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

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.