Enhancing recombinant mRNA translation via interactions with the 3-UTR for improved recombinant protein yields

A recently discovered addition to translational control is the existence of small (~22nt) non-coding microRNAs (miRNAs). These RNAs repress translation of mRNAs that have target binding sites in their 3' untranslated region (3'UTRs). Multiple miRNAs can act on a single gene, however one miRNA may act on many different genes, leading to complex combinatorial regulatory networks. Cap-dependent translation may be promoted by mRNA 'looping' mediated by interaction of the poly-A tail with the cap. In current models of miRNA function, mature miRNA complexed in micro-ribonucleoprotein particles bind to their cognate sequence in the 3'UTR of mRNA and interfere with this interaction, leading to translational repression. Under certain conditions, miRNA may also promote mRNA degradation. MicroRNAs are critical for cell proliferation, death and differentiation; all of these features are important parameters for the selection of high-producing cell lines that grow well in suspension culture. Very little information exists about the function of miRNAs in recombinant cell lines, including CHO cells, used for manufacturing therapeutic proteins. To our knowledge, only the induction of growth inhibitory miRNAs mIR-21 and -24 by temperature shift or during normal batch culture have been reported in CHO cells. At Lonza we recently undertook an extensive miRNA profiling study using the miRCURY LNA microarray and found that ~56 miRNA are significantly differentially expressed during the course of a state-of-the-art industrial fed-batch bioreactor culture. On the basis of the available data, we propose the following hypothesis: Over or under-expressed miRNA in antibody-producing cell lines are functional and are not just a phenotypic feature of host cell lines in general. We plan to demonstrate that miRNAs have a role in controlling the productivity of an antibody-producing CHOK1SV cell line and begin elucidating the mechanism(s) by which these work. We propose initially to investigate a subset of the 56 miRNA that we have already identified as being differentially expressed during fermentation of CHO cells. We will concentrate upon the 5 most up/down-regulated miRNAs (which we will confirm by qRT-PCR) to determine their effect on cell growth and recombinant gene expression. We will use commercially available anti-miRNA inhibitors and follow the effect on antibody and reporter gene (luciferase) expression in stable CHO cell lines. We will also determine the effect on antibody heavy/light chain and luciferase polypeptide synthetic rates (that reflect mRNA translation) in control cells and those transiently transfected with miRNA inhibitors by immunoprecipitation followed by SDS-PAGE/phosphorimaging. To detect changes in heavy/light chain and luciferase mRNA turnover we will use actinomycin D and qRT-PCR to calculate the half-life of these transcripts in the presence and absence of miRNA inhibitors. To confirm that any changes/improvements are due to knockdown of miRNAs we will use commercially available miRNA precursor molecules (Ambion) to up-regulate the expression of the specific miRNAs of interest. The effects on antibody mRNA translational rates will be determined as described above. To begin investigating the mechanism by which miRNAs may effect recombinant protein synthesis, we will use 35S-Met labelling of nascent polypeptide chains and 2D analysis/phosphorimaging to investigate the global effect on polypeptide synthesis. For those protein spots that are up/down-regulated in response to miRNA over-expression or knockdown, we will identify the protein by mass spectrometry and use qRT-PCR to confirm that the miRNA of interest does change the mRNA turnover and/or levels that code for the identified protein. The 3'-UTR will then be cloned into a luciferase reporter gene construct and transiently transfected into CHO cells. The effect upon reporter gene expression +/- the appropriate miRNA will be determine