miR‐143 targets MAPK7 in CHO cells and induces a hyperproductive phenotype to enhance production of difficult‐to‐express proteins

Abstract

In recent years, the number of complex but clinically effective biologicals such as multi-specific antibody formats and fusion proteins has increased dramatically. However, compared to classical monoclonal antibodies (mAbs), these rather artificially designed therapeutic proteins have never undergone millions of years of evolution and thus often turn out to be difficult-to-express using mammalian expression systems such as Chinese hamster ovary (CHO) cells. To provide access to these sophisticated but effective drugs, host cell engineering of CHO production cell lines represents a promising approach to overcome low production yields. MicroRNAs (miRNAs) have recently gained much attention as next-generation cell engineering tools. However, only very little is known about the capability of miRNAs to specifically increase production of difficult-to-express proteins. In a previous study we identified miR-143 amongst others to improve protein production in CHO cells. Thus, the aim of the present study was to examine if miR-143 might be suitable to improve production of low yield protein candidates. Both transient and stable overexpression of miR-143 significantly improved protein production without negatively affecting cell growth and viability of different recombinant CHO cells. In addition, mitogen-activated protein kinase 7 (MAPK7) was identified as a putative target gene of miR-143-3p in CHO cells. Finally, siRNA-mediated knock-down of MAPK7 could be demonstrated to phenocopy pro-productive effects of miR-143. In summary, our data suggest that miR-143 might represent a novel genetic element to enhance production of difficult-to-express proteins in CHO cells which may be partly mediated by down-regulation of MAPK7. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1046-1058, 2017.

Stable miRNA overexpression in human CAP cells: Engineering alternative production systems for advanced manufacturing of biologics using miR‐136 and miR‐3074

“…These results point toward a conserved mechanism of action of these miRNAs, which may be due to the high sequence homology of miRNAs found in different organisms, leading to pro‐productive effects in production cell lines irrespective of their background (CHO‐DG44 or CHO‐K1), species (CHO or CAP), or expressed product (SEAP, mAbs). This was previously observed for miR‐557 (Fischer et al, ) or miR‐143 (Schoellhorn et al, ). However, observed differences in the strength of productivity enhancement upon transient miRNA transfection into CHO and CAP cells (Figure ) may point toward possible differences in target gene expression or conservation of binding sequences, which may diverge between species.…”

“…For long‐term expression of miRNAs, the respective precursor sequences have to be integrated into the host cell genome. In previous studies, the pEGP‐miR vector has successfully been used for miRNA overexpression in CHO production cell lines (Fischer et al, ; Schoellhorn, Fischer, Wagner, Handrick, & Otte, ), where the pre‐miRNA including its genomic context is cloned into the ß‐globin intron upstream of the GFP‐PURO fusion protein under the control of the EF‐1 alpha promoter. To test its functionality in CAP cells, we initially transfected the vector expressing only the fusion protein (pEGP‐miR‐NULL).…”

“…Although transient transfection of miRNA mimics is frequently used as a powerful tool to elucidate the effect of a certain miRNA in production cells (Barron, Kumar et al, ; Fischer et al, ; Jadhav et al, ; Stiefel et al, ; Strotbek et al, ), for final cell line engineering the miRNA has to be stably overexpressed. Here, we used pEGP‐miR (Figure –), which was previously used to overexpress miRNAs in several different cell lines (Chang et al, ; Fischer et al, ; Larsen et al, ; Motiño et al, ; Schoellhorn et al, ). Surprisingly, in CAP cells gene expression driven by the EF‐1 alpha promoter was very weak (Supporting Figure S1).…”

Leaky Expression of the TET-On System Hinders Control of Endogenous miRNA Abundance

“…For the reasons already discussed inducible expression systems do not lend themselves to use as miRNA screening tools. Yet a plethora of miRNA with known biological function in CHO cells already exists, with functions ranging from; cell growth, cell cycle, metabolism, productivity, and inhibition of apoptosis . Stable manipulation in all above cases resulted in one specific phenotype, yet the inherent trade‐off in cell metabolism driving antagonistic desirable behaviors, growth, and protein production, still exists as a major bottleneck.…”

“…These developments have largely come in the form of culture media and feed formulation, along with on and off‐line monitoring systems. Genetic engineering of CHO cells has seen increased focus since publication of the first CHO‐K1 genome including several reports of miRNA manipulation leading to desirable cell phenotypes . In all cases, the miRNA up or down regulation was through constitutive intervention.…”