Cytidine acetylation yields a hypoinflammatory synthetic messenger RNA

Synthetic messenger RNA (mRNA) is an emerging therapeutic platform with important applications in oncology and infectious disease. Effective mRNA medicines must be translated by the ribosome but not trigger a strong nucleic acid-mediated immune response. To expand the medicinal chemistry toolbox for these agents, here they report the properties of the naturally occurring nucleobase N4-acetylcytidine (ac4C) in synthetic mRNAs. We find that ac4C is compatible with, but does not enhance, protein production in the context of synthetic mRNA reporters. However, replacement of cytidine with ac4C diminishes inflammatory gene expression in immune cells caused by synthetic mRNAs. Chemoproteomic capture indicates that ac4C alters the protein interactome of synthetic mRNAs, reducing binding to cytidine-binding proteins and an immune sensor. Overall, their studies illustrate the unique ability of ac4C to modulate RNA-protein interactions and provide a foundation for using N4-cytidine acylation to fine-tune the properties of nucleic acid therapeutics.

Synthetic messenger RNA (mRNA) is an emerging therapeutic modality with a wide range of clinical applications in cancer, genetic disorders, and infectious disease. The power and portability of these platforms is perhaps most well illustrated by their rapid deployment as vectors for the delivery of vaccines directed against SARS-CoV-2, the viral pathogen responsible for the COVID-19 pandemic. These synthetic mRNAs are composed of four key molecular elements: (1) a 5′ cap, which confers stability; (2) 5′ and 3′ untranslated regions (UTRs), important for translation initiation and stability; (3) a coding sequence (CDS) that determines the identity of the protein to be expressed; and (4) modified nucleobases, which can enhance an mRNA's protein production and immune evasion. Depending on the intended application, activation of the innate immune system by a synthetic mRNA may be either desirable (e.g., for a vaccine) or detrimental (e.g., for a therapy requiring repeated dosing), emphasizing the importance of defining structure-function relationships in this area.

Design of a synthetic mRNA panel

(A) Components of synthetic mRNA.

(B) Modified nucleobases, caps, and UTRs as graphical abbreviations used to describe mRNA structure.

(C) eGFP reporter mRNAs applied to assess the effects of ac4C. This mRNA panel allows comparison of cytidine, ac4C, 5fC, and Ψ in the context of the following non-coding elements: 14: 5′ cap, Kozak 5′ UTR. 58: 5′ cap, EMCV IRES 5′ UTR. 910: uncapped, Kozak 5′ UTR. 1112: uncapped, EMCV IRES 5′ UTR.

Substantial prior work has evaluated the impact of modified nucleosides on synthetic mRNA activity, revealing context-dependent effects. For example, early studies found that universal incorporation of pseudouridine (Ψ) and related analogs can increase protein production and decrease immune stimulation within certain synthetic mRNAs. More recently, a systematic study of modified uridine analogs in over 100 mRNAs found that modified uridines can cause higher, lower, or unchanged levels of protein expression compared with canonical uridine, depending on the protein encoded and synonymous codon sequence used. These examples illustrate a paradigm in which discovery research first defines the potential capabilities of modified nucleosides, providing a suite of chemical options that subsequent “medicinal chemistry-like” efforts to optimize individual therapeutic mRNAs can draw upon.

Many of the nucleoside analogs that have been explored in synthetic RNAs are derived from naturally occurring modifications. Compared with uridine, cytidine modifications have been less well explored in synthetic mRNAs, with the majority of studies focusing on 5-methylcytidine (mC) and its analogs. Considering modifications to expand the chemical diversity of synthetic mRNA, we were intrigued by N4-acetylcytidine. This RNA modification is universally conserved among all domains of life, suggesting the evolutionary potential for either immune recognition or tolerance. N4-acetylcytidine triphosphate (ac4CTP) is compatible with conventional in vitro transcription protocols used to prepare synthetic mRNAs. Finally, a recent study demonstrated that ac4C-containing luciferase reporter mRNAs can be translated by human cells. However, the quantitative interplay of ac4C with other mRNA elements (e.g., 5′ cap and UTR), as well as the immune regulatory potential of this modification, remains to be determined for any discrete coding sequence.

Kellie D. Nance, Supuni Thalalla Gamage, Md Masud Alam, Acong Yang, Michaella J. Levy, Courtney N. Link, Laurence Florens, Michael P. Washburn, Shuo Gu, Joost J. Oppenheim, Jordan L. Meier,

Cytidine acetylation yields a hypoinflammatory synthetic messenger RNA,

Cell Chemical Biology,