May 6-11, 2018
Genetic engineering of mammalian cells, in particular Chinese hamster ovary (CHO) cells, is of critical interest to the biopharmaceutical industry as a means to further boost the yields of therapeutic proteins. Complimentary to already in place advanced bioprocesses, stable overexpression of the pro-survival X-linked inhibitor of apoptosis (XIAP) is one example of the successful manipulation of CHO cell genetics resulting in prolonged culture survival, ultimately increasing recombinant protein productivity. However, saturation or burdening of the cells translational machinery can occur in instances of forced expression of a trans-gene thereby achieving the anticipated cellular phenotype without the associated improvement in productivity. Ribosomal footprint sequencing has demonstrated that ~15% of an IgG-producing CHO cell translatome is occupied by the Neomycin selection marker. microRNAs (miRNAs) have the ability to fine tune endogenous gene expression thereby achieving elevated gene levels without the excess that could negatively impact global gene expression. Additionally, not only does a single miRNA have the capacity to regulate multiple mRNA transcripts simultaneously but individual mRNAs can be regulated by a multitude of miRNAs at the post-transcriptional level. This can facilitate the maximal translation of an endogenous gene without surpassing the superphysiological threshold associated with diminished productivity. The promiscuous nature of miRNA represented by the variety of binding patterns associated with mRNA targeting limits the predictability of high confidence miRNA regulators of attractive engineering candidates. This results in a lengthy list of falsely predicted in-silico miRNA regulators for a single gene. We explored the identification of direct miRNA regulators of the pro-survival endogenous XIAP gene in CHO-K1 cells by using a miR-CATCH1 protocol. A biotin-tagged antisense DNA oligonucleotide was designed for an exposed predicted secondary structure loop of endogenous CHO XIAP. This mRNA anchor resulted in the pulldown of XIAP and all associated RNA/protein complexes thereby enriching for all bound miRNAs. Two miRNAs were chosen out of the 14 miRNAs identified for further validation, miR-124-3p and miR-19b-3p. Transient transfection of mimics for both resulted in the diminished translation of endogenous CHO XIAP protein whereas their inhibition increased XIAP protein levels (Fig. 1).
Please click Additional Files below to see the full abstract.
Paul S. Kelly, Alan Griffith, Sebastian Vencken, Nga T. Lao, Martin Clynes, and Niall Barron, "miR-CATCH identifies biologically active miRNA regulators of the pro-survival gene XIAP, in Chinese hamster ovary cells" in "Cell Culture Engineering XVI", A. Robinson, PhD, Tulane University R. Venkat, PhD, MedImmune E. Schaefer, ScD, J&J Janssen Eds, ECI Symposium Series, (2018). http://dc.engconfintl.org/ccexvi/245