Manipulation and exploitation of microRNAs for enhanced recombinant protein production in Chinese hamster ovary cells

MicroRNAs (miRs) are a class of non-coding RNA that function to regulate global mammalian gene expression by mediating the translational repression of mRNAs harbouring a complementary target region sequence within their 3'UTR. The RNA-RNA binding event between the miR seed region and mRNA target region disrupts mRNA translation and hence repression of protein synthesis from multiple mRNA targets encompassing a variety of cellular processes and pathways. In addition to this multiplicity, miRs also exhibit a high level of promiscuity as a single miR may silence many mRNA targets and a single mRNA transcript may be under the regulation of multiple miRs. miR activity may therefore be manipulated to favour/inhibit cellular processes/pathways of interest to yield desirable cell phenotypes such as enhanced secretion, metabolism or growth. The engineering of miR activity has been applied to enhancing the production of recombinant proteins in the industrially-relevant Chinese hamster ovary (CHO) cell line. In the industrial setting, the utilization of CHO cells has become the dominant mammalian system for manufacturing biotherapeutic recombinant proteins due to their aptitude for accurate protein folding, assembly and performing 'human like' post-translational modifications. A historical Lonza microarray conducted on a range of GS-CHOK1SV IgG-producing cell lines identified changes in miR abundance throughout culture that correlated with growth or recombinant IgG productivity. From this screen, three miRs in particular (miR-15b, -16-1 and -34c) were selected for further study with regard to the impact of targeted miR knockdown and engineered pri-miR over-expression in recombinant and host CHO cell lines and transfectants with respect to establishing whether changes in the amounts of these miRs impacted upon CHO cell growth and productivity. The studies undertaken here have shown that the engineered over-expression of pri-miRs can improve the longevity (e.g. when over-expressing pri-miR-16-1-34c and -15b-34c-16-1) and maximum viable cell concentration (e.g. when over-expressing pri-miR-15b, -16-1 and -16-1-34c) of a CHOK1SV-GSKO host cell line whilst the expression of miR-sponges for targeted miR knockdown can enhance the cell specific productivity (e.g. when expressing miR-sponge-S6) in a variety of GS-CHOK1SV IgG-producing cell lines. In particular, miR-sponges derived from the 3'UTR of the SRPR? mRNA, which should be targeted by miR-34c, reduced miR-mediated repression of SRPR? expression and hence an increase in SRPR? mRNA and protein expression was observed. Specifically, the application of a SRPR?-derived miR-sponge construct constituting 6 miR-binding site motifs was shown to be both functional in relieving endogenous SRPR? from miR-mediated translational repression as well as potentially enabling an increase in cell specific productivity in selected recombinant CHO cell lines, suggesting that secretory capacity was limited by the availability of SRPR?. In conclusion, the studies presented here have demonstrated that the exploitation of miR activity can be an effective tool for CHO cell engineering for the tuning of recombinant protein production without placing any additional translational burdens on the cell.