Synthetic G-quadruplex components for predictable, precise two-level control of mammalian recombinant protein expression

Melinda Pohle, Edward Curry, Suzanne Gibson, Adam Brown
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Abstract

Control of mammalian recombinant protein expression underpins the in vitro manufacture and in vivo performance of all biopharmaceutical products. However, routine optimization of protein expression levels in these applications is hampered by a paucity of genetic elements that function predictably across varying molecular formats and host cell contexts. Herein, we describe synthetic genetic components that are specifically built to simplify bioindustrial expression cassette design processes. Synthetic G-quadruplex elements with varying sequence feature compositions were systematically designed to exhibit a wide-range of regulatory activities, and inserted into identified optimal positions within a standardized, bioindustry compatible core promoter-5' UTR control unit. The resulting library tuned protein production rates over two orders of magnitude, where DNA and RNA G-quadruplexes could be deployed individually, or in combination to achieve synergistic two-level regulatory control. We demonstrate these components can predictably and precisely tailor protein expression levels in i) varying gene therapy and biomanufacturing cell hosts, and ii) both plasmid DNA and synthetic mRNA contexts. As an exemplar use-case, a vector design platform was created to facilitate rapid optimization of polypeptide expression ratios for difficult-to-express multichain products. Permitting simple, predictable titration of recombinant protein expression, this technology should prove useful for gene therapy and biopharmaceutical manufacturing applications.
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用于哺乳动物重组蛋白表达的可预测、精确的两级控制的合成 G-四链构件
对哺乳动物重组蛋白表达的控制是所有生物制药产品体外生产和体内表现的基础。然而,在这些应用中,蛋白质表达水平的常规优化却因缺乏可预测不同分子格式和宿主细胞环境下功能的遗传元件而受到阻碍。在此,我们将介绍专门用于简化生物工业表达盒设计过程的合成基因元件。我们系统地设计了具有不同序列特征组成的合成 G-四叠体元件,以显示出广泛的调控活性,并将其插入标准化的、生物产业兼容的核心启动子-5' UTR 控制单元中已确定的最佳位置。由此产生的文库将蛋白质生产率提高了两个数量级,其中 DNA 和 RNA G-quadruplexes 可单独使用,也可组合使用,以实现两级协同调控。我们证明,这些元件可以在 i) 不同的基因治疗和生物制造细胞宿主,以及 ii) 质粒 DNA 和合成 mRNA 的情况下,预测并精确定制蛋白质的表达水平。作为一个示例用例,我们创建了一个载体设计平台,用于快速优化难以表达的多链产品的多肽表达比例。该技术允许对重组蛋白的表达进行简单、可预测的滴定,因此在基因治疗和生物制药生产应用中应证明非常有用。
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