Microevolutionary dynamics of eccDNA in Chinese hamster ovary cells grown in fed-batch cultures under control and lactate-stressed conditions

Abstract

Chinese hamster ovary (CHO) cell lines are widely used to manufacture biopharmaceuticals. However, CHO cells are not an optimal expression host due to the intrinsic plasticity of the CHO genome. Genome plasticity can lead to chromosomal rearrangements, transgene exclusion, and phenotypic drift. A poorly understood genomic element of CHO cell line instability is extrachromosomal circular DNA (eccDNA) in gene expression and regulation. EccDNA can facilitate ultra-high gene expression and are found within many eukaryotes including humans, yeast, and plants. EccDNA confers genetic heterogeneity, providing selective advantages to individual cells in response to dynamic environments. In CHO cell cultures, maintaining genetic homogeneity is critical to ensuring consistent productivity and product quality. Understanding eccDNA structure, function, and microevolutionary dynamics under various culture conditions could reveal potential engineering targets for cell line optimization. In this study, eccDNA sequences were investigated at the beginning and end of two-week fed-batch cultures in an ambr^®250 bioreactor under control and lactate-stressed conditions. This work characterized structure and function of eccDNA in a CHO-K1 clone. Gene annotation identified 1551 unique eccDNA genes including cancer driver genes and genes involved in protein production. Furthermore, RNA-seq data is integrated to identify transcriptionally active eccDNA genes.

Figure 1

Growth characteristics for control and lactate-stressed CHO cell cultures. (a) Viable cell density (VCD) (10⁶ cells/mL) and cell viability (%), (b) Lactate (g/L), (c) Titer (g/L), and (d) Cell-specific productivity (pg/cell•day). Control cultures (purple circles) and stressed cultures (orange squares) N = 2. Error bars represent standard deviations.

Figure 2

Venn diagram of the genes identified on eccDNA using CIDER-Seq. Day 0 is blue, Control Day 12 is red, and Lactate-stressed Day 12 is yellow.

Figure 3

Network diagram of significantly enriched GO biological process terms (adjusted p-value < 0.1) for the human orthologs of Chinese hamster genes detected in Day 0 samples. The small gray nodes show individual genes and larger beige nodes indicate GO terms. The size of the beige nodes is proportional to the number of genes with that GO term and the colored lines indicate the GO category for which a gene belongs to.

Figure 4

Summary of literature mining results for eccDNA-relevant genes and genome instability linked genes known from literature: KEGG pathway enrichment and CHO culture eccDNA genes. (a) Top 30 of the 151 enriched KEGG pathways for the eccDNA-relevant genes texted mined from literature. The node size is proportional to the number of genes found in the pathway, while the node color indicates the pathway’s statistical significance. Pathways with significantly overlapping genes are connected by grey lines. (b) EccDNA-relevant genes identified via literature mining that were also found in CHO culture eccDNA sequences from one or more culture conditions: Day 0 (D0), Control Day 12 (C12), Lactate-stressed Day 12 (ST12). (c) GO biological process enrichment analysis of genome instability linked genes observed on CHO cell eccDNA. Gene ratio reflects the fraction of genes pertaining to a GO term and the color indicates the statistical significance.

Figure 5

Observed eccDNA biogenesis sites and characteristics. (a) Chromosome-scale heatmap of eccDNA sequences observed for the Day 0 samples. Frequency of observed eccDNA is shown in color; low (white) to high (red). Figure made with minimap2 and bedtools. (b) 500 kbp windows with the highest eccDNA mapping frequencies (Top 10 from Day 0 only). (c) Self-alignment of chromosome 9 biogenesis hot spot. Red lines indicate a direct repeat; blue lines indicate an inverted repeat. Plot made using Mummer.

Figure 6

Heat map of differentially expressed genes from RNA-seq analysis that were only observed in one culture condition. Control is abbreviated as C and Lactate-stress is abbreviated as ST. Numbers in abbreviations are indicative of the Day samples were taken. Cells with light levels of shading indicate low levels of transcript abundance while darker cells correspond to higher levels of transcript abundance.

Figure 7

Example of transcripts with regions containing SNPs that may have originated from an eccDNA template on chromosome 9 at base 14,641,267. Rows from top to bottom are: location of the sequence, coverage of the CHO-K1 reference assembly, the CHO-K1 reference genome, consensus eccDNA sequence for the Lactate-stressed Day 12 samples, coverage of the RNA data, RNA transcripts for the Lactate-stressed Day 12 cultures, and the Chinese hamster PICRH reference genome. The height of the gray bars in the coverage row reflects the amount of coverage for each nucleotide. Colored bars in the coverage row reflect SNPs relative to the reference genome. Nucleotides are shown as: adenine (A)—green, thymine (T)—red, guanine (G)—orange, and cytosine (C)—blue.