Evaluation of immunogenic properties of modified mRNA and self-replicating RNA based vaccines against cancer

Messenger RNA-based cancer vaccines have recentrly gained much scientific and clinical attention due to their promising performance and distinguished safety profile. An important advantage of mRNA vaccines over peptide- or cell-based therapy is their faster production, cheaper cost, and precisely defined composition. The main goal of this doctoral thesis was to improve mRNA by 1) optimizing mRNA delivery to target cells and 2) constructing more therapeutically effective mRNA. To this end, we studied the protein expression levels of non-viral delivery methods for mRNA and compared this to pDNA. Additionally, the molecular pathways behind the cytotoxic effects observed by mRNA delivery were unraveled in detail. Furthermore, together with our collaborators at the Synthetic biology at MIT (Cambridge, MA, USA), we are in the process of constructing next-generation RNA onstructs with optimized 5' and 3' UTRs (untranslated regions) and novel modified nucleotides that avoid detection of the RNA by pattern recognition receptors, which trigger the innate immune respons and dampens protein translation or alternatively induces cell death. Subsequently, in our quest for mRNA molecules that can provide more sophisticated behaviour, in collaboration with MIT, we assemble Venezuelan Equine Encephalitis (VEE)-based RNA constructs (replicons) that are capable of replicating in cells. This property enables the replicon to express proteins at much higher levels compared to traditional non-replicating mRNA. The VEE-based replicon to express proteins at much higher levels compared to traditional non-replicating mRNA. The VEE-based replicon, which does not integrate into the genome of the host cell, is inherently safer than DNA and thus more likely to obtain approval from regulatory agencies. As a therapeutic, the replicon is less laborious to product than protein, and retains the sustained expression benefits of a replicating vector system. As a final objective, model tumor antigens will be incorporated into these modified mRNA and replicon constructs and mice will be immunized with these RNAs. We will study the immune response after vaccincation with these RNA constructs as well as the vaccine's capacity to prevent tumor growth of B16-OVA melanoma cells and increase the mean survival time of the mice.