Complete Substitution with Modified Nucleotides in Self-Amplifying RNA Suppresses the Interferon Response and Increases Potency

Cell Biology Bioengineering Genetics Life Sciences
modified nucleotides self-amplifying RNA interferon response protein expression vaccine SARS-CoV-2

Introduction

Under the influence of the COVID-19 pandemic, mRNA vaccine technology has made significant advances. However, its short half-life and high dosage requirements have led to side effects and accessibility issues. To overcome these challenges, self-amplifying RNA (saRNA) has become a research hotspot. saRNA utilizes RNA-dependent RNA polymerase (RdRP) from alphaviruses for self-replication, theoretically enhancing vaccine efficacy and safety by reducing dosage and injection frequency. However, early studies on saRNA indicated that its strong induction of interferon (IFN) response suppresses antigen expression, lowering vaccine effectiveness. This study aims to inhibit the interferon response by fully replacing modified nucleotides, thereby enhancing the long-term protein expression and vaccine efficacy of saRNA.

Research Background

The research team is from Boston University, including the Departments of Biomedical Engineering, Biological Design Center, Virology, Immunology and Microbiology, National Emerging Infectious Diseases Laboratories, and the Department of Chemistry. The main authors, Joshua E. McGee, Jack R. Kirsch, and other co-authors published this study in the journal Nature Biotechnology, revealing the applications of modified nucleotides in saRNA and their significant benefits.

Research Process

The research mainly comprised the following steps:

  1. Construction and Synthesis of saRNA Library: A series of fully modified nucleotide substituted saRNA constructs encoding the fluorescent protein mCherry were synthesized via in vitro transcription (IVT) to evaluate their functionality.
  2. In Vitro Transfection and Screening: These saRNA constructs were transfected into HEK293T cells using lipofection. mCherry expression levels were detected via flow cytometry and fluorescence microscopy. Results showed significantly improved transfection efficiency with constructs fully substituted with 5-hydroxymethylcytidine (hm5C), 5-methylcytidine (m5C), or 5-methyluridine (m5U).
  3. Protein Expression Evaluation: Transfection experiments were conducted in HEK293T, C2C12, and Jurkat cells. Results indicated that m5C saRNA exhibited significantly higher protein expression levels than wild-type saRNA and n1-methylpseudouridine (n1mψ) mRNA across all three cell lines.
  4. Interferon Response Analysis: Experiments in human peripheral blood mononuclear cells (PBMCs) showed that m5C saRNA significantly reduced the expression of interferon-alpha (IFNα) and interferon-beta (IFNβ), demonstrating its effectiveness in suppressing early interferon responses.
  5. In Vivo Experiments: Protein expression levels and vaccine efficacy were evaluated in a mouse model, displaying that m5C saRNA provided significant protection at low doses and induced more sustained antibody responses compared to n1mψ mRNA and wild-type saRNA.

Research Results

In Vitro Transfection and Screening

The study demonstrated that saRNA fully substituted with modified nucleotides had significantly higher transfection efficiency in HEK293T cells compared to wild-type saRNA and n1mψ mRNA. Specifically, m5C saRNA’s transfection efficiency was 14 times greater, while hm5C and m5U increased by 10 and 8 times respectively. These results were further validated by flow cytometry and fluorescence microscopy analysis.

Protein Expression Evaluation

In HEK293T and C2C12 cells, the protein expression levels of m5C saRNA were 68 and 314 times higher than those of wild-type saRNA, respectively. Experiments in Jurkat cells also showed that m5C saRNA significantly increased protein expression even at low doses. These results suggest that m5C saRNA demonstrates excellent protein expression capacity in in vitro models.

Interferon Response Analysis

Experiments in human PBMCs showed that m5C saRNA could significantly suppress the expression of IFNα and IFNβ. Specifically, m5C saRNA reduced the expression of IFNα1 and IFNβ1 by 8.5 and 3 times, respectively. This indicates that m5C saRNA has a significant advantage in inhibiting early interferon responses, potentially improving vaccine efficacy and safety.

In Vivo Experiments

In a mouse model, m5C saRNA exhibited prolonged protein expression and significant vaccine protection. Mice injected with m5C saRNA showed high survival rates and significant antibody responses after being challenged with a lethal dose of SARS-CoV-2 virus. Particularly in the low-dose group, m5C saRNA induced antibody titers that were markedly higher than those induced by wild-type saRNA and n1mψ mRNA, further confirming its efficacy at low doses.

Conclusion

This study demonstrated that fully substituting modified nucleotides in saRNA can significantly suppress interferon response, enhance protein expression, and provide effective vaccine protection at low doses. This discovery not only helps improve the efficacy and safety of saRNA vaccines but also provides new perspectives for the potential of saRNA in gene therapy and other applications.

Research Highlights

  1. Innovative Approach: The study is the first to demonstrate that fully substituting modified nucleotides can significantly improve the transfection efficiency and protein expression levels of saRNA.
  2. Remarkable Protein Expression: m5C saRNA exhibited excellent protein expression capacity across various cell lines, especially demonstrating significant effects at low doses.
  3. Suppression of Interferon Response: m5C saRNA effectively suppressed early interferon responses, reducing inflammation and enhancing vaccine safety and efficacy.
  4. Long-lasting Vaccine Protection: In vivo experiments showed that m5C saRNA could provide long-lasting vaccine protection at low doses and induce strong antibody responses.

Research Value

This study provides important scientific insights into the development of saRNA technology, particularly in the fields of vaccines and gene therapy. By suppressing interferon response and enhancing protein expression, the application of modified nucleotides is expected to improve the efficacy and safety of saRNA vaccines, reduce dosage requirements and side effects, thereby promoting the broad adoption and development of saRNA technology in practical applications.

Future Prospects

Future research will further evaluate the safety and efficacy of m5C saRNA at a clinical level, especially in non-human primates and humans. Additionally, the compatibility and interactions of modified nucleotides in other RNA formats will be explored to further unlock the potential of saRNA technology. Through ongoing research and innovation, saRNA is expected to bring revolutionary advances in the fields of vaccines and gene therapy.