Improvement of the potency of a N1-methylpseudouridine-modified self-amplifying RNA through mutations in the RNA-dependent-RNA-polymerase

RNA vaccines are sensed as non-self molecules by the innate immune system, and balancing control of the immune activation and vaccine safety and efficacy has remained a challenge, especially for self-amplifying RNAs (saRNAs). Incorporation of modified nucleotides has been widely used to temper immune activation of RNA vaccines. However, it was previously reported that incorporation of modified nucleotides to saRNAs impeded antigen expression. Here, we used a reporter replicon of the attenuated TC-83 strain of Venezuelan equine encephalitis virus (VEEV) to investigate the impact of modified nucleotide incorporation on the replication capacity of the saRNA in transfected cells. {psi} and {psi}-modified molecules showed a profound defect in RNA synthesis compared to unmodified saRNA. Interestingly, the levels of RNA synthesis of m5C-modified RNAs were similar to unmodified molecules, positioning m5C as a promising candidate for saRNA modification. To overcome the impact of {psi} or m1{psi}-modified nucleotide incorporation in RNA synthesis, we explored two alternative approaches: engineering the UTR sequences and tuning polymerase fidelity. Our results uncover a previously unappreciated link between polymerase fidelity and saRNA amplification. Overall, we provide new insights for the design of saRNAs with high levels of heterologous protein expression and potential vaccine applications.