Awakening dormant glycosyltransferases in CHO cells with CRISPRa

Karottki, Karen Julie la Cour; Hefzi, Hooman; Xiong, Kai; Shamie, Isaac; Hansen, Anders Holmgaard; Li, Songyuan; Pedersen, Lasse Ebdrup; Li, Shangzhong; Lee, Jae Seong; Lee, Gyun Min; Kildegaard, Helene Faustrup; Lewis, Nathan E.

doi:10.1002/bit.27199

Cited by 34 publications

(38 citation statements)

References 21 publications

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“…The dramatically lowered HCP ppm values during purification of a mAb will facilitate downstream processes. These HCP-reduced knockouts can be combined with additional advantageous genetic modifications, such as the production of glycoengineered proteins [15][16][17][18] , higher viability during long culture times 19 , viral resistance or elimination [20][21][22] , and/or clones with higher protein production stability of mAbs 23 , to create predictable upstream and downstream processes with full control over critical process parameters.…”

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confidence: 99%

Multiplex secretome engineering enhances recombinant protein production and purity

et al. 2020

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Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a "clean" Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predict that the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyze the HCP content of 6-protein, 11-protein, and 14-protein knockout clones. These cell lines exhibit a substantial reduction in total HCP content (40%-70%). We also observe higher productivity and improved growth characteristics in specific clones. The reduced HCP content facilitates purification of a monoclonal antibody. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.

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confidence: 99%

Multiplex secretome engineering enhances recombinant protein production and purity

et al. 2020

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“…As chromatin states are defined by their histone modification patterns which in turn interact with DNA methylation (Du, Johnson, Jacobsen, & Patel, 2015; Zhao et al, 2016), the random epigenetic modulation applied in this study could also result in subtle changes in the chromatin states surrounding the integration site, thus enhancing transcription. Others have used tools for targeted alterations of histone modifications or for attracting transcription factors to enhance gene expression (Karottki et al, 2020). However, these changes are transient and in effect only for as long as the corresponding “writer” is active and provided to cells.…”

Section: Discussionmentioning

confidence: 99%

“…Thus one can rationally and reversibly switch on or off the expression of endogenous genes solely by changing the promoter DNA methylation of the target region (Marx et al, 2018; Morita et al, 2016; O'Geen et al, 2017). While many other approaches, such as the targeting of transcription factors (Agne et al, 2014; Karottki et al, 2020) typically are transient, changes in DNA methylation are maintained and inherited by progeny cells over many generations (Marx et al, 2018). The main limitation of this approach is that only genes with an already known impact on productivity or phenotype and only a limited number of genes can be targeted and the effect of targeting promoter regions is predominantly on/off, but does not achieve fine tuning of expression levels.…”

Section: Introductionmentioning

confidence: 99%

Random epigenetic modulation of CHO cells by repeated knockdown of DNA methyltransferases increases population diversity and enables sorting of cells with higher production capacities

Weinguny

Eisenhut

Klanert

et al. 2020

Biotech & Bioengineering

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Chinese hamster ovary (CHO) cells produce a large share of today's biopharmaceuticals. Still, the generation of satisfactory producer cell lines is a tedious undertaking. Recently, it was found that CHO cells, when exposed to new environmental conditions, modify their epigenome, suggesting that cells adapt their gene expression pattern to handle new challenges. The major aim of the present study was to employ artificially induced, random changes in the DNA-methylation pattern of CHO cells to diversify cell populations and consequently increase the finding of cell lines with improved cellular characteristics. To achieve this, DNA methyltransferases and/or the ten-eleven translocation enzymes were downregulated by RNA interference over a time span of ∼16 days. Methylation analysis of the resulting cell pools revealed that the knockdown of DNA methyltransferases was highly effective in randomly demethylating the genome. The same approach, when applied to stable CHO producer cells resulted in (a) an increased productivity diversity in the cell population, and (b) a higher number of outliers within the population, which resulted in higher specific productivity and titer in the sorted cells. These findings suggest that epigenetics play a previously underestimated, but actually important role in defining the overall cellular behavior of production clones.

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“…These methods mainly use gene knockout, knockdown, knock-in, overexpression, mutation, or small molecule suppression technologies to change the type and concentration of glycosidases and glycosyltransferases that are available inside these cells, thereby changing the glycosylation patterns of interested proteins expressed in them. Recent advances in gene editing tools, especially the CRISPR/Cas9 system, has enabled more rapid and cost-effective cell glycoengineering (Chan et al, 2016 ; Chung et al, 2017 ; Mabashi-Asazuma and Jarvis, 2017 ; Jansing et al, 2019 ; Karottki et al, 2020 ). Currently, the most widely used cells for protein glycoengineering are mammalian cells.…”

Section: Cell-based Protein Glycoengineeringmentioning

confidence: 99%

Protein Glycoengineering: An Approach for Improving Protein Properties

Guan

et al. 2020

Front. Chem.

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Natural proteins are an important source of therapeutic agents and industrial enzymes. While many of them have the potential to be used as highly effective medical treatments for a wide range of diseases or as catalysts for conversion of a range of molecules into important product types required by modern society, problems associated with poor biophysical and biological properties have limited their applications. Engineering proteins with reduced side-effects and/or improved biophysical and biological properties is therefore of great importance. As a common protein modification, glycosylation has the capacity to greatly influence these properties. Over the past three decades, research from many disciplines has established the importance of glycoengineering in overcoming the limitations of proteins. In this review, we will summarize the methods that have been used to glycoengineer proteins and briefly discuss some representative examples of these methods, with the goal of providing a general overview of this research area.

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Awakening dormant glycosyltransferases in CHO cells with CRISPRa

Cited by 34 publications

References 21 publications

Multiplex secretome engineering enhances recombinant protein production and purity

Multiplex secretome engineering enhances recombinant protein production and purity

Random epigenetic modulation of CHO cells by repeated knockdown of DNA methyltransferases increases population diversity and enables sorting of cells with higher production capacities

Protein Glycoengineering: An Approach for Improving Protein Properties

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