Engineering bacterial group I introns for efficient production of safe and durable nucleoside-modified circular mRNA therapeutics - PROJECT SUMMARY Synthetic messenger RNAs (mRNAs) represent a new class of biopharmaceuticals with broad clinical utility for a range of diseases. The incorporation of chemically modified uridine nucleosides, pseudouridine (Ψ) and N1- methylpseudouridine (m1Ψ), significantly reduces synthetic mRNA immunogenicity. Linear nucleoside-modified mRNAs are short-lived because they are susceptible to cellular exonucleases, hindering their broad clinical utility for a range of diseases. Synthetic circular mRNAs (circRNAs) evade cellular exonucleases, resulting in a longer half-life, but efficient circularization methods are incompatible with chemical modifications. The most efficient method of RNA circularization utilizes self-splicing group I introns. Proper folding—and thus activation—of the typical group I intron is disrupted by Ψ-modified nucleotides. A survey of self-splicing introns, however, identified a compact group I intron from the cyanobacterium Azoarcus that effectively circularizes short (~150 nt) Ψ- modified RNA. The Azoarcus intron can circularize long (~2000 nt) unmodified mRNA but not long Ψ-modified RNAs. This proposal seeks to test the hypothesis that Ψ and m1Ψ prevent circularization by disrupting tertiary interactions required for rapid and efficient intron splicing and to develop efficient circularization techniques compatible with Ψ and m1Ψ for safe and stable RNA therapeutics. Aim 1 will determine how uridine modifications affect the structure of Azoarcus group I intron. High throughput structural probing will be used to study how uridine modifications stabilize inactive conformations of the Azoarcus group I intron. Aim 2 will employ in vitro evolution to identify Azoarcus group I intron variants optimized to circularize Ψ and m1Ψ-modified RNA. The Ψ- and m1Ψ-modified circRNA will be tested in immune cells for their ability to induce an immune response. This study will provide structural insights into how nucleoside modifications change RNA structure and function and develop a straightforward methodology to prepare nucleoside-modified circular mRNAs, expanding the potential uses of mRNA therapeutics beyond vaccines. In addition, the proposed research will provide training in high- throughput sequencing, in vitro biochemistry, cellular RNA sensing, nucleic acid chemistry, and structural biology to prepare the fellow for a career as an independent investigator developing next-generation mRNA therapeutics.
