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

最近发现的翻译控制的另一个补充是存在小的(~22nt)非编码microRNAs(MiRNAs)。这些RNA抑制在其3‘非翻译区(3’UTRs)具有靶结合位点的mRNAs的翻译。多个miRNA可以作用于一个基因，而一个miRNA可能作用于多个不同的基因，从而形成复杂的组合调控网络。依赖于帽的翻译可能通过多聚A尾巴与帽的相互作用而介导的信使核糖核酸“环”来促进。在目前的miRNA功能模型中，成熟的miRNA与其3‘端非编码区的同源序列结合，干扰这种相互作用，导致翻译抑制。在一定条件下，miRNA还可以促进mRNA的降解。MicroRNAs对细胞的增殖、死亡和分化至关重要；所有这些特征都是选择在悬浮培养中生长良好的高产细胞系的重要参数。目前关于miRNAs在重组细胞系中的功能的信息很少，包括用于制造治疗性蛋白质的CHO细胞。据我们所知，在CHO细胞中，只有通过温度变化或在正常批次培养中诱导生长抑制miRNAs MIR-21和MIR-24的报道。在龙沙，我们最近使用miRCURY LNA微阵列进行了一项广泛的miRNA图谱研究，发现在最先进的工业补料分批生物反应器培养过程中，~56个miRNA显著差异表达。在现有数据的基础上，我们提出了以下假设：在抗体产生细胞系中，miRNA的过度或低表达是有功能的，而不仅仅是宿主细胞系的一般表型特征。我们计划证明miRNAs在控制产生抗体的CHOK1SV细胞系的生产力中具有作用，并开始阐明这些作用的机制(S)。我们最初建议研究我们已经确定在CHO细胞发酵过程中差异表达的56个miRNA的一个子集。我们将集中于5个上调/下调最多的miRNAs(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。我们将使用商用的抗miRNA抑制剂，并观察其对稳定的CHO细胞系中抗体和报告基因(荧光素酶)表达的影响。我们还将通过免疫沉淀和SDS-PAGE/P成像来确定对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/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/改进都是由于miRNAs的敲除，我们将使用商业上可用的miRNA前体分子(Ambion)来上调目标特定miRNAs的表达。对抗体信使核糖核酸转化率的影响将如上所述确定。为了开始研究miRNAs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/磷成像来研究miRNAs对多肽合成的全球影响。对于那些对miRNA过度表达或击倒做出反应而上调/下调的蛋白质点，我们将通过质谱仪鉴定蛋白质，并使用qRT-PCR来确认目标miRNA确实改变了所识别蛋白质的mRNA周转和/或编码水平。然后将3‘-非编码区克隆到荧光素酶报告基因构建体中，并瞬时导入CHO细胞。将确定适当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