Leveraging cytoplasmic transcription to develop self-amplifying DNA vaccines

项目摘要 新冠肺炎疫苗发展的空前速度，彰显了疫苗平台的价值。 特别是，事实证明，mrna疫苗在引发强烈的免疫反应方面出人意料地成功，同时具有 考虑到这个系统的新颖性，这是一个非凡的安全概况。同样重要的是非凡的速度 以及它们的生产规模。尽管取得了巨大的成功，但信使核糖核酸疫苗仍面临着重大限制。 信使核糖核酸是一种天生不稳定的分子。这一特性的一个结果是，mrna疫苗需要 储存在冰柜中，它们的保质期是以解冻后的小时来衡量的。这些存储 要求被认为即使在医疗保健系统发达的国家也很难确保，而且 这在世界上许多其他地方都是极具问题的。信使核糖核酸疫苗的第二个限制是 生产不同于任何其他生物制造过程。因此，它受到严重缺乏基础设施的限制 和专业知识。新冠肺炎基因疫苗提供了想象下一代核的动力 更容易规模化生产并分发到全国各地医疗系统的酸性疫苗 世界。 这一提议假设，基于DNA的疫苗可以使设计和部署安全和 规模化生产更快、更容易、更便宜的有效疫苗。生产的临床-- 级DNA依赖于一些最简单、最快、最便宜的生物制造工艺 行业中的流程。然而，到目前为止，DNA疫苗未能引发保护性免疫反应。 因为只有一小部分进入细胞的DNA分子被运输到细胞核，在那里它们 可以转录。 在这款R21中，研究人员将通过应用来测试开发新一代DNA疫苗的可行性 从合成生物学引入允许抗原表达的遗传电路的方法 在细胞质中。自我放大的DNA疫苗将包括几个病毒起源的基因，这些基因将转录 位于细胞质中的质粒的抗原序列。此外，这些向量将包括其他 酶可以修饰信使核糖核酸分子，以提高其稳定性和翻译效率。通过介绍几个 扩增水平，抗原的表达预计会比 用传统的DNA疫苗能达到什么效果。该项目将经历八次迭代 设计-建造-测试-学习循环，利用细胞培养中的基因表达数据合理地改进疫苗设计。 如果成功，未来的研究将测试该平台与广泛抗原的兼容性，优化 提供基于DNA的疫苗，并在动物实验中分析候选疫苗的安全性和有效性。

Project Summary The unprecedented speed of COVID-19 vaccine development has demonstrated the value of vaccine platforms. In particular, mRNA vaccines proved surprisingly successful at eliciting a strong immune response while having a remarkable safety profile considering the novelty of this system. Just as important are the remarkable speed and scale of their production. Despite their spectacular success, mRNA vaccines suffer from major limitations. mRNA is an inherently unstable molecule. One consequence of this property is that mRNA vaccines need to be stored in freezers and their shelf-life is measured in hours after they have been thawed. These storage requirements are considered difficult to ensure even in countries with developed healthcare systems and are extremely problematic in many other parts of the world. The second limitation of mRNA vaccines is that their production is unlike any other biomanufacturing process. As a result, it is limited by a critical lack of infrastructure and expertise. The COVID-19 mRNA vaccines provided an incentive to imagine the next generation of nucleic acid vaccines that would be easier to manufacture at scale and distribute to healthcare systems throughout the world. This proposal hypothesizes that a DNA-based vaccine could enable the design and deployment of safe and effective vaccines that would be faster, easier, and cheaper to manufacture at scale. The production of clinical- grade DNA relies on biomanufacturing processes that are some of the simplest, fastest, and most inexpensive processes in the industry. However, DNA vaccines have failed to elicit a protective immune response so far because only a small fraction of the DNA molecules entering a cell are transported to the nucleus where they can be transcribed. In this R21, researchers will test the feasibility of developing a new generation of DNA vaccines by applying methods from synthetic biology to introduce genetic circuits allowing the expression of the antigen to take place in the cytoplasm. Self-amplifying DNA vaccines will include several genes of viral origins that will transcribe the antigenic sequences from plasmids located in the cell cytoplasm. In addition, these vectors will include additional enzymes to modify mRNA molecules to increase their stability and translation efficiency. By introducing several levels of amplification, the expression of the antigen is expected to be several orders of magnitude higher than what can be achieved with traditional DNA vaccines. The project will proceed through eight iterations of the design-build-test-learn cycle to rationally improve vaccine designs using gene expression data in cell culture. If successful, future studies will test the platform compatibility with a broad range of antigens, optimize the delivery of DNA-based vaccines, and analyze the safety and efficacy of candidate vaccines in animal studies.