mRNA vaccines represent a promising alternative to conventional vaccine approaches because of their high potency, capacity for rapid development and potential for low-cost manufacture and safe administration. However, their application has until recently been restricted by the instability and inefficient in vivo delivery of mRNA. Recent technological advances have now largely overcome these issues, and multiple mRNA vaccine platforms against infectious diseases and several types of cancer have demonstrated encouraging results in both animal models and humans. This Review provides a detailed overview of mRNA vaccines and considers future directions and challenges in advancing this promising vaccine platform to widespread therapeutic use.

DNA and RNA stimulate the mammalian innate immune system through activation of Toll-like receptors (TLRs). DNA containing methylated CpG motifs, however, is not stimulatory. Selected nucleosides in naturally occurring RNA are also methylated or otherwise modified, but the immunomodulatory effects of these alterations remain untested. We show that RNA signals through human TLR3, TLR7, and TLR8, but incorporation of modified nucleosides m5C, m6A, m5U, s2U, or pseudouridine ablates activity. Dendritic cells (DCs) exposed to such modified RNA express significantly less cytokines and activation markers than those treated with unmodified RNA. DCs and TLR-expressing cells are potently activated by bacterial and mitochondrial RNA, but not by mammalian total RNA, which is abundant in modified nucleosides. We conclude that nucleoside modifications suppress the potential of RNA to activate DCs. The innate immune system may therefore detect RNA lacking nucleoside modification as a means of selectively responding to bacteria or necrotic tissue.

In vitro–transcribed mRNAs encoding physiologically important proteins have considerable potential for therapeutic applications. However, in its present form, mRNA is unfeasible for clinical use because of its labile and immunogenic nature. Here, we investigated whether incorporation of naturally modified nucleotides into transcripts would confer enhanced biological properties to mRNA. We found that mRNAs containing pseudouridines have a higher translational capacity than unmodified mRNAs when tested in mammalian cells and lysates or administered intravenously into mice at 0.015–0.15 mg/kg doses. The delivered mRNA and the encoded protein could be detected in the spleen at 1, 4, and 24 hours after the injection, where both products were at significantly higher levels when pseudouridine-containing mRNA was administered. Even at higher doses, only the unmodified mRNA was immunogenic, inducing high serum levels of interferon-α (IFN-α). These findings indicate that nucleoside modification is an effective approach to enhance stability and translational capacity of mRNA while diminishing its immunogenicity in vivo. Improved properties conferred by pseudouridine make such mRNA a promising tool for both gene replacement and vaccination.

Toll-like receptors (TLRs) are the basic signaling receptors of the innate immune system. They are activated by molecules associated with pathogens or injured host cells and tissue. TLR3 has been shown to respond to double stranded (ds) RNA, a replication intermediary for many viruses. Here we present evidence that heterologous RNA released from or associated with necrotic cells or generated by in vitro transcription also stimulates TLR3 and induces immune activation. To assess RNA-mediated TLR3 activation, human embryonic kidney 293 cells stably expressing TLR3 and containing a nuclear factor-B-dependent luciferase reporter were generated. Exposing these cells to in vitro transcribed RNA resulted in a TLR3-dependent induction of luciferase activity and interleukin-8 secretion. Treatment with in vitro transcribed mRNA activated nuclear factor-B via TLR3 through a process that was dose-dependent and involved tyrosine phosphorylation. Furthermore, in vitro transcribed natural or 2-fluoro-substituted mRNA induced the expression of TLR3, interferon regulatory factor-1, tumor necrosis factor-␣, and interleukin-1 receptor-associated kinase-M mRNA in human dendritic cells (DCs). DCs responded to mRNA treatment by expressing activation markers, and this maturation was inhibited by antagonistic TLR3-specific antibody. Endogenous RNA released from or associated with necrotic cells also stimulated DCs, leading to interferon-␣ secretion, which could be abolished by pretreatment of necrotic cells with RNase. These results demonstrate that RNA, likely through secondary structure, is a potent host-derived activator of TLR3. This finding has potential physiologic relevance because RNA escaping from damaged tissue or contained within endocytosed cells could serve as an endogenous ligand for TLR3 that induces or otherwise modulates immune responses.

In vitro-transcribed mRNA has great therapeutic potential to transiently express the encoded protein without the adverse effects of viral and DNA-based constructs. Mammalian cells, however, contain RNA sensors of the innate immune system that must be considered in the generation of therapeutic RNA. Incorporation of modified nucleosides both reduces innate immune activation and increases translation of mRNA, but residual induction of type I interferons (IFNs) and proinflammatory cytokines remains. We identify that contaminants, including double-stranded RNA, in nucleoside-modified in vitro-transcribed RNA are responsible for innate immune activation and their removal by high performance liquid chromatography (HPLC) results in mRNA that does not induce IFNs and inflammatory cytokines and is translated at 10- to 1000-fold greater levels in primary cells. Although unmodified mRNAs were translated significantly better following purification, they still induced high levels of cytokine secretion. HPLC purified nucleoside-modified mRNA is a powerful vector for applications ranging from ex vivo stem cell generation to in vivo gene therapy.