Multidimensional approaches to understand and improve RNA therapeutic design and delivery

PROJECT SUMMARY: RNA therapeutics are experiencing a renaissance with the clinical successes of the COVID-19 messenger RNA (mRNA) vaccines. In parallel, nanomaterials have emerged as highly promising vehicles for RNA delivery. However, despite considerable scientific advances in nanoparticle therapeutics over the last several decades, few nanoparticle-based RNA therapeutics have been clinically approved. As the RNA therapeutics landscape expands, there remain key understanding gaps that must be addressed to inform the rational design of RNA therapeutic systems across molecular, cellular, and organismal levels and enable broad clinical translation: (1) how RNA cargo parameters, carrier properties, and their combinations govern functional in vivo RNA delivery; (2) how the biological environment of the host alters RNA nanocarriers and their in vivo functionalities; and (3) in turn, how RNA therapeutic systems modify the host. Further exacerbating these knowledge gaps is a lack of high-sensitivity, high-throughput tools for interrogation of functional in vivo RNA delivery, nanoparticle-biomolecule interactions, and the host response. The proposed program will adopt multi- pronged strategies to address these challenges, by integrating our complementary expertise in ultrasensitive biomolecule detection, RNA engineering, and nanoparticle drug delivery. Using mRNA as a representative RNA drug, we seek to elucidate design rules for both RNA nanocarrier and cargo for functional RNA delivery. Leveraging our technology for multiplexed single-molecule detection of low abundance biomolecules, we will pursue orthogonal focus areas: (1) develop and apply an ultrasensitive screening platform for pooled in vivo analysis of mRNA therapeutic systems, to identify RNA cargo and carrier determinants of functional mRNA delivery; (2) understand and predict the host response to RNA therapeutic systems via high-multiplex single- molecule protein detection; and (3) probe the biomolecular interactions of nanocarriers and their effects on in vivo functionality via high-throughput, high-resolution profiling of biomolecule adsorption. These three independent yet synergistic directions align well with NIGMS mission objectives in the application of innovative physical methodologies and quantitative approaches to establish foundations for disease treatment. If successful, this program will build an integrated understanding of RNA therapeutic system design rules and host factors that govern RNA delivery and individual response. The ultrasensitive profiling tools developed in this work will be of broad utility across diverse RNA therapeutic systems and disease applications.