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

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的疗法预计也将对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产生重大影响。尽管它们被证明是有效的和巨大的潜力，但基于RNA的疗法是出了名的难以推广。由于这些治疗药物本身就很脆弱，因此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，该技术可以在任何地点现场和按需生产mRNA，同时保护产品免受降解，避免超冷储存和运输的需要。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来减少温室气体排放。具体而言，拟议项目将开发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mRNA的新方法。DReAM利用含有水层和油层的反应膜。mRNA在水中酶促产生，然后提取到油中，在油中mRNA是稳定的，并被保护免于降解。DReAM技术可以通过在各种环境中现场生产其他药品，支持太空探索，国防和自然灾害恢复，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，以发展和多样化博士STEM劳动力。此外，该团队将专注于向K-12学生和公众传播DReAM的愿景，以激发他们对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努力的成功将解决阻碍更有效和公平地提供先进医疗保健的重大挑战。我们的愿景是颠覆RNA制造和分销领域，同时影响国家和全球对公平分配和管理拯救生命的治疗药物的需求，包括关键的基于mRNA的疫苗。该EFRI项目将开发一种使用双连续界面堵塞乳液凝胶（bijels）的分布式核糖核酸制造（DReAM）的变革过程，这是一种由研究团队成员开发的微结构膜，可以同时进行RNA合成和分离。DReAM过程将使分布式连续生产RNA的需求，并改变制药行业目前的模式，集中批量处理仍然是常态。我们建议利用DNA作为遗传模板的固有稳定性，通过RNA聚合酶的活性在油-水界面产生mRNA，同时从水相进料DNA。在DNA转录后，mRNA将通过脂质介导的相间转移选择性地隔离在油相中。将mRNA分配到有机相中将在原位从试剂流中分离mRNA，并稳定mRNA以防止有害水解，从而避免了对低温运输的需要，这将极大地改变该领域。将通过体外翻译和基于细胞的测定来证实DReAM产生的mRNA的生物等效性。为了实现这一愿景，实验，计算和建模方法将被整合到解决拥挤的表面活性颗粒和分子的界面动力学，纳米颗粒固定化酶的活性在流体-流体界面的影响，界面微观结构，和机制的运输和分配的生物大分子在化学上异质性，拓扑结构复杂的结构。分子建模将被整合到项目的各个方面，包括基本表征，宏观建模和DReAM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知识价值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.