How to train your cell - Towards controlling phenotypes by harnessing the epigenome of Chinese hamster ovary production cell lines

Marx, Nicolas; Eisenhut, Peter; Weinguny, Marcus; Klanert, Gerald; Borth, Nicole

doi:10.1016/j.biotechadv.2022.107924

Cited by 10 publications

(11 citation statements)

References 229 publications

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“…An initial limitation considers the relevance of the clonal effect during our cell line development (CLD) and engineering processes. Given the nature of single cell cloning, cells undergo changes (at genetic, epigenetic and transcriptomic levels) that might have affected their capacity to growth and secrete the r-proteins 52 . Although the limited number of clones evaluated for each cell line (3 for each engineering approach) might not provide a full separation of the consequences of overexpressing these two TFs’ from the clonal effect, our data showed clear correlations between the expression levels of MYC and XBP1s with cell growth and EPO titres, respectively, in CHO cells.…”

Section: Discussionmentioning

confidence: 99%

Engineering of Chinese hamster ovary cells for co-overexpressing MYC and XBP1s increased cell proliferation and recombinant EPO production

Latorre

Torres

Vergara

et al. 2023

Sci Rep

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Improving the cellular capacity of Chinese hamster ovary (CHO) cells to produce large amounts of therapeutic proteins remains a major challenge for the biopharmaceutical industry. In previous studies, we observed strong correlations between the performance of CHO cells and expression of two transcription factors (TFs), MYC and XBP1s. Here, we have evaluated the effective of overexpression of these two TFs on CHO cell productivity. To address this goal, we generated an EPO-producing cell line (CHOEPO) using a targeted integration approach, and subsequently engineered it to co-overexpress MYC and XBP1s (a cell line referred to as CHOCXEPO). Cells overexpressing MYC and XBP1s increased simultaneously viable cell densities and EPO production, leading to an enhanced overall performance in cultures. These improvements resulted from the individual effect of each TF in the cell behaviour (i.e., MYC-growth and XBP1s-productivity). An evaluation of the CHOCXEPO cells under different environmental conditions (temperature and media glucose concentration) indicated that CHOCXEPO cells increased cell productivity in high glucose concentration. This study showed the potential of combining TF-based cell engineering and process optimisation for increasing CHO cell productivity.

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Section: Discussionmentioning

confidence: 99%

Engineering of Chinese hamster ovary cells for co-overexpressing MYC and XBP1s increased cell proliferation and recombinant EPO production

Latorre

Torres

Vergara

et al. 2023

Sci Rep

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“…7 ). Further epigenomic characterisation may help establish the optimal combination of histone marks to facilitate development of synthetic UCOEs that effectively stabilise high transgene expression [15] , [48] , [49] . Both the A2UCOE and the Rsp3 UCOE have demonstrated ability to improve transgene expression within multiple cell systems, including both viral and non-viral vectors, suggesting potential application for gene therapies.…”

Section: Discussionmentioning

confidence: 99%

“…Epigenetic silencing of the recombinant gene of interest (GOI) is a major contributor to productivity loss over time [3] , [8] , [9] , [10] , [11] , [12] , [13] , [14] , [15] . This involves non-mutational gene inactivation through repressive covalent modifications, including methylation of cytosine in DNA and posttranslational modifications of histones, which can act independently or together to determine the transcriptional state of a gene [16] , [17] , [18] , [19] .…”

Section: Introductionmentioning

confidence: 99%

Use of ubiquitous chromatin opening elements (UCOE) as tools to maintain transgene expression in biotechnology

Sizer

White

2023

Computational and Structural Biotechnology Journal

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“…Following a selection step and optionally subsequent gene amplification, extensive screening processes are applied to identify and characterize stable producer cell clones. Since the epigenetic environment around the inserted expression vectors (EVs) largely regulates the functionality of the transcriptional machinery and thus transgene expression (Marx et al, 2022), (semi-)targeted integration (TI) has emerged as an alternative cell engineering approach to overcome issues related to RI. Instead of uncontrolled, RI of EVs into the CHO genome, insertion mediated either by transposases or RNAguided genome editing tools, such as CRISPR-Cas9, allows sitespecific TI in highly transcribed regions or single genomic loci (Lee et al, 2015(Lee et al, , 2016Schmieder et al, 2022;Zhao et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

Nanopore Cas9‐targeted sequencing enables accurate and simultaneous identification of transgene integration sites, their structure and epigenetic status in recombinant Chinese hamster ovary cells

Leitner

Motheramgari

Borth

et al. 2023

Biotech & Bioengineering

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The integration of a transgene expression construct into the host genome is the initial step for the generation of recombinant cell lines used for biopharmaceutical production. The stability and level of recombinant gene expression in Chinese hamster ovary (CHO) can be correlated to the copy number, its integration site as well as the epigenetic context of the transgene vector. Also, undesired integration events, such as concatemers, truncated, and inverted vector repeats, are impacting the stability of recombinant cell lines. Thus, to characterize cell clones and to isolate the most promising candidates, it is crucial to obtain information on the site of integration, the structure of integrated sequence and the epigenetic status. Current sequencing techniques allow to gather this information separately but do not offer a comprehensive and simultaneous resolution. In this study, we present a fast and robust nanopore Cas9‐targeted sequencing (nCats) pipeline to identify integration sites, the composition of the integrated sequence as well as its DNA methylation status in CHO cells that can be obtained simultaneously from the same sequencing run. A Cas9‐enrichment step during library preparation enables targeted and directional nanopore sequencing with up to 724× median on‐target coverage and up to 153 kb long reads. The data generated by nCats provides sensitive, detailed, and correct information on the transgene integration sites and the expression vector structure, which could only be partly produced by traditional Targeted Locus Amplification‐seq data. Moreover, with nCats the DNA methylation status can be analyzed from the same raw data without prior DNA amplification.

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How to train your cell - Towards controlling phenotypes by harnessing the epigenome of Chinese hamster ovary production cell lines

Cited by 10 publications

References 229 publications

Engineering of Chinese hamster ovary cells for co-overexpressing MYC and XBP1s increased cell proliferation and recombinant EPO production

Engineering of Chinese hamster ovary cells for co-overexpressing MYC and XBP1s increased cell proliferation and recombinant EPO production

Use of ubiquitous chromatin opening elements (UCOE) as tools to maintain transgene expression in biotechnology

Nanopore Cas9‐targeted sequencing enables accurate and simultaneous identification of transgene integration sites, their structure and epigenetic status in recombinant Chinese hamster ovary cells

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