Overexpression of MiR-32 in Chinese Hamster Ovary Cells Increases Production of Fc-fusion Protein

Abstract

The demand for industrial genetically modified host cells has been 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 has been demonstrated that overexpression or downregulation of some microRNAs in Chinese Hamster Ovary (CHO) cells as the most often employed host cell in biopharmaceutical manufacturing, can improve their productivity. MicroRNAs are small non-coding RNAs that play a key role in controlling cellular mechanisms through their binding to different mRNA targets and negatively regulating gene expression. In efforts to increase the host cell's productivity through microRNA engineering of the cells, some microRNA targets have been selected based on their previously identified role in human cancers. MicroRNA-32 (miR-32), which is conserved between humans and hamsters (Crisetulus griseus), has been 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 overexcretion on cell productivity, growth and viability in compare with scrambled control. Our findings highlight the application of miRNAs as CHO cell engineering tools and indicated that miR-32 could be a target for engineering CHO cells to increase cell productivity.

Kinetic and functional analysis of abundant microRNAs in extracellular vesicles from normal and stressed cultures of Chinese hamster ovary cells

“…Namely, let‐7b (common in CHO EVs from stressed cultures) may induce CHO cell apoptosis, while miR‐92a (common in CHO EVs from unstressed, exponential‐phase cultures) may promote CHO cell survival. A more robust study stably overexpressing these miRs in CHO cells will be required to fully understand the role these miRs have on industrially relevant parameters such as cell growth, viability, apoptosis, titer, and product quality (Bazaz et al, 2023; Leroux et al, 2021). Studies have revealed that specific combinations of miRs can result in phenotypic changes (Cursons et al, 2018; Kao et al, 2022), suggesting that the highly abundant miRs identified in CHO EVs may need to be examined in combination to identify any potential phenotypes.…”

Kinetic and functional analysis of abundant microRNAs in extracellular vesicles from normal and stressed cultures of Chinese Hamster Ovary (CHO) cells

“…Namely, let-7b (common in CHO EVs from stressed cultures) may induce CHO cell apoptosis, while miR-92a (common in CHO MPs from unstressed, exponential-phase cultures) may promote CHO cell survival. A more robust study stably overexpressing these miRs in CHO cells will be required to fully understand the role these miRs have on industrially-relevant parameters such as cell growth, viability, apoptosis, titer, and product quality (Bazaz et al, 2023;Leroux et al, 2021). Studies have revealed that specific combinations of miRs can result in phenotypic changes (Cursons et al, 2018;Kao et al, 2022), suggesting that the highly abundant miRs identified in CHO EVs may need to be examined in combination in order to identify any potential phenotypes.…”