Manipulating phenotypes by epigenetic mechanism

For many years, the plasticity and variation of phenotypes observed in CHO cell lines was attributed to genomic variation. However, while individual mutations of single genes may certainly contribute to a defined phenotype, it typically is the adaptation of the expression pattern of multiple genes which together then modulate and define cellular behavior. Such changes in the transcription pattern are defined by several layers of epigenetic regulation that act on short term and long term, serving both as rapid response mechanisms and as cellular “memory”1, 2. These include differential DNA-methylation, predominantly in promoter regions, but also in regulatory regions of the genome. These co-operate and are co-regulated with modifications of histones which change the state of chromatin and thus the accessibility for the transcriptional machinery. On top of these, there are interactions between specific genomic regions and triplex-forming long-non-coding RNAs that can both upor downregulate transcription by attracting or blocking off transcription factors. The later can serve as very rapid and very strong regulators of transcription. Such detailed understanding of the underlying mechanisms can be used to advantage to enhance our control over phenotypes both by specifically altering the expression level of individual genes (to the degree of turning them ON or OFF3) and by altering the global transcriptome to achieve enhanced cellular performance. Likewise, directed evolution and adaptation protocols also result in a new transcriptome defined by epigenic memory that lays down altered cellular behavior1. Ultimately, these tools offer new possibilities for metabolic or cellular engineering, which have the advantage of being fully reversible and dosable, as no changes in the genome sequence are required. Such epigenetic control mechanisms could be used in two directions: i) to increase the phenotypic diversity within a population, for instance during cell line development, to enable isolation of rare variants with superior properties; and ii) to stabilize an already selected phenotype such that more reproducible process outcomes are achieved. 1. Feichtinger et al. (2016) Comprehensive genome and epigenome characterization of CHO cells in response to evolutionary pressures and over time. Biotechn. Bioeng. 113:10:2241-2253 2. Hernandez et al. (2019) Epigenetic regulation of gene expression in CHO cells in response to the changing environment of a batch culture. Biotechn. Bioeng, https:\\doi.org/10.1002/bit.26891 3. Marx et al. (2018) CRISPR-based targeted epigenetic editing enables gene expression modulation of the silenced beta-galactoside alpha-2,6-sialyltransferase 1 in CHO cells. Biotechn J 13:10, 170021

To clone or not to clone? – Wrong question! An investigation on genome heterogeneity and stability and on what controls cell behavior

The most striking characteristic of CHO cells is their adaptability, which enables efficient production of proteins as well as growth under a variety of culture conditions, but also results in genomic and phenotypic instability. Potential causes include i) the high number of chromosomal rearrangements, including variation in chromosome numbers observed in CHO or any other rapidly growing cell line; ii) mutations including SNPs or InDels that change the activity or function of enzymes; iii) epigenetic changes that alter the gene expression pattern of a cell without impacting the genome sequence itself. To understand the relative contribution of these towards phenotype evolution, full genome sequences and methylomes of 6 related cell lines were analysed for changes in genome sequence and in DNA-methylation patterns. In addition, histone modifications and DNA-methylation patterns at several time points of a batch culture were determined. Finally, different methods to assess genomic stability over time were tested, including the distribution and spread of chromosome counts per cell in a population, and the analysis of large scale rearrangements by chromosome painting and amplified fragment length polymorphism (AFLP). In summary, our results reveal the following picture: On the epigenetic level, short term adaptation of gene expression patterns to alterations in the environment (such as changes in nutrient availability or waste concentrations during a process) are predominantly controlled by modifications of histones and resulting changes in chromatin states. Long term adaptation to altered culture conditions, such as the transition from adherent to suspension culture, adaptation to different media or selection of specific phenotypes, are controlled by more stable changes in DNA-methylation patterns which are largely inherited by daughter cells as long as conditions remain constant. Genomic variants including SNPs, InDels, translocations, copy number variation and inversions, occur (and disappear) on a continuous basis, even over time. These variants happen on a random basis, they may contribute to phenotype if they provide a growth advantage, however, due to their continuous occurrence they are difficult to stabilize and/or control and may well be unavoidable. The majority of SNPs (99%) have no impact on coding sequence. All populations analysed, whether subclone or pool, contained a comparable absolute number of variants and at a similar frequency distribution within the population. The effect of subcloning on genome homogeneity is thus lost by the time cells are expanded to sufficient numbers for an MCB. Due to the variation present in each population, methods to assess genomic identity or stability are severely hampered by background noise, making the use of AFLP and probably other methods such as STR or DNA fingerprinting difficult. Nevertheless, genomic changes can be followed and semi-quantified by these methods in combination with rigid statistic tests. Counting the number of chromosomes per nucleus reveals a large spread in numbers, with typically only 30-50% of cells forming a peak at a dominant chromosome count. Again, cells with aberrant chromosome counts appear and disappear on a continuous basis, as subcloning does not lead to more homogenous count distributions. Over time, chromosome counts become more divergent, frequently near-tetraploid counts appear. Chromosome painting reveals frequent, large scale rearrangements, with aberrant chromosomes present from start, and/or appearing and disappearing over time. In view of these results, the question arises whether subcloning is a suitable step to ensure genomic homogeneity and stability and whether, rather than proving that a clone is actually derived from a single cell, efforts should not be directed towards developing tools and methods that enable reliable and rapid characterization of subclones/cell lines in terms of homogeneous behavior rather than genome. While subcloning may be required for assurance of a single gene integration site or for selection of specific behavior that may be energetically more demanding and thus requires protection from outgrowth by faster growing cells, the common expectation that subclones are genomically homogenous needs to be challenged for a rapidly growing mammalian cell line

Applications of cell sorting in biotechnology

Due to its unique capability to analyze a large number of single cells for several parameters simultaneously, flow cytometry has changed our understanding of the behavior of cells in culture and of the population dynamics even of clonal populations. The potential of this method for biotechnological research, which is based on populations of living cells, was soon appreciated. Sorting applications, however, are still less frequent than one would expect with regard to their potential. This review highlights important contributions where flow cytometric cell sorting was used for physiological research, protein engineering, cell engineering, specifically emphasizing selection of overproducing cell lines. Finally conclusions are drawn concerning the impact of cell sorting on inverse metabolic engineering and systems biology

Comprehensive meta-analysis of the CHO coding transcriptome

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Implementation and evaluation of a high-throughput siRNA screening system for suspension CHO cells

Chinese Hamster Ovary (CHO) cells are the most frequently used mammalian cell factory for the production of human-like recombinant proteins. Due to existing limitations in growth and protein production, genetic optimization of CHO cell lines may significantly enhance bioprocess productivities. Knockdown of genes by siRNAs is a standard method to identify genes involved in a desirable phenotype, either because their knockdown improves or degenerates the property. As at least 13000 different transcripts are present in a cell at any time, it is of interest to develop a method that is able to efficiently test the effect of gene knockdown at an appropriate throughput and scale. Here we describe the implementation of a high-throughput and small scale siRNA screening assay for suspension CHO cells that produce a secreted fluorescent protein. First, growth of CHO cells in 384 well plates was optimized. Second, a suitable method to deliver siRNAs into CHO cells was implemented and optimized. The optimization procedure was conducted by varying initial cell number, lipofection reagent concentration, media composition and incubation time with the help of several control siRNAs. Laser cytometry was used to detect the number of cells, the amount of fluorescent protein per cell and the total fluorescence per well. In addition, cell viability was determined afterwards by the CellTiter Glo® Luminescent Cell Viability Assay. The screening system was evaluated by a pilot screen, consisting of a set of kinome-targeting siRNAs (n=2112). For assessment of reproducibility, this entire screen was conducted twice. While the viability assay shows bad reproducibility, questioning its suitability, the cell number, amount of fluorescent protein per cell, and the total fluorescence per well show a good correlation between the two screens. Target genes, capable of enhancing the phenotype of CHO cells towards a higher growth and/or productivity upon their siRNA-induced knockdown were identified. This indicates the suitability of this high-throughput siRNA screening system to identify genes that are involved in the enhancement of growth and/or productivity in CHO cells

Enhancement by reduction - Pushing the N-glycosylation capacity of CHO cells by cleaning up the Golgi

CHO cells have gained their position as the most commonly used production system for complex biological therapeutics for a variety of reasons, including their ability to produce human like N-glycosylation patterns: correct N-glycan structures ensure that a product performs adequately in terms of efficacy and without the risk of eliciting immunogenic reactions. Nevertheless, N-glycosylation in CHO cells has posed several challenges. Many cell-engineering approaches have tackled the problem of low levels of sialylation and the lack of α-2,6-linked sialic acid by introducing additional sialyltransferases as well as boosting the sialic acid pathway and the transport of CMP-NA precursors into the Golgi. Various reports show that the process of N-glycan maturation can run into limitations when the production load is high, a problem of increasing relevance as the boundaries of productivity of CHO cells are being pushed further and further. In this regard, the link between high productivity and reduced sialylation and galactosylation as well as the occurrence of high-mannose structures has been established. This observation can be partially explained by lack of sugar-precursors due to the depletion of nutrients towards the end of a bioprocess, but it has also been proposed that there is a limited capacity of the Golgi for N-glycan processing. With more glycoprotein traversing through the secretory pathway, the abundance of glycosyltransferases in the Golgi membrane might not be sufficient to act upon all N-glycan molecules. Our strategy is based on the knock-out of multiple galactosyltransferases and sialyltransferases that have no or only a minor role in N-glycosylation of recombinant proteins to generate free space in the Golgi membrane, which can then be re-populated with the most effective isoenzymes to ensure high levels of glycan maturation even at high production rates. For sialylation, ST3GAL4 was previously identified as the key player. Out of the four galactosyltransferases involved in N-glycosylation, B4GALT1 has been proposed to be the dominant isoform, but published results vary concerning the contributions of the other isoenzymes. Therefore, we studied the activity of each of these four galactosyltransferases individually by removing the respective other three isoenzymes using CRISPR and a paired sgRNA deletion strategy. Three different glycoproteins (Epo-Fc, IFNG and a heavily glycosylated Fc fusion protein) were produced transiently and analysed by mass spectrometry for site specific N-glycans. The results clearly show that B4GALT1 alone is sufficient for high levels of galactosylation of all model proteins. B4GALT2 and B4GALT3 contribute to different extents but only yield low levels of galactosylation when acting alone, with slightly protein and site-dependent effects. To enhance N-glycosylation ST3GAL4 and B4GALT1 will be overexpressed in a cell line with a thus “cleaned-up” Golgi, referring to the lack of the other isoenzymes. The superiority of this system will be validated with a transient expression system based on mRNA transfection to obtain high productivity mimicking the high production load of industrial cell lines

Assessment of genomic instability in Chinese Hamster ovary (CHO) cells

CHO cells are the number one production system for therapeutic proteins due to their ease of handling, their fast growth in suspension culture and their capability to perform complex protein folding and human-like post-translational modifications. This flexibility is in part due to, but at the same time set off by the frequent occurrence of chromosomal rearrangements and other genomic variants, which influences individual cell line performance and the stability of industrial producer cell lines, resulting in prolonged screening phases in order to isolate cells with sufficiently stable properties. Furthermore producer cell properties are also frequently lost again over time and properties within clones derived of the same cell population may vary significantly. The present work focuses on methods for quantification of the rate of chromosomal rearrangements in a given cell line over time in culture. The methods tested include Amplified Fragment Length Polymorphism (AFLP), Chromosome Painting and Chromosome Counting. The principle of AFLP is a restriction enzyme digest of genomic DNA, followed by ligation of the fragments to adapters with a predefined sequence. DNA amplification of restriction fragments is performed using selective AFLP primers complementary to the annealed adapter sequence, but containing extra nucleotides. An initial pattern of bands of digested genomic DNA is defined which allows quantification of chromosomal changes over time using sophisticated statistical techniques. The second technique used is Chromosome Counting of metaphase spreads from a statistically significant number of cells (50-100) in a CHO cell population, with a focus on the spread of counts and ploidy and on how that changes over time. Finally, using chromosome painting, translocations within and across chromosomes and the variation in individual cells within a population can be observed in fine detail. A variety of CHO host cell lines, both pools and subclones were analyzed over a period of six-months in culture. With AFLP we could identify genomic rearrangements for each cell line over time revealing different rates of genomic changes in the analyzed cell lines as well as degrees of relationship between the cell lines and clones at the starting point. Chromosome Counting indicated that the chromosome number and its variation in a CHO cell population differs not only within a population over time, but also between different CHO cell lines. Furthermore the chromosome number of a CHO cell culture changes over time. The older a culture, the more variation and diversity within the population is observed, frequently with a clear tetraploid sub-population appearing after several months in culture. Chromosome painting reveals appearance of new chromosome variants over time, but typically not within the entire population. Overall we can conclude that CHO cells are highly rearranged and that the genomic stability over a production process cannot be guaranteed

Identification of transgene integration sites, their structure and epigenetic status with Cas9-targeted nanopore sequencing in CHO cells

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Generation of a Chinese Hamster Ovary cell genome-wide deletion library

Nowadays, around 70% of all industrially produced biopharmaceuticals are generated from Chinese Hamster Ovary (CHO) cells showing the high interest for further characterization and optimization of this cell line and its derivates. Despite their importance, the connection between the CHO cell genome sequence and function has not been explored in detail so far. Forward genetic screens are the state-of-the-art approach to investigate the link between genotype and phenotype using the CRISPR system as an efficient tool for this purpose. These screens are usually focusing on the ~ 28,000 protein coding genes, which cover only ~ 3 % of the genome. Our approach aims to correlate larger functional regions of the genome, including coding and non-coding sequences, with process relevant cell behavior, such as growth and productivity. To this end, we designed a deletion library approach that targets larger genomic regions of 100 – 150 kb using paired CRISPR gRNAs. So far, we demonstrated successful and efficient deletions up to 150 kb, resulting in proper loss-of-function mutations. These modifications were analyzed on genome and phenotype level, demonstrating that deletion efficiencies are size independent. Furthermore, to enable the presence of active gRNA pairs in each individual cell, we implemented bicistronic transcription of gRNAs separated by a tRNA sequence that unequivocally links each pair. Additionally, we determined CRISPR Cpf1 – an alternative CRISPR enzyme – activity in CHO with no cross-interaction to the CRISPR/Cas9 system, providing the possibility to use the two systems in parallel, one for targeted insertion of the gRNA pair into the genome for later identification of the deleted region, the other for deletion of the corresponding genomic region itself. Currently we are working on the generation of a first smallscale deletion library targeting lncRNAs in CHO for the implementation of the strategy before going genomewide. This will then open the opportunity both of generating large scale gene knockout libraries and of characterizing non-coding genomic regions, gene clusters or regulatory elements