NAR molecular medicineNov 19, 2025

Self-amplifying RNAs with modified building blocks produce strong protein levels and immune response in living organisms

mRNA Technology Weekly Brief ↗PubMed ↗DOI ↗OA ↗

Updated Mar 18, 2026

Abstract

Synthetic (saRNA) incorporating noncanonical nucleotides like (m5C) and (m5U) demonstrates sustained gene expression.

saRNA can achieve higher and more sustained expression compared to standard messenger RNA in therapeutic applications.
The inclusion of modified nucleotides in synthetic mRNA enhances RNA stability, reduces immune activation, and improves gene expression.
For the first time, noncanonical nucleotides m5C and m5U have been shown to be compatible with a specific replicon for protein translation.
saRNA utilizing m5C or m5U was found to mediate sustained expression of the luciferase reporter gene.
saRNA incorporating m5U resulted in more prolonged gene expression than those using m5C.
Modified saRNA induced distinct antigen-specific immune responses when encoding the model antigen ovalbumin.

Simplified

When utilized in therapeutic applications, synthetic (saRNA) can lead to higher and more sustained expression than standard messenger RNA (mRNA). This feature is particularly important for gene replacement therapy applications where prolonged expression could reduce the dose and frequency of treatments. The inclusion of modified nucleotides in synthetic non-amplifying mRNA has been shown to increase RNA stability, reduce immune activation and enhance gene expression. Preclinical and clinical studies with saRNA have so far exclusively relied on RNA containing the canonical nucleotides adenosine, cytidine, guanosine and uridine. For the first time, we show that noncanonical nucleotides, such as (m5C) and (m5U), are sufficiently compatible with a replicon derived from Venezuelan equine encephalitis alphavirus mediating protein translation, while those containing-methylpseudouridine in place of uridine show no detectable expression. When administered, saRNA generated with m5C or m5U mediates sustained gene expression of the luciferase reporter gene, with those incorporating m5U appearing to lead to more prolonged expression. Finally, distinct antigen-specific humoral and cellular immune responses were induced by modified saRNA encoding the model antigen ovalbumin. The use of modified nucleotides with saRNA-based platforms could enhance their potential to be used effectively in a variety of applications. in vitro N in vivo1

Key numbers

4–5×

Increase in luciferase expression

Measured at Days 14 and 17 post-injection.

10×

IgG titer increase

Observed between Days 21 and 28 post-vaccination.

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Self-amplifying RNAs with modified building blocks produce strong protein levels and immune response in living organisms

Abstract

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