Overexpression of miR-32 in Chinese hamster ovary cells increases production of Fc-fusion protein

Abstract

The demand for industrial genetically modified host cells were increased with the growth of the biopharmaceutical market. Numerous studies on improving host cell productivity have shown that altering host cell growth and viability through genetic engineering can increase recombinant protein production. During the last decades, it was demonstrated that overexpression or downregulation of some microRNAs in Chinese Hamster Ovary (CHO) cells as the host cell in biopharmaceutical manufacturing, can improve their productivity. The selection of microRNA targets has been based on their previously identified role in human cancers. MicroRNA-32 (miR-32), which is conserved between humans and hamsters (Crisetulus griseus), was shown to play a role in the regulation of cell proliferation and apoptosis in some human cancers. In this study, we investigated the effect of miR-32 overexpression on the productivity of CHO-VEGF-trap cells. Our results indicated that stable overexpression of miR-32 could dramatically increase the productivity of CHO cells by 1.8-fold. It also significantly increases cell viability, batch culture longevity, and cell growth. To achieve these results, following the construction of a single clone producing an Fc-fusion protein, we transfected cells with a pLexJRed-miR-32 plasmid to stably produce the microRNA and evaluate the impact of mir-32 overexpression on cell productivity, growth and viability in compare with scrambled control. Our findings highlight the application of miRNAs as engineering tools and indicated that miR-32 could be a target for engineering CHO cells to increase cell productivity.

Keywords: CHO cell engineering; Fc-fusion; Productivity; miR-32; microRNA.

Fig. 1

Expression of FC-fusion in CHO-K1 cells and clonal selection results. a Schematic view of the pTracer-CMV2-VEGF-trap construct. b Indicates the result of fluorescent imaging of pTracerCMV2-Aflibercept transfected cells after antibiotic selection of cell pool. c Indicates the derivative single clones after limiting dilution by their Fc-fusion expression and population heterogeneity based on percentage of GFP positive population. More details of flow cytometry results are shown in Additional file 1: Fig. S1. Error bars represent standard deviation of technical replicate (n = 3) of ELISA test

Fig. 2

VEGF-ELISA and SDS-PAGE results. a Indicates the result of VEGF-ELISA. The standard aflibercept and CHO-K1 supernatant have been used as positive and negative control respectively. Error bars represent standard deviation of technical replicate (n = 3). The stars denote the significance of differences (p < 0.05). b Indicates the reduced SDS-PAGE gel result of purified VEGF-trap produced by selected single clone. The samples were subjected to reducing (R) SDS-PAGE assay and the band of approximately 70 kDa was observed, as expected, L1 and L2 are different purified fractions, L4 standard aflibercept, L6, protein marker (10–250 kDa) L3 and L5 are empty

Fig. 3

Overexpression of miR-32 in CHO-VEGF-trap Cells. a Indicates fluorescent microscopy of miR-32 transfected Cells. b Indicates the result of comparative analysis of miR-32 expression level using qRT-PCR in miR-32 transfected Cells in compare with non-transfected (NT) and scrambled transfected (Sc) cells, the miRNA expression is illustrated as fold-change relative to the controls at day 6 of batch cultivation and normalized to U6 snoRNA. Error bars represent standard deviation of technical replicate (n = 3). The significance of differences between miR-32 transfected cells and controls was verified (p < 0.05)

Fig. 4

Comparative analysis of productivity, cell growth and viability. a A comparative analysis of productivity of mir-32 transfected CHO cells with NT and Sc controls reveals 1.8-fold increase in productivity of miR-32 overexpressed cells. Error bars represent standard deviation of technical replicate (n = 3). The significance of differences between the experimental and control groups was verified (p < 0.05). b, c The effect of stable overexpression of miR-32 on growth rate and viability of VEGF-trap expressing CHO cells during 10-days batch culture, respectively. Each point in b and c represents viable cell density and cell viability of an individual day, respectively