Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.
Metab Eng. 2013 Nov;20:157-66. doi: 10.1016/j.ymben.2013.10.005. Epub 2013 Oct 18.
MicroRNAs (miRNAs) are short non-coding RNAs that post-transcriptionally regulate the expression of different target genes and, thus, enable engineered gene networks to achieve complex phenotypic changes in mammalian cells. We hypothesized that exploiting this feature of miRNAs could improve therapeutic protein production processes by increasing viable cell densities and/or productivity of the mammalian cells used for manufacturing. To identify miRNAs that increase the productivity of producer cells, we performed a genome wide functional miRNA screen by transient transfection of Chinese hamster ovary (CHO) cells stably expressing an IgG1 antibody (CHO-IgG1). Using this approach, we identified nine human miRNAs that improved the productivities not only of the CHO-IgG1 cells but also of CHO cells expressing recombinant human serum albumin (HSA), demonstrating that the miRNAs act in a product-independent manner. We selected two miRNAs (miR-557 and miR-1287) positively impacting the viable cell density and the specific productivity, respectively, and then stably co-expressed them in IgG1 expressing CHO cells. In these cells, higher IgG1 titers were observed in fed-batch cultures whilst product quality was conserved, demonstrating that miRNA-based cell line engineering provides an attractive approach toward the genetic optimization of CHO producer cells for industrial applications.
微小 RNA(miRNA)是短的非编码 RNA,它们在后转录水平上调节不同靶基因的表达,从而使工程基因网络能够在哺乳动物细胞中实现复杂的表型变化。我们假设利用 miRNA 的这一特性可以通过提高哺乳动物细胞的活细胞密度和/或生产效率来改善治疗性蛋白生产工艺。为了确定增加生产细胞生产率的 miRNA,我们通过瞬时转染稳定表达 IgG1 抗体的中国仓鼠卵巢(CHO)细胞(CHO-IgG1)进行了全基因组功能 miRNA 筛选。通过这种方法,我们鉴定了 9 种人 miRNA,它们不仅提高了 CHO-IgG1 细胞的生产率,还提高了表达重组人血清白蛋白(HSA)的 CHO 细胞的生产率,表明这些 miRNA 以非产物依赖的方式发挥作用。我们选择了两种 miRNA(miR-557 和 miR-1287),分别对活细胞密度和特异性生产率有积极影响,然后在 IgG1 表达的 CHO 细胞中稳定共表达它们。在这些细胞中,在补料分批培养中观察到更高的 IgG1 滴度,而产品质量得以保留,这表明基于 miRNA 的细胞系工程为工业应用提供了一种有吸引力的 CHO 生产细胞遗传优化方法。