in the presence of curcuma extract and enhanced isoflavone transport rates mediated by curcuma and ginger extracts. Combinations of the plant extracts did not lead to any additional increase in uptake or transport rates. By investigation of mixed micelle incorporation efficiency, we could dismiss changes in bioaccessibility as a potential enhancing mechanism in our experimental setup. We further conducted a rhodamine 123 efflux assay and discovered inhibition of P-glycoproteins by the ginger and black pepper extracts, highlighting a plausible route of action leading to increased isoflavone bioavailability

Extracts Prepared from Feed Supplements Containing Wood Lignans Improve Intestinal Health by Strengthening Barrier Integrity and Reducing Inflammation

Lignans are known to exhibit a broad spectrum of biological activities, indicating their potential as constituents of feed supplements. This study investigated two extracts derived from the feed supplements ‘ROI’ and ‘Protect’—which contain the wood lignans magnolol and honokiol (‘ROI’), or soluble tannins additional to the aforementioned lignans (‘Protect’)—and their impact on selected parameters of intestinal functionality. The antioxidant and anti-inflammatory properties of the extracts were determined by measuring their effects on reactive oxygen species (ROS) and pro-inflammatory cytokine production in vitro. The impact on intestinal barrier integrity was evaluated in Caco-2 cells and Drosophila melanogaster by examining leaky gut formation. Furthermore, a feeding trial using infected piglets was conducted to study the impact on the levels of superoxide dismutase, glutathione and lipid peroxidation. The Protect extract lowered ROS production in Caco-2 cells and reversed the stress-induced weakening of barrier integrity. The ROI extract inhibited the expression or secretion of interleukin-8 (IL-8), interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα). Moreover, the ROI extract decreased leaky gut formation and mortality rates in Drosophila melanogaster. Dietary supplementation with Protect improved the antioxidant status and barrier integrity of the intestines of infected piglets. In conclusion, wood lignan-enriched feed supplements are valuable tools that support intestinal health by exerting antioxidant, anti-inflammatory and barrier-strengthening effects