miR‐CATCH Identifies Biologically Active miRNA Regulators of the Pro‐Survival Gene XIAP, in Chinese Hamster Ovary Cells

Abstract

Genetic engineering of mammalian cells is of interest as a means to boost bio-therapeutic protein yield. X-linked inhibitor of apoptosis (XIAP) overexpression has previously been shown to enhance CHO cell growth and prolong culture longevity while additionally boosting productivity. The authors confirmed this across a range of recombinant products (SEAP, EPO, and IgG). However, stable overexpression of an engineering transgene competes for the cells translational machinery potentially compromising product titre. MicroRNAs are attractive genetic engineering candidates given their non-coding nature and ability to regulate multiple genes simultaneously, thereby relieving the translational burden associated with stable overexpression of a protein-encoding gene. The large number of potential targets of a single miRNA, falsely predicted in silico, presents difficulties in identifying those that could be useful engineering tools. The authors explored the identification of direct miRNA regulators of the pro-survival endogenous XIAP gene in CHO-K1 cells by using a miR-CATCH protocol. A biotin-tagged antisense DNA oligonucleotide for XIAP mRNA is designed and used to pull down and capture bound miRNAs. Two miRNAs are chosen out of the 14 miRNAs identified for further validation, miR-124-3p and miR-19b-3p. Transient transfection of mimics for both results in the diminished translation of endogenous CHO XIAP protein whereas their inhibition increases XIAP protein levels. A 3'UTR reporter assay confirms miR-124-3p to be a bona fide regulator of XIAP in CHO-K1 cells. This method demonstrates a useful approach to finding miRNA candidates for CHO cell engineering without competing for the cellular translational machinery.

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.…”

Systematic evaluation of the microRNAome through miR-CATCHv2.0 identifies positive and negative regulators of BRAF-X1 mRNA

“…Furthermore, we ensured that this enrichment is robust by comparing it with the one obtained by performing a capture with a scrambled probe that does not recognize any sequence in the human transcriptome (SCR, Figure 1(b), upper right). Interestingly, in both cases the enrichment of target mRNA is several orders of magnitude higher than that obtained using only one probe and no sonication [10,[12][13][14], confirming the effectiveness of the variations that have been introduced in the experimental protocol.…”

“…In such methods, the 3ʹUTR under study is exogenously administered and used as bait, either as a 3ʹ-end biotinylated molecule [7] or as a chimerical transcript containing MS2 RNA hairpin motifs [8,9]. Alternatively, in the miR-CATCH method we previously developed, a cross-linking step using formaldehyde coupled with an affinity-purification step using a biotinylated DNA antisense probe are used to pull down the endogenous target mRNA of interest, together with all the microRNA species bound to it [10][11][12][13][14].…”

Enhancing CHO by Systems Biotechnology

“…Pristovšek et al* provide a new approach to efficiently select for highest yield in bioprocesses from early stages of clone selection by combining measurements for growth and titer, while Griffith et al explore a new method to fine‐tune the overexpression of engineered genes so as not to compromise the overall capacity of the translational machinery of the cell. Ha et al present data on relieving oxidative stress of CHO cells in culture, resulting in increased productivity, while Schmieder et al* present new engineering tools that increase the efficiency and applicability of genome editing by CRISPR, enabling the simultaneous deletion of multiple genes and the combination of multiple endonucleases without interference.…”