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

最近发现的一个翻译控制的补充是小的（~22nt）非编码microrna （mirna）的存在。这些rna抑制在其3‘非翻译区（3’ utr）具有目标结合位点的mrna的翻译。多个miRNA可以作用于单个基因，但一个miRNA可以作用于许多不同的基因，导致复杂的组合调控网络。在目前的miRNA功能模型中，成熟的miRNA在微核糖核蛋白颗粒中与mRNA的3'UTR同源序列结合，并干扰这种相互作用，导致翻译抑制。在一定条件下，miRNA也可能促进mRNA的降解。microrna对细胞增殖、死亡和分化至关重要；所有这些特征都是选择悬浮培养高产细胞系的重要参数。关于mirna在重组细胞系（包括CHO细胞）中用于制造治疗性蛋白的功能的信息很少。据我们所知，只有在CHO细胞中通过温度变化或正常批量培养诱导生长抑制mirna mIR-21和-24的报道。在龙沙，我们最近使用miRCURY rna微阵列进行了广泛的miRNA分析研究，发现在最先进的工业补料批式生物反应器培养过程中，约56种miRNA的表达存在显著差异。根据现有数据，我们提出以下假设：在产生抗体的细胞系中，miRNA的过表达或过低表达是功能性的，而不仅仅是宿主细胞系的一种表型特征。我们计划证明mirna在控制产生抗体的CHOK1SV细胞系的生产力中起作用，并开始阐明这些作用的机制。我们建议首先研究56个miRNA中的一个子集，我们已经确定在CHO细胞发酵过程中存在差异表达。我们将集中研究5个上调/下调最多的mirna（我们将通过qRT-PCR确认），以确定它们对细胞生长和重组基因表达的影响。我们将使用市售的抗mirna抑制剂，并在稳定的CHO细胞系中跟踪对抗体和报告基因（荧光素酶）表达的影响。我们还将通过免疫沉淀和SDS-PAGE/磷酸化成像来确定对照细胞和瞬时转染miRNA抑制剂的细胞中抗体重链/轻链和荧光素酶多肽合成率（反映mRNA翻译）的影响。为了检测重链/轻链和荧光素酶mRNA周转的变化，我们将使用放线菌素D和qRT-PCR来计算这些转录本在存在和不存在miRNA抑制剂的情况下的半衰期。为了确认任何变化/改善是由于miRNA的敲低，我们将使用市售的miRNA前体分子（Ambion）来上调感兴趣的特定miRNA的表达。对抗体mRNA翻译率的影响将如上所述确定。为了开始研究mirna影响重组蛋白合成的机制，我们将使用新生多肽链的35S-Met标记和2D分析/磷酸化成像来研究对多肽合成的整体影响。对于那些因miRNA过表达或敲低而上调/下调的蛋白质点，我们将通过质谱法鉴定蛋白质，并使用qRT-PCR来确认感兴趣的miRNA确实改变了编码所鉴定蛋白质的mRNA的周转和/或水平。然后将3'-UTR克隆到荧光素酶报告基因结构中，并瞬间转染到CHO细胞中。将确定+/-合适的miRNA对报告基因表达的影响

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