Strategies for controlling the innate immune responses to in vitro transcribed messenger RNAs

Abstract

Synthetic messenger RNA (mRNA) produced via in vitro transcription (IVT mRNA) has emerged as an appealing tool for the transient introduction of genes, particularly for vaccination applications. The interaction that IVT mRNA has with the innate immune system is centrally important to its performance as a vaccine. These innate immune responses can both interfere with the expression of the encoded antigenic protein and direct development of adaptive immunity. The objective of this thesis is to investigate the innate immune responses to IVT mRNA and to identify strategies to modulate these immune responses. We first demonstrated that substitution of the modified bases 5-methylcytosine and pseudouridine in IVT mRNA consistently reduces antiviral cytokine responses but affects transgene expression in a gene-specific manner. To assess the pathogen recognition receptors involved in detection of IVT mRNA, we developed proximity ligation assays, which allowed histological identification of PRR signaling complexes. We used these assays to identify that nanoparticle-mediated delivery modified PRR-activation following intramuscular delivery compared to delivery of the naked IVT mRNA molecule. Lastly, we developed a strategy to program the immunostimulatory properties of IVT mRNA by tethering adjuvants directly to the molecule. We show that upon intramuscular injection, the combination delivery of a TLR7 adjuvant and IVT mRNA lead to heightened local antiviral responses when delivered tethered, rather than as a cocktail. This work provides a foundation for the modulation and systematic study of IVT mRNA’s interaction with the innate immune system. Insights gained from this work may help direct and advance the design of IVT mRNA sequences for vaccination applications.