EFRI DCheM: Distributed Ribonucleic Acid (RNA) Manufacturing via Continuous Enzymatic Reaction and Separation in Biphasic Liquid Media

EFRI DCheM：通过双相液体介质中的连续酶促反应和分离进行分布式核糖核酸 (RNA) 制造

• 批准号: 2132141
• 负责人: Daeyeon Lee
• 金额: $ 200万
• 依托单位: University of Pennsylvania
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2132141&HistoricalAwards=false
• 关键词: EFRI; DCheM; Distributed; Ribonucleic; Acid

Vaccines based on messenger RNA (mRNA) have played a crucial role in changing the trajectory of the COVID-19 pandemic and will become increasingly important in developing new vaccines for future diseases. RNA-based therapies are also projected to have a major impact in formulating new cancer treatments as well as regenerative medicines that enable repair and regrowth of damaged tissues. Despite their proven effectiveness and enormous potential, RNA-based therapies are notoriously difficult to distribute. Because these therapeutics are inherently fragile, they require ultracold storage and shipping. This project will overcome this challenge by developing a novel technology to produce mRNA on-site and on-demand in any location while protecting the product from degradation, obviating the need for ultracold storage and transportation. Furthermore, this technology will lower the cost of production and distribution, minimize energy consumption, and reduce greenhouse gas emissions by simplifying the vaccine supply chain. Specifically, the proposed project will develop a transformative process for distributed ribonucleic acid manufacturing (DReAM) based on a novel approach to produce and stabilize mRNA in a single processing step. DReAM exploits reactive membranes that contain a water layer and an oil layer. The mRNA is enzymatically produced in the water and then extracted into the oil, where the mRNA is stable and protected from degradation. The DReAM technology can further serve national interests by enabling on-site production of other pharmaceutical products in a wide range of settings, supporting space exploration, national defense, and recovery from natural disasters. This project will draw graduate and undergraduate students from geographically diverse locations, with emphasis on institutions serving students underrepresented in STEM, to grow and diversify the doctoral STEM workforce. Furthermore, the team will focus on disseminating DReAM’s vision to K-12 students and the public to generate interest in STEM-related careers. The DReAM team will create a framework that identifies the expertise and infrastructure needed to maximize economic growth and employment opportunities. More importantly, success of the DReAM effort will address the grand challenge that has stymied progress in delivering advanced healthcare more efficiently and equitably. Our vision is to disrupt the field of RNA manufacturing and distribution while impacting the national and global need for equitable distribution and administration of life-saving therapeutics, including critical mRNA-based vaccines. This EFRI project will develop a transformative process for distributed ribonucleic acid manufacturing (DReAM) using bicontinuous interfacially-jammed emulsion gels (bijels), a microstructured membrane developed by members of the research team, which allow for simultaneous RNA synthesis and separation. The DReAM process will enable distributed continuous production of RNA on-demand and shift the current paradigm in the pharmaceutical industry where centralized batch processes remain the norm. We propose to leverage the inherent stability of DNA as a genetic template to produce mRNA at the oil-aqueous interface through the activity of RNA polymerase while feeding DNA from the aqueous phase. Upon transcription of the DNA, the mRNA will be selectively sequestered in the oil phase via lipid-mediated interphase transfer. Partitioning of the mRNA into the organic phase will isolate mRNA from the reagent stream in situ and stabilize mRNA against deleterious hydrolysis, obviating the need for cryogenic transportation, which will dramatically transform the field. The bioequivalence of mRNA produced by DReAM will be confirmed through in vitro translation and cell-based assays. To realize this vision, experimental, computational, and modeling approaches will be integrated to address the effects of crowding of surface-active particles and molecules on interfacial dynamics, the activity of nanoparticle-immobilized enzymes at the fluid-fluid interface as affected by the interface microstructure, and the mechanisms for transport and partitioning of biomacromolecules in chemically heterogeneous, topologically complex structures. Molecular modeling will be integrated into all aspects of the project including fundamental characterization, macroscopic modeling, and control of the DReAM process.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

基于信使 RNA (mRNA) 的疫苗在改变 COVID-19 大流行的轨迹方面发挥了至关重要的作用，并且在开发针对未来疾病的新疫苗方面将变得越来越重要，预计也会对制定疫苗产生重大影响。新的癌症治疗方法以及能够修复和再生受损组织的再生药物，尽管基于 RNA 的疗法已被证明有效且具有巨大的潜力，但由于这些疗法本质上是脆弱的，因此它们需要超冷储存和运输。项目将克服这一挑战通过开发一种新技术，可以在任何地点现场按需生产 mRNA，同时保护产品免遭降解，无需超冷储存和运输。此外，该技术将降低生产和分销成本，最大限度地减少能源消耗。具体来说，该项目将开发一种分布式核糖核酸制造 (DReAM) 的变革性工艺，该工艺基于一种在单个处理步骤中生产和稳定 mRNA 的新方法。膜包含水层和油层的 mRNA 在水中通过酶法产生，然后提取到油中，其中 mRNA 是稳定的并且不会被降解，DReAM 技术可以通过实现其他物质的现场生产来进一步服务于国家利益。该项目将吸引来自不同地理位置的研究生和本科生，重点关注为 STEM 中代表性不足的学生提供服务的机构，以促进其发展和多样化。博士STEM此外，该团队将重点向 K-12 学生和公众传播 DReAM 的愿景，以激发对 STEM 相关职业的兴趣。 DReAM 团队将创建一个框架，确定最大限度地提高经济增长和就业机会所需的专业知识和基础设施。更重要的是，DReAM 努力的成功将更有效、更公平地解决阻碍先进医疗保健进步的巨大挑战，我们的愿景是颠覆 RNA 制造和分配领域，同时影响国家和全球对公平分配和管理的需求。该 EFRI 项目将开发一种利用双连续界面堵塞乳液凝胶（bijels）进行分布式核糖核酸制造（DReAM）的变革性工艺，bijels 是一种由研究团队成员开发的微结构膜。 ，它允许同时进行 RNA 合成和分离，DReAM 工艺将实现 RNA 按需的分布式连续生产，并改变制药行业当前集中式批量工艺仍然是常态的模式。提出利用DNA的固有稳定性作为遗传模板，通过RNA聚合酶的活性在油-水界面产生mRNA，同时从水相供给DNA。DNA转录后，mRNA将被选择性地隔离在油中。通过脂质介导的相间转移将 mRNA 分配到有机相中，将 mRNA 从试剂流中原位分离出来，并稳定 mRNA 免受有害的水解，从而无需低温运输，这将极大地改变该领域的生物等效性。 DReAM 产生的 mRNA 将通过体外翻译和基于细胞的测定得到证实，为了实现这一愿景，将整合实验、计算和建模方法，以解决表面活性颗粒和分子拥挤对界面动力学的影响。纳米粒子固定化酶在流体-流体界面上受界面微观结构影响的活性，以及​​化学异质、拓扑复杂结构中生物大分子的运输和分配机制将被整合到其中。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。