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 在哺乳动物细胞中大规模生产重组蛋白是制造蛋白质的过程 生物制剂作为医学疗法，用于获得足够数量的蛋白质用于大规模筛选，以及 如果满足以下条件，则许多蛋白质必须在哺乳动物细胞（而不是细菌）中产生。 该蛋白质需要特定模式的糖基化、二硫键形成或其他修饰 只能在哺乳动物细胞中形成，但是哺乳动物细胞中的蛋白质生产效率要低得多。 这主要是因为编码转基因的 mRNA 只是一种蛋白质。 相比之下，在细菌细胞中，转基因只占总细胞 mRNA 的一小部分。 mRNA 最多可占细胞总 mRNA 的 50%。在本申请中，我们描述了一种增加细胞 mRNA 的策略。 与目前水平相比，哺乳动物细胞中的转录水平提高了 50-100 倍。 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。 将转基因表达为环状 RNA 的新颖“龙卷风”技术。 与相应的线性相比异常稳定并且可以累积到异常高的水平 Chimerna 科学家已经生成了关键的概念验证数据，证明了其表达能力。 含有与蛋白质编码开放阅读框大小相似的插入片段的RNA环。 在哺乳动物细胞中高水平表达蛋白质的全新方法，该提案的具体目标 是： (1) 优化来自 Tornado 的环状 RNA 的蛋白质表达，可编码环状 RNA。 蛋白质，如果它们含有内部核糖体进入位点（IRES）。该目的的目标是表征蛋白质。 最佳 IRES、插入片段大小和适合使用 Tornado 表达的环状 RNA 进行蛋白质翻译的细胞系。 (2) 蛋白质比较 Tornado 衍生的环状 RNA 和线性 mRNA 的输出 为此目的，我们将比较蛋白质输出。 对于胞质蛋白和分泌蛋白，我们将直接比较线性 RNA 和 Tornado 编码的环状 RNA。 并确定使用 Tornado 系统实现的高水平环状 RNA 生产是否会导致 增加培养细胞中的蛋白质产量，这些实验将使我们能够检验以下想法： 基因编码的环状RNA可以作为哺乳动物高水平蛋白质表达的新平台 该表达系统可能对生物医学研究和蛋白质制造产生重大影响。 降低蛋白质制造成本，提高蛋白质产量并简化蛋白质表达。