Codon optimality modulates cellular stress and innate immune responses triggered by exogenous RNAs.

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• The success of COVID-19 mRNA vaccines highlights the potential of mRNA therapeutics.
• Limited data exists on how synonymous codon usage impacts cellular stress and innate immunity.
• Understanding these factors is crucial for designing effective mRNA-based therapies.

Purpose of the Study:

• To investigate the impact of synonymous codon optimization on mRNA translatability, cellular stress, and innate immune responses.
• To explore how RNA modifications (nucleoside modification, circularization) affect these outcomes.
• To provide insights into engineering mRNA sequences for improved therapeutic applications.

Main Methods:

• Developed a proprietary codon optimality matrix to re-engineer luciferase reporter mRNA sequences.
• Assessed luciferase activity in various cell lines using dual-luciferase assays.
• Analyzed innate immune pathway activation and integrated stress response markers (e.g., eIF2α phosphorylation).
• Investigated the effects of nucleoside modification and RNA circularization.

Main Results:

• Optimally engineered mRNAs showed significantly higher luciferase activity compared to non-optimal sequences.
• Non-optimal mRNAs triggered innate immune pathways and the integrated stress response.
• Nucleoside modification and circularization partially or fully abrogated these adverse responses.
• Circular RNA formation enhanced RNA stability and protein expression durability.

Conclusions:

• RNA sequence, composition, and structure are critical determinants of translatability and immunogenicity.
• Suboptimal codon usage in mRNA can induce cellular stress and immune activation.
• A combined approach involving sequence optimization, chemical modification, and topological engineering is necessary for favorable therapeutic outcomes in mRNA medicine.