Biology subjects

Tzani, I.

Publications and source records attributed to Tzani, I..

5 recordsLinked to original sources

bioRxiv2022

Understanding the transcriptional response to ER stress in Chinese hamster ovary cells using multiplexed single cell RNA-seq

Single cell RNA-seq (scRNA-seq) has recently been shown to provide a powerful method for the analysis of transcriptional heterogeneity in Chinese hamster ovary (CHO) cells. A potential drawback of current scRNA-seq platforms is that the cost can limit the complexity of experimental design and therefore the utility of the approach. In this manuscript, we report the use of oligonucleotide barcoding to perform multiplexed CHO cell scRNA-seq to study the impact of tunicamycin (TM), an inducer of the unfolded protein response (UPR). For this experiment, we treated a CHO-K1 GS cell line with 10g/ml tunicamycin and acquired samples at 1, 2, 4 and 8 hr post-treatment as well as a non-treated TM-control. We transfected cells with sample-specific polyadenylated ssDNA oligonucleotide barcodes enabling us to pool all cells for scRNA-seq. The sample from which each cell originated was subsequently determined by the oligonucleotide barcode sequence. Visualisation of the transcriptome data in a reduced dimensional space confirmed that cells were not only separable by sample but were also distributed according to time post-treatment. These data were subsequently utilised to perform weighted gene co-expression analysis (WGCNA) and uncovered groups of genes associated with TM treatment. For example, the expression of one group of coexpressed genes was found to increase over the time course and were enriched for biological processes associated with ER stress. The use of multiplexed single cell RNA-seq has the potential to reduce the cost associated with higher sample numbers and avoid batch effects for future studies of CHO cell biology. HighlightsO_LIPolyadenylated ssDNA oligonucleotide labelling is a viable strategy for multiplexed CHO cell scRNA-seq analysis. C_LIO_LITo demonstrate the effectiveness of the method we conducted an experiment to study the CHO cell response to tunicamycin treatment. C_LIO_LIscRNA-seq was carried out on an untreated control and at 4 time points post tunicamycin treatment. Cells from each sample were transfected with a unique oligonucleotide barcode and pooled for single cell transcriptomics. C_LIO_LIEach sample was demultiplexed post-sequencing and gene expression profiles of > 5,300 cells were obtained across the experiment. Following dimensionality reduction and visualisation, the cells were distributed according to sample identity. C_LIO_LIAnalysis of the resulting data enabled improved understanding of the transcriptional response to tunicamycin treatment. Three gene coexpression modules were found to be correlated with the tunicamycin time course. Gene set enrichment analysis revealed the over representation of genes related to biological processes associated with ER stress, and protein misfolding in one of these groups of coexpressed genes. C_LIO_LIFurther use of this approach will enable the CHO cell biology community to perform increasingly complex single cell experiments in a cost-effective manner. C_LI

cell biology↗

bioRxiv2022

Found in translation: Microproteins are a new class of potential host cell impurity in mAb drug products

Chinese hamster ovary (CHO) cells are used to produce almost 90% of therapeutic monoclonal antibodies (mAbs). The annotation of non-canonical translation events in these cellular factories remains incomplete, limiting not only our ability to study CHO cell biology but also detect host cell protein (HCP) contaminants in the final mAb drug product. We utilised ribosome footprint profiling (Ribo-seq) to identify novel open reading frames (ORFs) including N-terminal extensions and thousands of short ORFs (sORFs) predicted to encode microproteins. Mass spectrometry-based HCP analysis of four commercial mAb drug products using the extended protein sequence database revealed the presence of microprotein impurities for the first time. We also show that microprotein abundance varies with growth phase and can be affected by the cell culture environment. In addition, our work provides a vital resource to facilitate future studies of non-canonical translation as well as the regulation of protein synthesis in CHO cell lines.

genomics↗

bioRxiv2020

Tracing production instability in a clonally-derived CHO cell line using single cell transcriptomics

A variety of mechanisms including transcriptional silencing, gene copy loss and increased susceptibility to cellular stress have been associated with a sudden or gradual loss of monoclonal antibody (mAb) production in Chinese hamster ovary (CHO) cell lines. In this study, we utilised single cell RNA-seq (scRNA-seq) to study a clonally-derived CHO cell line that underwent production instability leading to a dramatic reduction of the levels of mAb produced. From the scRNA-seq data we identified sub clusters associated with variations in the mAb transgenes and observed that heavy chain gene expression was significantly lower than that of the light chain across the population. Using trajectory inference, the evolution of the cell line was reconstructed and was found to correlate with a reduction in heavy and light chain gene expression. Genes encoding for proteins involved in the response to oxidative stress and apoptosis were found to increase in expression as cells progressed along the trajectory. Future studies of CHO cell lines using this technology have the potential to dramatically enhance our understanding of the characteristics underpinning efficient manufacturing performance as well as product quality. HighlightsO_LIA clonally-derived CHO cell line in our laboratory had undergone production instability - in that the amount of intact monoclonal antibody had reduced dramatically to levels at which reliable quantitation was no longer possible. We were, however, able to detect mAb heavy and light chain protein, as well as dimerised light chain species in the cell culture media. C_LIO_LISingle cell RNA-seq was utilised to capture > 3,800 gene expression profiles from the cell line at 72hrs post seeding. C_LIO_LIAnalyses of the scRNA-seq data uncovered transcriptional heterogeneity and revealed the presence of multiple intra cell line clusters. The heavy chain transcript was detected at a significantly lower level in comparison to light chain transcripts. Light chain gene expression was not only more abundant, but also expressed more uniformly across the cell population. C_LIO_LIUsing unsupervised trajectory analysis, the emergence of heterogeneity in the cell population was traced from those cells most similar to the original isolated clone to those where transcription of the mAb heavy and light chain was undetectable. C_LIO_LISubsequent analysis of CHO cell gene expression patterns revealed a correlation between the progression of cells along the trajectory and the upregulation of genes involved in the cellular response to oxidative stress. C_LI

cell biology↗

bioRxiv2019

Sub physiological temperature induces pervasive alternative splicing in Chinese hamster ovary cells

RNA sequencing (RNASeq) has been widely used to associate alterations in Chinese hamster ovary (CHO) cell gene expression with bioprocess phenotypes, however alternative mRNA splicing, has thus far, received little attention. In this study, we utilised RNASeq for transcriptomic analysis of a monoclonal antibody producing CHOK1 cell line subjected to a temperature shift. More than 2,365 instances of differential splicing were observed 24hrs after the reduction of cell culture temperature. 1,163 of these alternative splicing events were identified in genes where no changes in abundance were detected by standard differential expression analysis. Ten examples of alternative splicing were selected for independent validation using qPCR in the monoclonal antibody producing CHOK1 line used for RNASeq and a further 2 CHOK1 cell lines. This analysis provided evidence that exon skipping and mutually exclusive splicing events occur in genes linked to the cellular response to changes in temperature and mitochondrial function. While further work is required to determine the impact of changes in mRNA sequence on cellular phenotype, this study demonstrates that alternative splicing analysis can be utilised to gain a deeper understanding of post-transcriptional regulation in CHO cells during biopharmaceutical production.

cell biology↗

bioRxiv2019

Expanding the Chinese hamster ovary cell long non-coding RNA transcriptome using RNASeq

Our ability to study Chinese hamster ovary (CHO) cell biology has been revolutionised over the last decade with the development of next generation sequencing and the publication of reference DNA sequences for CHO cells and the Chinese hamster. RNA sequencing has not only enabled the association of transcript expression with bioreactor conditions and desirable bioprocess phenotypes but played a key role in the characterisation of protein coding and small non-coding RNAs. The annotation of long non-coding RNAs, and therefore our understanding of their role in CHO cell biology, has been limited to date. In this manuscript, we use high resolution RNASeq data to more than double the number of annotated lncRNA transcripts for the CHOK1 genome. In addition, the utilisation of strand specific sequencing enabled the identification of more than 1,000 new lncRNAs located antisense to protein coding genes. The utility of monitoring lncRNA expression is demonstrated through an analysis of the transcriptomic response to a reduction of cell culture temperature and identification of simultaneous sense/antisense differential expression for the first time in CHO cells. To enable further studies of lncRNAs, the transcripts annotated in this study have been made available for the CHO cell biology community.

cell biology↗