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

总结 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，用于获得足够量的蛋白质用于大规模筛选，以及用于 产生蛋白质生物缀合物。许多蛋白质必须在哺乳动物细胞中制造（与细菌相反）， 蛋白质需要特定的糖基化模式、二硫键形成或其它修饰， 只在哺乳动物细胞中形成。然而，哺乳动物细胞中的蛋白质生产效率要低得多 比细菌细胞中的蛋白质产量更高。这在很大程度上是因为编码转基因的mRNA仅是一种 哺乳动物细胞中总细胞mRNA的一小部分。相反，在细菌细胞中， mRNA可以占总细胞mRNA的50%。在本申请中，我们描述了一种策略， 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。这一巨大的飞跃 表达水平可以从根本上改变哺乳动物细胞中的蛋白质生产。为此，我们将使用 新的“龙卷风”技术，用于将转基因表达为环状RNA。这些环状RNA是 与相应的线性浓度相比， mRNA。Chimerna科学家已经生成了关键的概念验证数据，证明了表达能力 RNA环包含大小与蛋白质编码开放阅读框相似的插入片段。为了开发 从根本上说，新的方式来表达蛋白质在哺乳动物细胞中的高水平，这一建议的具体目标， （1）优化Tornado环状RNA的蛋白表达。环状RNA可以编码 蛋白质，如果它们含有内部核糖体进入位点（IRES）。这一目标的目的是描述 最佳IRES、插入物大小和适合于使用Tornado表达的环状RNA进行蛋白质翻译的细胞系。 我们将系统地测试这些特征中的每一个并表征蛋白质输出。(2)为了比较蛋白质 Tornado衍生的环状RNA和线性mRNA的输出。在这个目标中，我们将比较蛋白质产量 细胞溶质蛋白和分泌蛋白的基因。我们将直接比较线性和龙卷风编码的环状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。总之，这些实验将使我们能够测试这个想法， 基因编码的环状RNA可以作为哺乳动物中高水平蛋白表达的新平台， 细胞这种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， 降低蛋白质生产成本、增加蛋白质产量和简化蛋白质表达。

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.