Leveraging cytoplasmic transcription to develop self-amplifying DNA vaccines

利用细胞质转录开发自我扩增 DNA 疫苗

• 批准号: 10579667
• 负责人: Jean M Peccoud
• 金额: $ 20.77万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-07 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10579667
• 关键词: 2019-nCoV; Address; Animals; Antigens; Architecture; Bacteriophage T7; Biological Assay; Biology; Biomanufacturing; COVID-19; COVID-19 pandemic; COVID-19 vaccine; Cell Culture Techniques; Cell Nucleus; Cell Survival; Cells; Clinical; Code; Cold Chains; Complex; Country; Cytoplasm; DNA; DNA Vaccines; DNA amplification; DNA cassette; DNA delivery; Data; Development; Emergency Situation; Emerging Communicable Diseases; Ensure; Enzymes; Event; Feedback; Flow Cytometry; Fluorescent in Situ Hybridization; Freezing; Frequencies; Future; Gene Amplification; Gene Expression; Generations; Genes; Genetic; Genetic Transcription; Genomics; Healthcare Systems; Hour; Human; Immune response; Incentives; Industry; Inferior; Infrastructure; Investigation; Learning; Life; Location; Measures; Messenger RNA; Methods; Nucleic Acid Vaccines; Plasmids; Process; Production; Program Development; Property; RNA; RNA amplification; RNA vaccine; Research Personnel; Safety; Speed; Structure; System; T7 RNA polymerase; Technology; Temperature; Testing; Time; Transcription Initiation; Transfection; Translations; Vaccine Design; Vaccines; Variant; Viral Genes; Virus; design; design and construction; design,build,test; dosage; experimental study; expression vector; feasibility testing; future epidemic; immunogenic; immunogenicity; improved; innovation; interest; mRNA capping; manufacture; manufacturing facility; manufacturing process; next generation; novel; novel vaccines; plasmid DNA; preservation; prevent; promoter; scaffold; single molecule; socioeconomics; success; synthetic biology; trend; vaccine access; vaccine candidate; vaccine development; vaccine platform; vector; vector vaccine

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.

项目摘要 COVID-19疫苗开发的空前速度证明了疫苗平台的价值。 特别地，mRNA疫苗被证明在引发强烈的免疫应答方面令人惊讶地成功，同时具有 考虑到这个系统的新颖性，这是一个显著的安全性特征。同样重要的是 和生产规模。尽管mRNA疫苗取得了巨大的成功，但它们仍受到很大的限制。 mRNA是一种固有的不稳定分子。这一特性的一个结果是mRNA疫苗需要 储存在冰箱中，它们的保质期是在解冻后的几小时内测量的。这些存储 即使在医疗保健系统发达的国家， 在世界上的许多其他地方都是非常严重的问题。mRNA疫苗的第二个局限性是， 生产不同于任何其他生物制造过程。因此，它受到基础设施严重缺乏的限制 和专业知识。COVID-19 mRNA疫苗为想象下一代核酸疫苗提供了动力。 酸性疫苗更容易大规模生产，并分发给整个卫生保健系统。 世界 该提案假设基于DNA的疫苗可以设计和部署安全且 更快、更容易、更便宜的大规模生产。临床生产- 级DNA依赖于生物制造过程，这些过程是最简单、最快和最便宜的 工业中的过程。然而，DNA疫苗迄今未能引发保护性免疫应答 因为只有一小部分进入细胞的DNA分子被运送到细胞核， 可以被转录。 在R21中，研究人员将测试开发新一代DNA疫苗的可行性， 从合成生物学的方法，引入遗传电路，允许抗原的表达发生 在细胞质中。自扩增DNA疫苗将包括几个病毒来源的基因，这些基因将转录 来自位于细胞质中的质粒的抗原序列。此外，这些载体将包括额外的 酶修饰mRNA分子以增加其稳定性和翻译效率。通过引入几个 当扩增水平达到一定程度时，预期抗原的表达比扩增水平高几个数量级。 传统的DNA疫苗所能达到的效果。该项目将通过八个迭代进行， 设计-构建-测试-学习循环，利用细胞培养中的基因表达数据合理改进疫苗设计。 如果成功，未来的研究将测试平台与广泛抗原的相容性，优化免疫原性。 提供基于DNA的疫苗，并在动物研究中分析候选疫苗的安全性和有效性。