From UltraCold to ambient: what’s actually working for mRNA/saRNA stability

Cold-chain constraints emerged as a critical bottleneck for the first mRNA COVID-19 vaccines, catalyzing an intense effort to extend the shelflife of RNA–nanoparticle products from ultracold toward refrigerated and ambient conditions. This Expert Insight distils the strategies that reliably improve stability for messenger RNA (mRNA) and self-amplifying RNA (saRNA). Main chemical and physical degradation pathways of RNA and lipids are addressed and practical solutions are summarized, while highlighting the inherent differences in fragility dependent on sequence length (siRNA < mRNA < saRNA) and the strong influence of lipid and buffer choices. In addition to the use of lipids, polymers are mentioned as promising alternative carriers for RNA delivery, with promising stability profiles and potential cryopreservative properties. However, while computational RNA sequence design and rational carrier and buffer choices increase stability, lyophilisation is generally used to enable long‑term storage of RNA‑nanoparticles at non‑freezing/refrigerator temperatures, and in some cases even ambient temperature. As lyophilisation is generally time‑intensive and costly, other drying techniques are also highlighted. In addition, the stability and expression of circRNA is compared to linear mRNA and saRNA, underlining the importance of direct comparisons of different RNA‑nanoparticles with each other, something that is still lacking in stability studies. Moreover, given their unique properties, a ‘top‑down selection framework’ is suggested in which the intended use, and consequent required expression profile and storage conditions, guide the choice of RNA modality, carrier and drying procedure. Finally, a call to prioritize rigorous comparability studies, validated accelerated/forced-degradation models, and robust industrial freeze‑drying controls is included to accelerate the development of next-generation, thermostable RNA modalities.

Sequence design, lipid optimization, and lyophilisation are moving mRNA/saRNA storage from ultracold toward ambient – but stability challenges scale with RNA length.

What you will learn

01

Why fragility increases with RNA length

02

Which formulation and drying strategies work

03

How to match RNA modality to intended use

Stability improvement pathway

1

Sequence & nucleotide design

2

Lipid & buffer optimzation

3

Lyophilisation with cryoprotectants

4

Ambient/refrigerated storage

Key findings

Fragility follows length: siRNA < mRNA < saRNA – a single strand break anywhere in a 10,000-nt saRNA abolishes function

Lyophilisation is the key enabler of refrigerated and ambient storage; saRNA-LNP cycles can take up to 4.5 days due to bound water

Polymer carriers offer inherent cryoprotection and simpler freeze-drying than LNPs; polymer–LNP hybrids show promise

A top-down framework – use case first, then RNA modality, carrier, and drying method – is proposed to guide future design

Key interests

mRNA stability

saRNA

Lyophilisation

LNP formulation

Cold chain

Cryoprotectants

RNA vaccines

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