Modified Nucleotides Boost saRNA Vaccine Efficacy and Protection Against SARS-CoV-2

By Dr. Chinta SidharthanReviewed by Lily Ramsey, LLMSep 18 2024

The use of modified nucleotides such as N¹-methylpseudouridine (N1Mψ) in messenger ribonucleic acid (mRNA) vaccines has helped mitigate systemic inflammatory responses and improve the vaccine's efficacy. However, factors such as a short half-life and adverse reactions impact the efficacy of mRNA vaccines.

In a recent study published in Nature Biotechnology, scientists from Boston University examined whether the use of modified nucleotides in self-amplifying RNA could address the shortcomings of mRNA vaccines and effectively protect against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

​​​​​​​Study: Complete substitution with modified nucleotides in self-amplifying RNA suppresses the interferon response and increases potency. Image Credit: KomootP/Shutterstock.com

Background

Chemically modified nucleotides have been used in mRNA vaccines to improve their ability to enter cells, make the mRNA molecules more stable, and lower inflammatory immune responses. Modified nucleotides were extensively used in developing mRNA vaccines against coronavirus disease 2019 (COVID-19).

Compared to normal RNA, the use of N1mΨ in mRNA vaccines prevents the activation of toll-like receptors and other immune sensors and increases the effectiveness of the vaccine. However, mRNA vaccines have a short half-life, which necessitates larger and more frequent doses, increasing the risk of adverse reactions.

Self-amplifying RNA, or saRNA, can replicate inside the body, with small doses producing large amounts of proteins, making it an ideal replacement for mRNA in vaccines. However, saRNAs trigger early immune responses, which lower the efficacy of saRNA vaccines.

Although the use of chemically modified nucleotides in saRNAs could be a potential solution, the method has not yet been effective.

About the Study

In the present study, the researchers explored the use of various modified nucleotides in saRNA constructs to improve protein expression and transfection efficiency and enhance the efficacy of gene therapies and vaccines.

A library of saRNA constructs was synthesized, with modified nucleotides 5-methyluridine (m5U), 5-methylcytidine (m5C), and 5-hydroxymethylcytidine (hm5C) substituted in them through in vitro transcription. A reporter protein was also encoded into the saRNA.

The researchers then compared the efficiencies of hm5C and m5C modified saRNAs with that of unmodified saRNA and N1mΨ-modified mRNA. The modified saRNA constructs were introduced into the human embryonic kidney cell line HEK293T.

Cationic lipofection, where positively charged lipids are complexed with the saRNA to facilitate entry, was used to transfect the constructs into the cells and evaluate the transfection efficacy.

Additionally, the saRNAs were also loaded into lipid nanoparticles and transfected into multiple cell types, including the immortalized human T lymphocyte Jurkat cells and the mouse myoblast C2C12 cells.

After in vitro transcription, cellulose chromatography was used to purify the RNA preparations and remove double-stranded RNA impurities to lower the undesirable immune responses.

The impact of the modified and unmodified saRNAs on the immune system was measured by culturing peripheral blood mononuclear cells with lipid nanoparticles loaded with saRNA constructs.

The immune responses were evaluated by analyzing the expression of cytokines and interferon-related genes, especially interferon α and interferon β expression. Additionally, in vivo experiments were conducted using murine models where lipid nanoparticle-encapsulated saRNAs were administered to mice through intramuscular injections.

The saRNA construct was encoded with the firefly luciferase gene, which allowed the protein expression to be temporally monitored using bioluminescent imaging. Furthermore, serum samples from the mice were analyzed periodically to measure the interferon levels and evaluate the systemic immune responses.

The dose-response to a saRNA vaccine was also tested by encoding the SARS-CoV-2 spike protein gene into the saRNA construct. Mice that were administered varying doses of the saRNA vaccine were challenged with a lethal SARS-CoV-2 variant, and the efficacy of the vaccine was tested by measuring the levels of neutralizing antibodies in the blood.

Major Findings

The study found that the modified saRNAs containing hm5C and m5C showed significantly higher transfection efficiency than standard, unmodified saRNA constructs. The saRNA with m5C had 4.9-fold higher protein expression levels than the unmodified saRNA and 68 times higher protein expression than N1mΨ-modified mRNA in the HEK293T cells.

The expression of the m5C-modified saRNA was also found to be stable and lasted for a week after transfection. The protein expression of the m5C-modified saRNA was significantly better than that of the unmodified saRNA and N1mΨ-modified mRNA in all three cell lines.

The in vivo experiments also demonstrated that the m5C-modified saRNA showed stable protein expression for 28 days, along with lowered inflammatory immune responses compared to the unmodified saRNA.

The m5C-modified saRNA vaccine for SARS-CoV-2 also resulted in better survival rates even at low doses, with significantly higher neutralizing antibody titers elicited as compared to the unmodified saRNA and N1mΨ-modified mRNA.

Conclusions

Overall, the study found that the use of the chemically modified nucleotide m5C in saRNA constructs significantly improved the transfection efficiency and protein expression. Furthermore, the use of m5C-modified saRNA encoding the SARS-CoV-2 spike protein as a vaccine resulted in better survival rates and lower inflammatory responses than unmodified saRNA.

These findings highlight the potential use of m5C-modified saRNA for developing low-dose vaccines with prolonged efficacy and reduced adverse effects.