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A Historical Overview on mRNA Vaccine Development

Rein Verbeke1, 2, Miffy H.Y. Cheng2, and Pieter R. Cullis2

1Ghent University, Ghent Research Group on Nanomedicines, Faculty of Pharmaceutical Sciences, Ottergemsesteenweg 460, 9000 Ghent, Belgium

2University of British Columbia, Department of Biochemistry and Molecular Biology, Health Sciences Mall 2350, BC V6T 1Z4 Vancouver, Canada

1.1 Introduction

In less than one year after the COVID-19 pandemic outbreak, two mRNA vaccines received the first emergency use authorization from the Food and Drug Administration (FDA) and the European Medicines Agency (EMA), i.e. BNT162b2 (Comirnaty) from Pfizer/BioNTech and mRNA-1273 (Spikevax) from Moderna. In Phase 3 clinical trials, these mRNA vaccines were found to be generally safe and up to 95% efficacious after the second dose of vaccination in preventing symptomatic SARS-CoV-2 infection [1, 2]. The outstanding efficacy and unprecedented speed with which these mRNA vaccines were produced and distributed, strongly helped to curtail the burden of the pandemic and prevented millions of deaths [3, 4].

Now proven against COVID-19, there is explosive growth in research and investments in mRNA technology. Most notably, the platform of nucleoside-modified mRNA encapsulated in lipid nanoparticles (LNPs) that is utilized in today's COVID-19 mRNA vaccines is poised to have a rapid transformative effect on the future of medicine. Vaccines based on this platform are now being tested in Phase 3 clinical trials for several viral diseases other than COVID-19, such as against influenza (BNT161 and mRNA-1010), cytomegalovirus (mRNA-1647), and respiratory syncytial virus (mRNA-1345), while many other mRNA vaccines are being (pre)clinically studied to target diseases, such as bacterial and parasitic infections, cancer, and autoimmune diseases. In addition, the ability of the mRNA-LNP platform to deliver genetic information for the temporal production of proteins inside cells makes it a potential key technology to enable gene editing, protein replacement, and other immunotherapeutic approaches [5].

Figure 1.1 Discoveries and milestones in the development of mRNA-based vaccines are subdivided into three parallel timelines of mRNA-, LNP design, and clinical development.

It may seem that the COVID-19 mRNA vaccines came out of the blue, but in fact, decades of research were needed to develop this novel vaccine technology. To understand why it took so long for mRNA vaccines to breakthrough, we need to appreciate the collective efforts made by many scientists and the various problems they have tackled and solved that ultimately led to the development of this first generation of mRNA vaccines. As elucidated by Dowdy, the fundamental problem is that a billion years of evolutionary defenses need to be tackled to successfully deliver RNA. This includes both cellular barriers that have kept foreign RNAs on the outside of cells from invading the inside of cells, as well as the many innate immune defense mechanisms evolved to recognize and destroy foreign RNA [6]. Nonetheless, mRNA represents a most excellent vaccine modality to mimic viral infections, this is to trick the immune system to develop memory against the encoded, pathogen antigen. Indeed, when successfully delivered, mRNA has the potential to process and present encoded antigens through the same cellular machinery as occurring during viral infections, while it may also benefit from the specifically designed immune mechanisms against viruses to prime and promote durable adaptive immune responses, i.e. the immune adjuvant potential of mRNA.

In this first chapter section, a short historical overview is given of the development of mRNA vaccines (Figure 1.1).

1.2 The Path of mRNA as an Unstable and Toxic Product to a New Class of Medicine

1.2.1 The Discovery and In Vitro Production of mRNA

In 2015, Cobb wrote an essay that addressed the question of who discovered mRNA. By reconstructing the collective insights and different kinds of evidence gathered during the 1950s through the 1960s, Cobb concluded that mRNA was the product of many years of work by a community of researchers [7]. Ultimately, this research process gained momentum in the summer of 1961, when the nature and properties of mRNA were for the first time described in a theoretical model by Jacob and Monod [8]. In this review article on genetic regulation of protein synthesis, they proposed the existence of an intermediate molecule, or "messenger ribonucleotide" that is produced from DNA and that brings the genetic information to the ribosomes for protein synthesis. At about the same time, experimental support for the existence of mRNA was provided by two different research teams [9, 10]. Both research teams had succeeded in isolating mRNA and demonstrating its association with "pre-existing" ribosomes. This replaced the prevailing theory of the time that new specialized ribosomes are synthesized from the gene, and that these ribosomes are specific for the production of the corresponding protein, i.e. the "one gene - one ribosome - one protein" hypothesis. For detailed information about the remarkable series of events and some of the outstanding experiments that led to the discovery of mRNA, see reference [11].

In 1969, protein synthesis from mRNA inside ribosomes was first demonstrated in cell-free systems. In these experiments, RNA fractions purified from reticulocytes dictated the synthesis of globin when incubated with ribosomes obtained from Escherichia coli [12] or a different mammalian species [13]. Later on, the translation of mRNA into hemoglobin was also proven in living cells after the microinjection of 9 s RNA from rabbit reticulocytes into frog oocytes [14, 15]. While these studies might have sparked one's imagination to use mRNA for therapeutic applications, at the time, the focus was solely on understanding its biological function.

In 1984, a simple and efficient method was established for in vitro mRNA synthesis using template DNA and a bacteriophage SP6 polymerase that initiates transcription at an SP6 promoter located upstream of the gene of interest [16]. In the following years, T7 and T3 RNA polymerases were also reported for successful in vitro transcribed (IVT) RNA synthesis [17-20]. These methods still represent the foundation of how mRNA is manufactured in today's COVID-19 mRNA vaccines. However, a limitation of IVT mRNA production using phage polymerases is that it can give rise to multiple contaminants in the form of short and long double-stranded RNA (dsRNA) strands [21, 22]. These dsRNA byproducts have been shown to be largely responsible for the innate immune response to IVT mRNA, and when not controlled, have the potential to jeopardize the safety and functionality of mRNA vaccines (discussed in more detail below). There is, therefore, continuing interest in optimizing the IVT process of mRNA in order to reduce the formation of dsRNA byproducts, as well as in finding (more) cost-effective purification methods [23-25].

Decades of basic research into the structural characteristics of mRNA and the biological interactions of mRNA with numerous proteins inside the cell not only brought new insights into mRNA metabolism and mRNA translation process but also leveraged IVT mRNA to reach a more optimal design [26]. The genetic information in mRNA is encoded in a codon sequence, where a triplet of adjacent nucleotides specifies an amino acid to be incorporated in a protein, also referred to as the open reading frame (ORF). The ORF is flanked at the 5´ and 3´ positions with start and stop codons. Because most amino acids are encoded by more than one codon, the codon usage in the ORF can be varied, also referred to as synonymous codon usage. Over the years, several strategies have been proposed to optimize codons so as to improve the translation and half-life of the mRNA, including methods of adjusting codons to match host transfer RNA abundances [27] for the enrichment of GC content [28, 29], and to optimize mRNA folds in the construct [30, 31]. It is important, however, to consider that these methods may have unintended effects on the performance of mRNA vaccines, such as altering protein folding and changing the sites of posttranslational modifications that may affect the immunogenicity and function of the encoded antigen, reviewed in [32].

Different nontranslated structural elements are present in eukaryotic mRNA, which were found to have essential roles in different stages of the mRNA life cycle. In 1975, the cap structure was discovered, which is an N7-methylated guanosine linked to the first nucleotide of the RNA via a reverse 5´ to 5´ triphosphate linkage [33-36]. By binding to the eukaryotic initiation factor (eIF) 4E, the cap structure enables the recruitment of translating ribosomes to mRNA. The cap structure and its methylation state are also significant determinants of how the host cell distinguishes itself from nonself RNA, as well as they are important for mRNA stability. At the 3´...