Optimization of the production and function of self-amplifying RNA vaccines

Abstract

Recently, RNA-based vaccines have become highly attractive, with proven ability to induce protective immunes responses against COVID-19, matched with their rapid and relatively simple production. Self-amplifying RNA (saRNA) may provide additional advantages due to its ability to: i) self-replicate facilitating exponential antigen expression; ii) induce equivalent protection with lower doses compared to conventional mRNA vaccines; and iii) elicit longer antigen expression in vivo. saRNA is a single stranded RNA derived from a positive strand virus genome such as Alphaviruses, where the structural proteins are replaced by a gene of interest (GOI). In this thesis, we sought to test the hypothesis that saRNA could be improved through directed evolution and/or nucleotide modifications. First, we optimized the production of saRNA through a design of experiment (DoE) approach to produce a maximal RNA yield and validated the improved IVT method on various sizes of RNA. We then used the optimal IVT method to produce saRNA for the optimization of the function of saRNA where we utilized an iterative directed evolution approach in order to evolve the replicase of the saRNA such that mutations would either confer interferon resistance and/or will allow higher antigen expression in cells. We observed no increase in transgene expression; however, we were able to establish a system and understand the parameters needed to support an iterative directed evolution approach. Lastly, we incorporated different modified nucleosides during IVT and determined whether in vitro antigen expression and in vivo immunogenicity could be improved. We found that N1-Methylpseudouridine incorporated saRNA did not enhance antigen expression in vitro. However, 2’-O-Methyl-GTP incorporation significantly enhanced luciferase and green fluorescent protein (eGFP) expression in interferon-competent HELA and THP-1 cells. Most importantly, we observed that 2’-O-Methyl-GTP modified saRNA encoding for influenza HA elicited significantly stronger antibody responses compared to unmodified saRNA in vivo after a prime-boost regimen.