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

基于信使核糖核酸的疫苗在改变新冠肺炎大流行的轨迹方面发挥了关键作用，并将在开发针对未来疾病的新疫苗方面变得越来越重要。基于RNA的疗法预计也将在制定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方面产生重大影响。尽管基于RNA的疗法已经证明了有效性和巨大的潜力，但众所周知，它们很难分发。由于这些疗法天生就很脆弱，它们需要超低温储存和运输。该项目将通过开发一种新技术来克服这一挑战，在任何地点现场按需生产信使核糖核酸，同时保护产品不被降解，从而消除对超低温存储和运输的需求。此外，这项技术将降低生产和分销成本，最大限度地减少能源消耗，并通过简化疫苗供应链减少温室气体排放。具体地说，拟议的项目将开发一种用于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一加工步骤中生产和稳定信使核糖核酸的新方法。Dream利用含有水层和油层的反应膜。这种信使核糖核酸在水中被酶解产生，然后被提取到油中，在油中信使核糖核酸是稳定的，并且不会被降解。梦想技术可以在广泛的环境中实现其他医药产品的现场生产，支持太空探索、国防和自然灾害恢复，从而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，重点是为STEM中代表性不足的学生提供服务的机构，以增长和多样化STEM博士劳动力。此外，该团队将专注于向K-12学生和公众传播梦想的愿景，以激发人们对STEM相关职业的兴趣。梦想团队将创建一个框架，确定最大化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，梦想努力的成功将解决阻碍更高效、更公平地提供先进医疗服务方面取得进展的重大挑战。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公平分配和管理救命治疗药物的需求，包括关键的基于RNA的疫苗。该EFRI项目将开发一种用于分布式核糖核酸制造(DREAM)的变革性工艺，使用双连续界面堵塞的乳胶凝胶(Bijels)，这是一种由研究团队成员开发的微结构膜，允许同时合成和分离RNA。DREAM过程将使按需分布式连续生产RNA成为可能，并改变制药行业目前的模式，在制药行业，集中式批处理仍然是标准。我们建议利用DNA固有的稳定性作为遗传模板，通过RNA聚合酶的活性在油-水界面产生信使核糖核酸，同时从水相喂入DNA。在DNA转录后，通过脂质介导的相间转移，mRNA将选择性地隔离在油相中。将信使核糖核酸分配到有机相将在原位将信使核糖核酸从试剂流中分离出来，并稳定信使其免受有害的水解，从而消除了对低温运输的需要，这将极大地改变该领域。由DREAM产生的信使核糖核酸的生物等效性将通过体外翻译和基于细胞的分析来证实。为了实现这一愿景，将整合实验、计算和建模方法，以解决表面活性粒子和分子的拥挤对界面动力学的影响，纳米粒子固定化的酶在流体-液体界面上的活性受界面微结构的影响，以及生物大分子在化学非均质、拓扑复杂的结构中的传输和分配机制。分子建模将被整合到项目的所有方面，包括基本表征、宏观建模和梦想过程的控制。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.