Therapeutic in vitro transcribed (IVT) messenger RNAs (mRNAs) have become a revolutionary tool in the realm of medicine, particularly in the development of mRNA-based therapies and vaccines.
A crucial aspect of these therapeutic mRNAs is the incorporation of modified ribonucleotides. These modifications aim to mitigate innate immunogenicity and enhance mRNA stability, critical factors for the success of mRNA-based therapies and vaccines. One such modification, N1-methylpseudouridine (1-methylΨ), is present in clinically approved SARS-CoV-2 mRNA vaccines, highlighting its significance in the field.
Background: The specific impact of various modified ribonucleotides, including 5-methoxyuridine (5-methoxyU), 5-methylcytidine (5-methylC), and 1-methylΨ, on mRNA translation fidelity remains largely unexplored. While these modifications have been utilized to boost recombinant protein synthesis in vitro and validate the proof of concept for IVT mRNA-based therapies, the detailed effects on translation fidelity have not been comprehensively investigated.
Experimental Design: To unravel the nuances of how ribonucleotide modifications influence mRNA translation, a series of in vitro experiments were conducted. The researchers designed and synthesized IVT mRNAs, specifically Fluc+1FS, to serve as reporters for out-of-frame protein synthesis. These mRNAs encoded segments of firefly luciferase with modifications such as 5-methoxyU, 5-methylC, and 1-methylΨ. The impact of these modifications on translation fidelity was rigorously assessed through a combination of in vitro translation assays and cellular transfection experiments using HeLa cells.
Results: The study revealed that while 1-methylΨ and 5-methylC modifications did not significantly affect translation efficiency individually, their combination led to decreased translation of the unmodified mRNA. Intriguingly, 5-methoxyU alone or in combination with 5-methylC significantly reduced translation efficiency. Moreover, the incorporation of 1-methylΨ notably increased ribosomal +1 frameshifting during mRNA translation.
Immunogenicity Implications: Given the clinical relevance of 1-methylΨ in licensed SARS-CoV-2 mRNA vaccines, the researchers explored its impact on immunogenicity in vivo. Mice vaccinated with BNT162b2, a SARS-CoV-2 mRNA vaccine containing 1-methylΨ, exhibited an increased T cell response to +1 frameshifted spike peptides. This effect was not observed in mice vaccinated with a control mRNA vaccine, suggesting that 1-methylΨ can elicit off-target cellular immune responses.
Mechanistic Insights: To understand the molecular mechanisms underlying +1 ribosomal frameshifting induced by 1-methylΨ, the researchers performed detailed analyses, including western blotting and mass spectrometry. The results demonstrated that 1-methylΨ led to the synthesis of +1 frameshifted polypeptides, providing mechanistic insights into the phenomenon. Importantly, high-throughput RNA sequencing ruled out transcriptional errors as the cause, confirming that the frameshifted products resulted from bona fide ribosomal +1 frameshifting.
Translation Elongation and Stalling: The study delved into the kinetics of translation elongation during 1-methylΨ mRNA translation. The findings indicated that translation of 1-methylΨ mRNA exhibited slower elongation rates, with evidence of ribosome stalling. Further experiments with the aminoglycoside paromomycin suggested that altered aminoacyl-tRNA binding kinetics contributed to the observed ribosome stalling.
Sequence Design for Mitigation: Considering the potential limitations posed by ribosomal frameshifting in therapeutic applications, the researchers explored mRNA sequence modifications to reduce such events. Using a reporter mRNA system, they identified specific ribosome slippery sequences associated with +1 frameshifting. By synonymously mutating these sequences, the researchers demonstrated the feasibility of altering mRNA sequences to mitigate ribosome frameshifting.
The findings presented in this study unveil a novel and significant impact of 1-methylΨ, a modified ribonucleotide, on +1 ribosomal frameshifting during mRNA translation. This represents a groundbreaking revelation as it marks the first report demonstrating the influence of mRNA modification on ribosomal frameshifting. The repercussions of this frameshifting extend beyond the realm of translational dynamics, encompassing profound implications for host T cell immunity and the potential generation of new B cell antigens.
- 1-methylΨ: This is a modified version of the usual uridine building block in RNA.
- Ribosomal frameshifting: This is a rare event where the ribosome “slips” while reading the mRNA code, resulting in a different protein being made.
- Impact: The study found that 1-methylΨ significantly increases +1 frameshifting, meaning the ribosome is more likely to slip forward by one nucleotide.
- Implications: This has important consequences for both protein production and the immune system:
- Protein production: Making different proteins can change how cells behave.
- Immune system: Frameshifting can create new antigens, which the immune system can learn to recognize and attack. This could be beneficial for developing new vaccines.
This is a significant discovery that could have wide-ranging implications for understanding how genes work and developing new medical therapies.
Host Immune Response and Off-Target Effects: One of the central observations is the substantial increase in +1 ribosomal frameshifting induced by 1-methylΨ, leading to the activation of cellular immunity against +1 frameshifted products following vaccination with mRNA containing this modification. This raises significant implications for vaccine design and suggests that the off-target effects of ribosomal frameshifting may extend to increased production of B cell antigens. The interplay between mRNA modifications and the host immune response introduces a new layer of complexity in the evaluation and optimization of mRNA-based vaccines.
Comparison with Other Modification Strategies: In the broader landscape of ribonucleotide modification strategies, the study contrasts the impact of 1-methylΨ with other modifications, such as 5-methoxyU. While 1-methylΨ significantly increases frameshifting, the incorporation of 5-methoxyU leads to a decrease in translation efficiency of in vitro transcribed mRNAs. This divergence in effects underscores the need for meticulous consideration of the choice of ribonucleotide modifications in the development of mRNA therapeutics, recognizing the potential limitations and advantages associated with each modification strategy.
Exploration of Mistranslation Events: While the focus of this study has been on +1 ribosomal frameshifting induced by 1-methylΨ, the researchers acknowledge the possibility of other mistranslation events, such as leaky scanning, contributing to T cell responses against +1 frameshifted peptide antigens. This acknowledgment broadens the spectrum of potential immune responses triggered by mRNA modifications and highlights the complexity inherent in the host-cell recognition of modified mRNA products.
Nucleotide Insertions and Deletions: An integral aspect of the investigation pertains to the nucleotide insertions and deletions within in vitro transcribed mRNAs. The data reveal that the incorporation of 1-methylΨ does not alter the frequency of these events, indicating a stability in the overall fidelity of the transcription process. This assurance provides a foundation for attributing the frameshifted products to post-transcriptional mechanisms rather than transcriptional errors.
Mechanistic Insights and Ribosome Function: The study provides mechanistic insights into the observed frameshifting phenomenon. The slower translation elongation during 1-methylΨ mRNA translation is attributed to ribosome stalling, a process facilitated by altered aminoacyl-tRNA binding. This aligns with the established understanding that both ribosome stalling and slippery sequences are prerequisites for productive +1 ribosome frameshifting. The mechanistic data presented here align with previous observations in naturally occurring mRNAs, reinforcing the importance of ribosome stalling and slippery sequences in the context of frameshifting.
Implications for Future Therapeutics: The fundamental understanding gained from this study is of paramount importance for the future design and optimization of mRNA-based therapeutics. The potential for mistranslation events to decrease therapeutic efficacy or increase toxicity underscores the necessity for meticulous sequence design. As the field of mRNA-based therapeutics continues to evolve, the insights provided by this study serve as a critical guide for navigating the complexities associated with ribonucleotide modification strategies and their impact on mRNA translation fidelity.
reference link :https://www.nature.com/articles/s41586-023-06800-3#Sec2