Nucleotide modifications enhance self-amplifying RNA safety and payload expression

Introduction: : Messenger RNA (mRNA) provides a revolutionary approach to vaccine development. Lipid nanoparticles (LNPs) stabilize mRNA and promote cellular internalization of mRNA and subsequent expression of viral antigens to trigger immune responses against infection. However, current RNA technologies often require frequent or higher dosing to achieve efficacy due to low transfection efficiency in vivo and transient antigen expression. Self-amplifying RNA (saRNA) is a strategy to prolong antigen expression with less frequent dosing. Inspired by an RNA virus, a saRNA transcript encodes both the target antigen and a virus-derived RNA-dependent RNA polymerase (RdRp). The RdRp replicates and amplifies the RNA template encoding the cargo, thereby increasing the antigen expression level and duration of the expression. However, replication of wild-type (WT) saRNA and antigen expression are often inhibited by strong innate interferon responses against the saRNA. The modified nucleotides used to reduce interferon responses in mRNA vaccines, such as N1-methylpseudouridine, are not saRNA-compatible. We hypothesized that substitution with appropriate modified nucleotides will lower the interferon responses compared to WT saRNA and lead to higher and more durable cargo expression than conventional (N1-methylpseudouridine-modified) mRNA and WT saRNA.

Materials and

Methods: : Conventional mRNA and Venezuelan equine encephalitis virus-derived saRNA encoding mCherry or firefly luciferase were synthesized by in vitro transcription. Modified nucleotides including 5-methylcytidine (m5C) and 5-hydroxymethylcytidine (hm5C) were incorporated during in vitro transcription to produce modified saRNA. Double-stranded RNA (dsRNA) was removed after RNA synthesis by cellulose chromatography, and removal was confirmed by a dot-blot analysis. To screen for compatible modified nucleotides, we transfected different modified saRNAs encoding mCherry in HEK293T cells using Lipofectamine MessengerMax Transfection Reagent, and we determined transfection efficiency by flow cytometry. Conventional mRNA, WT and modified saRNAs were encapsulated in LNPs composed of 1.5 mol% DMG-PEG2000, 38.5 mol% cholesterol, 10 mol% phospholipids (2 types) and 50 mol% ionizable lipids (14 types). LNP size and polydispersity index were characterized by dynamic light scattering. RNA encapsulation efficiency was quantified by the QuantiFluor RNA System. We co-cultured LNPs in C2C12 murine myoblasts to determine the optimal formulation for cell transfection. We injected mRNA, WT or modified saRNAs encoding firefly luciferase loaded in the optimized LNPs in 6-8 week old female C57BL/6 mice intramuscularly in the hindlimb. One day after vaccination, we collected blood to determine serum cytokine levels by ELISA. We monitored luciferase expressions over time at the injection site by IVIS imaging. We monitored weight loss in mice as an indicator of adverse responses to the RNA vaccines.

Results, Conclusions, and Discussions:: m5C-modified and hm5C-modified saRNAs led to the highest transfection efficiency among the modified nucleotides screened. 1,2-dioleoyl-sn-glycero-3-PE as the phospholipid and SM-102 as the ionizable lipid was the optimal formulation for C2C12 cell transfection. The LNPs had a diameter of ~100 nm, low polydispersity index (~0.1 or less) and RNA encapsulation efficiency >90%. m5C-modified saRNA mediates a 314x luciferase expression compared to mRNA, and a 3.5x expression compared to WT saRNA in C2C12 cells in vitro. At a 2.5 μg dose, m5C-modified saRNA LNPs attained a peak luciferase expression 3.1x higher than mRNA LNPs, and high level of luciferase expression was maintained for greater than 4 weeks in the mice. In contrast, the expression from mRNA LNPs declined ~100-fold within a week. Moreover, m5C-modified saRNA reduced IFN-α response by more than 50% compared to WT saRNA in vivo. WT saRNA with or without dsRNA removal showed similar expression kinetics, but dsRNA removal significantly reduced weight loss, suggesting the importance of dsRNA removal in mitigating the adverse responses. Lastly, we administered mRNA, WT, hm5C-modified and m5C-modified saRNAs at 3, 10 or 30 μg doses to determine the dose-response relationship and safety profile of the RNAs. All saRNAs induced comparable peak expression levels and areas under the curve across all doses, whereas mRNA reached peak expression and area under the curve at the intermediate 10 μg dose. Furthermore, at the 30 μg dose, both modified saRNAs reduced weight loss and significantly accelerated weight recovery in mice than WT saRNA, but the weight change was not significantly different between mice of the two modified saRNA groups. Increasing the dose from 3 to 30 μg significantly elevated the blood IFN-α response in the mRNA group, but in neither of the saRNA groups. Taken together, substituting native nucleotides with compatible modified nucleotides in saRNA significantly prolongs payload expression and reduces interferon responses. Modified saRNAs maintain high payload expression and are safe in vivo even at a high dose.

Acknowledgements and/or References (Optional):: The work was supported by the NSF Trailblazer Award and Boston University. We also thank the BU IVIS Imaging Core (1S10RR024523-01) for support.

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