Using highly expressed circular RNAs to substantially enhance protein expression yields in mammalian cells

使用高表达的环状 RNA 显着提高哺乳动物细胞中的蛋白质表达产量

• 批准号: 10081544
• 负责人: Jacob Litke
• 金额: $ 22.5万
• 依托单位: CHIMERNA THERAPEUTICS INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10081544
• 关键词: Bacteria; Biological; Biomedical Research; Cell Line; Cells; Code; Cultured Cells; Data; Exonuclease; Goals; Internal Ribosome Entry Site; Mammalian Cell; Medical; Messenger RNA; Modification; Open Reading Frames; Output; Pattern; Production; Proteins; RNA; Recombinant Proteins; Resistance; Scientist; System; Technology; Testing; Tornadoes; Transcript; Transgenes; Transgenic Organisms; Translations; circular RNA; cost; disulfide bond; experimental study; glycosylation; large scale production; manufacturing process; novel; promoter; protein expression; quantum

SUMMARY Large-scale production of recombinant proteins in mammalian cells is the process for manufacturing protein biologics as medical therapies, for obtaining sufficient quantities of proteins for large-scale screens, and for creating protein bioconjugates. Many proteins must be made in mammalian cells (as opposed to bacteria) if the protein requires specific patterns of glycosylation, disulfide bond formation, or other modifications that can only be formed in mammalian cells. However, protein production in mammalian cells is much less efficient than protein production in bacterial cells. This is largely because the mRNA encoding the transgene is only a small fraction of the total cellular mRNA in mammalian cells. In contrast, in bacterial cells, the transgene mRNA can account for up to 50% of total cellular mRNA. In this application, we describe a strategy to increase transcript levels in mammalian cells by 50-100-fold compared to current levels. This quantum leap in transcript expression levels could fundamentally change protein production in mammalian cells. To do this, we will use the novel “Tornado” technology for expressing transgenes as circular RNAs. These circular RNAs are exceptionally stable and can accumulate to exceptionally high levels compared to corresponding linear mRNAs. Chimerna scientists have generated key proof-of-concept data demonstrating the ability to express RNA circles containing inserts similar in size to protein-coding open reading frames. In order to develop a fundamentally new way to express proteins in mammalian cells at high levels, the specific aims of this proposal are: (1) To optimize protein expression from Tornado-derived circular RNAs. Circular RNAs can encode proteins if they contain an internal ribosome entry site (IRES). The goal of this aim is to characterize the optimal IRES, insert size, and cell line suitable for protein translation using Tornado-expressed circular RNA. We will systematically test each of these features and characterize protein output. (2) To compare protein output from Tornado-derived circular RNA and linear mRNAs. In this aim, we will compare protein output for cytosolic proteins and secreted proteins. We will directly compare linear to Tornado-encoded circular RNA and determine if the high-level circular RNA production achieved using the Tornado system results in increased protein production in cultured cells. Together, these experiments will allow us to test the idea that genetically encoded circular RNAs can serve as a new platform for high-level protein expression in mammalian cells. This expression system could have a major effect on biomedical research and protein manufacturing by reducing costs for protein manufacturing, increasing protein yields and simplifying protein expression.

概括 哺乳动物细胞中重组蛋白的大规模生产是生产蛋白质的过程 生物制剂作为医学疗法，用于获得大量筛查的足够数量的蛋白质以及 创建蛋白质生物缀合物。如果在哺乳动物细胞（与细菌相对）中必须制造许多蛋白质 该蛋白质需要特定的糖基化，二硫键形成或其他可以的修饰的特定模式 仅在哺乳动物细胞中形成。但是，哺乳动物细胞中的蛋白质产生的效率要低得多 比细菌细胞中的蛋白质产生。这很大，因为编码转换的mRNA只是一个 哺乳动物细胞中总细胞mRNA的小部分。相反，在细菌细胞中，转化 mRNA最多可占总细胞mRNA的50％。在此应用程序中，我们描述了一种增加的策略 与电流水平相比，哺乳动物细胞中的转录水平比50-100倍。笔录中的这个量子飞跃 表达水平可以从根本上改变哺乳动物细胞中的蛋白质产生。为此，我们将使用 小说“龙卷风”技术，用于表达翻译为圆形RNA。这些圆形RNA是 与相应的线性相比，异常稳定，可以累积到异常高的水平 mrnas。 Chimerna科学家产生了关键的概念证明数据，证明了表达能力 RNA电路包含插入物的大小与蛋白质编码开放式阅读框相似。为了发展一个 从根本上说，在高水平的哺乳动物细胞中表达蛋白质的新方法，该提案的具体目的 为：（1）从龙卷风衍生的圆形RNA中优化蛋白质表达。圆形RNA可以编码 如果蛋白质包含内部核糖体入口位点（IRES），则它们。这个目标的目的是表征 最佳IRES，插入尺寸和细胞系适用于使用龙卷风表达的圆形RNA蛋白质翻译。 我们将系统地测试这些特征，并表征蛋白质输出。 （2）比较蛋白质 来自龙卷风衍生的圆形RNA和线性mRNA的输出。在此目标中，我们将比较蛋白质输出 用于胞质蛋白和分泌蛋白。我们将直接比较线性与龙卷风编码的圆形RNA 并确定使用龙卷风系统实现的高级圆形RNA产生是否会导致 培养细胞中蛋白质产生增加。这些实验将使我们能够测试这样的想法 遗传编码的圆形RNA可以用作哺乳动物中高级蛋白表达的新平台 细胞。该表达系统可能会对生物医学研究和蛋白质制造产生重大影响 降低蛋白质生产的成本，增加蛋白质产量并简化蛋白质表达。