In vitro- transcribed (IVT) mRNA mimics the structure of endogenous mRNA, consisting of 5 components from 5ʹ to 3ʹ: 5ʹ cap, 5ʹ untranslated region (UTR), an open reading frame that encodes the antigen, 3ʹ UTR and a poly(A) tail (Figure 1)^[1].

Tips：

(1) The 5' cap structure contains a 7-methylguanosine nucleoside. It is linked through a triphosphate bridge to the 5' end of mRNA. In mammals, the first or second nucleotide from the 5’ end is methylated on the 2’ hydroxyl of the ribose, known as 2’-O-methylation.

(2) This methylation helps avoid recognition by viral RNA sensors in the cytosol, thus preventing unintended immune responses. Additionally, the 5’ cap protects the mRNA from degradation by exonucleases. Further, poly(A) binding proteins and translation initiation factor proteins interact at the 3’ end of the mRNA. They help to circularize mRNA and recruit ribosomes for initiating translation. The length of the poly(A) tail indirectly controls both mRNA translation and half-life. A sufficiently long tail, between 100-150 base pairs, is required. This tail interacts with poly(A) binding proteins to form complexes necessary for initiating translation. It also protects the cap from degradation by decapping enzymes^[1].

(3) The 5ʹ and 3ʹ UTRs flanking the coding region control mRNA translation, half-life, and subcellular localization. The open reading frame of the mRNA vaccine contains the coding sequence that is translated into protein. This sequence can be optimized to increase translation without changing the protein sequence. This can be done by substituting rarely used codons with more frequently occurring codons that encode the same amino acid residue. For example, CureVac AG, a biopharmaceutical company, found that human mRNA codons rarely have A or U at the third position. They patented a strategy that replaces A or U at the third position in the open reading frame with G or C^[1].

mRNA vaccines do not require complex cell culture and purification systems, as they can be directly synthesized in the laboratory, enabling rapid large-scale production. These advantages have allowed mRNA vaccines to play a crucial role in the prevention and control of the COVID-19 pandemic. However, the development process of mRNA vaccines has not been without challenges. mRNA is unfeasible for clinical use because of its labile and immunogenic nature. Initially, many scientists held a negative attitude towards mRNA vaccine technology. Breakthroughs in two key technologies have propelled the advancement of mRNA vaccine technology.

Lipid Nanoparticle Delivery System

Naked mRNA molecules are rapidly degraded in biological fluids, do not accumulate in target tissues following systemic administration, and cannot penetrate into target cells even if they get to the target tissue. Further, the immune system is exquisitely designed to recognize and destroy vectors containing genetic information. Therefore, the safe and efficient delivery of mRNA is crucial for the widespread use of mRNA vaccines. The emergence of lipid nanoparticles has ingeniously addressed this issue.

Lipid nanoparticles (LNPs), are spherical vesicles consisting of one (unilamellar) or more (multilamellar) phospholipid bilayers. LNPs are typically composed of four components: ionizable cationic lipids, helper lipids (phospholipids), cholesterol and PEG-lipids (pegylated lipids)^[2][3]. LNPs protect the nucleic acids from degradation, maximize delivery to on- target cells and minimize exposure to off- target cells.

Modified Nucleosides

Katalin Karikó and Drew Weissman discovered that dendritic cells and TLR-expressing cells were potently activated by bacterial and mitochondrial RNA, but not by mammalian total RNA, which is rich in modified nucleosides^[4]. Therefore, they hypothesized that nucleoside modifications inhibit the immune response of RNA. Based on this assumption, they stimulated dendritic cells with RNA containing modified nucleosides and found that the modified nucleosides can prevent recognition by TLR receptors, especially modified uridines, leading to a significant reduction in the expression of TNF-α (Figure 3)^[4]. Both the Moderna and Pfizer–BioNTech SARS- CoV-2 vaccines contain nucleoside-modified mRNA and have demonstrated an efficacy of over 94% in phase 3 clinical trials^[1].

In subsequent research, Katalin Karikó and Drew Weissman demonstrated that modified mRNA has a higher translation capacity in experimental mice compared to unmodified mRNA, significantly increasing protein synthesis^[5]. With this, the key obstacle in clinical applications of mRNA was overcome. It is precisely based on these groundbreaking discoveries by Katalin Karikó and Drew Weissman that the Nobel Committee awarded them this year's Nobel Prize in Physiology or Medicine.

The majority of mRNA vaccines currently in preclinical trials and in clinical use are administered as a bolus injection into the skin, muscle or subcutaneous space, where they are taken up by immune or non- immune cells and translated into antigens that are displayed to T and B cells.

(1) Injected mRNA vaccines are endocytosed by antigen- presenting cells.

(2) After escaping the endosome and entering the cytosol, mRNA is translated into protein by the ribosome. The translated antigenic protein can stimulate the immune system in several ways.

(3) Intracellular antigen is broken down into smaller fragments by the proteasome complex, and the fragments are displayed on the cell surface to cytotoxic T cells by major histocompatibility complex (MHC) class I proteins.

(4) Activated cytotoxic T cells kill infected cells by secreting cytolytic molecules, such as perforin and granzyme.

(5) Additionally, secreted antigens can be taken up by cells, degraded inside endosomes and presented on the cell surface to helper T cells by MHC class II proteins.

(6) Helper T cells facilitate the clearance of circulating pathogens by stimulating B cells to produce neutralizing antibodies, and by activating phagocytes, such as macrophages, through inflammatory cytokines^[1].

The recent success of cancer immunotherapies has fueled interest in using mRNA therapies for similar applications (Table 1)^[6]. In the case of mRNA cancer immunotherapies, one method involves modifying the tumor microenvironment. This modification is achieved through expressing deficient or altered tumor suppressor proteins^[6]. There is also an increasing emphasis on using mRNA as a therapeutic vaccine. The goal of this vaccine would be to instruct the immune system to identify and eliminate cancer cells.

The development of therapeutic cancer vaccines faces several challenges for successful clinical translation. Unlike preventative vaccines for infectious diseases, therapeutic cancer vaccines must ensure a strong cytotoxic CD8⁺ T cell response to eradicate cancer cells. This is different as protection against infection in other diseases is largely conferred by a robust humoral response^[6].

Selecting appropriate antigens that can induce highly tumor-specific immune responses is another challenge. This is due to the high variability of antigens across different individuals. The increasing trend of patient-specific new antigens aims to address this issue. However, even if an antigen can induce a cellular immune response, the suppressive tumor microenvironment might prevent T cell infiltration into tumors and could lead to T cell exhaustion. Therefore, therapeutic vaccines may need to be administered with another therapy designed to overcome the suppressive microenvironment^[6].

mRNA vaccines offer high efficacy, rapid development capabilities, and the potential for low-cost manufacturing. However, their application has long been constrained by the instability and inefficiency of mRNA delivery in vivo. Advances in nucleoside modification and lipid nanoparticle technology have largely overcome these challenges, with various mRNA vaccine platforms targeting infectious diseases and several cancers demonstrating promising therapeutic effects in animal models and humans. mRNA vaccines hold great promise to replace traditional vaccine approaches in the future.