CHO cell culture longevity and recombinant protein yield are enhanced by depletion of miR‐7 activity via sponge decoy vectors

Abstract

Improving the efficiency of recombinant protein production by CHO cells is highly desirable as more complex proteins (MAbs, fusion proteins, blood/clotting factors, etc.) go into development and come onto the market. Previous reports have shown that microRNA (miRNA)-7 overexpression arrests the growth of CHO cells and that its depletion increases the proliferation of various cell types. In this study we generated stable CHO clones that overexpressed a miR-7-specific decoy transcript (sponge) downstream of a green fluorescent protein reporter gene. The miR-7 sponge efficiently diverted miR-7 away from its endogenous targets as exemplified by the increased expression of CDC7. Although the sponge effectively sequestered miR-7, it also appeared to protect the bound miRNA sequence from degradation in the cell, as exemplified by the apparent increase in mature miR-7 levels without any change in primary transcription. Phenotypically, CHO clones with sequestered miR-7 displayed improved maximum cell density (40%), significantly improved viability and an almost two-fold increase in yield of secreted protein in a fed-batch culture. These findings demonstrate that miRNA sponge transcripts could potentially be used in cell line development projects to generate producer clones that grow to higher densities and last longer in the bioreactor - thereby improving product yield.

miRNA engineering of CHO cells facilitates production of difficult‐to‐express proteins and increases success in cell line development

“…Furthermore, individual miRNAs are capable of regulating entire cellular pathways (Fischer et al, ; Hackl et al, ), and thus might have a substantial impact on CHO cell phenotypes. Currently available literature on miRNA research in CHO cells mostly focusses on the identification of miRNAs to improve recombinant protein production (Emmerling et al, ; Fischer et al, , ; Jadhav et al, ; Kelly et al, ; Loh et al, ), or cell growth (Barron et al, ; Druz et al, ; Sanchez et al, ), while other studies were dedicated to basic principles of miRNA biogenesis and function (Diendorfer et al, ; Fischer et al, ,; Hackl et al, , ; Hernandez Bort et al, ). We have previously identified miR‐557 to increase productivity of a recombinant easy‐to‐express mAb in CHO cells (Strotbek et al, ).…”

Rapid high-throughput characterisation, classification and selection of recombinant mammalian cell line phenotypes using intact cell MALDI-ToF mass spectrometry fingerprinting and PLS-DA modelling

“…In order to address this issue, a number of proteomic, transcriptomic and metabolic based studies have now been undertaken and the subsequent data used to develop models to predict the phenotype of a given cell line (e.g. Clarke et al, 2011;Clarke et al, 2012;Doolan et al, 2013;Jacob et al, 2010;Mead et al, 2009;Mead et al, 2012;Meleady et al, 2011;Sanchez et al, 2014;Selvarasu et al, 2012), however these models are usually developed from cell line data at the end of the cell line development process.…”

“…In order to address this issue, a number of proteomic, transcriptomic and metabolic based studies have now been undertaken and the subsequent data used to develop models to predict the phenotype of a given cell line (e.g. Clarke et al, 2011;Clarke et al, 2012;Doolan et al, 2013;Jacob et al, 2010;Mead et al, 2009;Mead et al, 2012;Meleady et al, 2011;Sanchez et al, 2014;Selvarasu et al, 2012), however these models are usually developed from cell line data at the end of the cell line development process.The goal of this work was to develop a screening system that would allow the selection of highly productive cell lines for monoclonal antibody (mAb) production early in the cell line development process that would use substantially less resource to achieve the same or better success rate as current methods. The vision was to be able to select a small number of cell lines based upon the analysis of data generated in multi-well plates, and take these straight to a lab-scale bioreactorevaluation stage (10 L) with a high probability that the selected cell lines were highly productive.…”

Identifying HIPK1 as Target of miR‐22‐3p Enhancing Recombinant Protein Production From HEK 293 Cell by Using Microarray and HTP siRNA Screen

“…The search for the specific affected genes was done by using microarrays, high‐throughput screening, next generation sequencing, and/or quantitative reverse transcription PCR (qRT‐PCR) . While these studies did not describe the specific mechanism by which the microRNA acted, they identified genes and pathways involved in apoptosis, HDAC5 modulation, and the ubiquitin pathway …”