PROJECT SUMMARY Messenger RNA, or mRNA, and its translation into protein lies at the heart of the central dogma of molecular biology. Converting this basic cellular mechanism into a therapeutic opportunity was the basis of the first two successful COVID-19 vaccines. This technology has the potential to be further advanced into much broader therapeutic modalities, such as a gene replacement medicine for genetic diseases. Currently, mRNA molecules for human therapeutics are generated from biological enzymatic reactions. While this process can create large amounts of material, it suffers from several drawbacks. These include multiple steps in manufacturing, purity, and patient safety. However, the greatest shortcoming is the rapid turnover of mRNA in the body, which severely limits its duration of effect and tunability for a genetic medicine. Unless addressed, this shortcoming will handicap mRNA therapeutics from ever becoming more than a vaccine technology. Chemical modification was the missing ingredient and final piece necessary for the realization of other recently FDA-approved nucleic acid drugs, including antisense oligonucleotides and small interfering RNAs. Chemical modifications enabled nuclease protection, significantly extended drug half-lives, and predictable pharmacological tuning. Likewise, realizing the full potential of mRNA as a human therapeutic will ultimately come down to chemistry. RNA can be chemically synthesized in small fragments. However, no technology exists to easily create long chemically defined translation-competent mRNA molecules. In addition, most of the chemical modifications extensively characterized for their beneficial properties for other nucleic acid therapeutics have not been explored in mRNA research, and certainly not in a therapeutic context. This project proposes to tackle these challenges by generating full-length mRNAs from chemically synthesized fragments, investigating the impact of diverse chemical modifications on mRNA translation, and applying new synthetic chemical methods to make longer mRNAs suitable for human therapeutics. The aims of this proposal are to 1) evaluate the impact of specific nucleotide modifications on model mRNA translation in cells and in vitro, 2) assess the compatibility of triazole linkages with mRNA translation and on-resin “click” chemistry for solid-phase chemical synthesis of longer mRNA, and 3) demonstrate long mRNA chemical synthesis and its potential for therapeutic development in cells and in vivo. The results of this focused project should pioneer a paradigm-shifting approach to mRNA therapeutic development and open new possibilities for conferring better control over the drug properties of mRNA.

Project Narrative Although messenger RNA (mRNA) as a therapeutic holds tremendous promise, bottlenecks to development, application, and manufacturing arise due to current enzyme-based production methods. Solid-phase synthesis of chemically defined mRNAs offers an ideal solution but also presents its own challenges, including compatibility with translation and mRNA synthesis size limits. To address these challenges and unlock a new generation of mRNA therapeutics, this project creates and studies solid- phase synthesized mRNAs with diverse chemical modifications and unnatural linkages.