RNAylated proteins as a platform to develop next-generation RNA therapeutics

Recent advances in RNA technology have paved the way for the development of RNA-based therapeutics, a rapidly expanding class of treatments that revolutionize the standard care for a wide range of diseases. These innovative therapies offer a promising solution for personalized medicine, leveraging the unique properties of RNA to target previously undruggable pathways with unprecedented precision and efficacy. Moreover, RNA therapeutics are not only cost- effective and relatively simple to manufacture, but also offer a significant advantage over traditional treatments in terms of their ability to precisely modulate gene expression and target specific disease mechanisms. Despite the significant advances in recent years, there are still several limitations to overcome to unlock the full potential of RNA therapeutics, including low stability and translation efficiency of exogenously delivered RNA in mammalian systems. To address the existing challenges, one strategy is to introduce strategic RNA modifications, such as pseudouridine (Ψ), to enhance the stability and translation efficiency of nucleic acids while preventing immune response activation. The COVID-19 vaccines utilize a modified version of pseudouridine (N1-methylpseudouridine) to achieve enhanced stability, high translation efficiency, and low immunogenicity. Despite significant advancements, current therapeutics still require high and repetitive doses to be able to show therapeutic potency. To overcome the existing challenges, this thesis presents a strategy harnessing a novel RNA modification named RNAylation, catalyzed by a bacteriophage T4 ADP-ribosyltransferase ModB during Escherichia coli infection. RNAylation covalently links RNA with a protein by a natural N-glycosidic bond in a site-specific manner. Therefore, RNAylation leads to the generation of RNA-protein conjugates. Given that the covalent attachment of protein to RNA can enhance the stability of the attached RNA, we have envisioned developing a platform by employing this novel RNA modification to produce RNA-protein conjugates to improve the therapeutic potential of RNA by addressing current limitations in the RNA therapeutics field. This study establishes a robust ModB-mediated conjugation platform, defining design principles for generating a versatile platform of nucleic acid-protein conjugates with broad applicability. Therefore, this work lays the foundation to tailor RNA-protein conjugates with therapeutic properties to develop next-generation RNA therapeutics with higher stability and functionality.