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

• Published in: Biotechnology journal Vol. 9; no. 3; pp. 396 - 404
• Main Authors: Sanchez, Noelia, Kelly, Paul, Gallagher, Clair, Lao, Nga T., Clarke, Colin, Clynes, Martin, Barron, Niall
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01-03-2014; WILEY‐VCH Verlag
• Subjects: Animals; Antibodies, Monoclonal; Biopharmaceutical production; Bioreactors; Cell Count; Cell Culture Techniques - methods; Chinese hamster ovary; CHO Cells; Cricetinae; Cricetulus; Genetic Vectors; Longevity - genetics; MicroRNAs - antagonists & inhibitors; MicroRNAs - genetics; miRNA; miRNA sponge; Recombinant Proteins - biosynthesis; Recombinant Proteins - metabolism; Target mediated miRNA protection; miRNA sponge; miRNA; Chinese hamster ovary; Target mediated miRNA protection; Biopharmaceutical production
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.201300325

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.
miRNAs have the ability to regulate the expression of multiple target genes in a cell. Preventing miRNA‐7 from binding its endogenous mRNA targets in Chinese Hamster Ovary cells by overexpressing a decoy sponge results in higher cell density and prolonged cellular viability. This results in less apoptosis and therefore improved recombinant protein yield from cells in the bioreactor.