Messenger ribonucleic acid (mRNA)-based drugs, notably mRNA vaccines, have been widely proven as a promising treatment strategy in immune therapeutics. The extraordinary advantages associated with mRNA vaccines, including their high efficacy, a relatively low severity of side effects, and low attainment costs, have enabled them to become prevalent in pre-clinical and clinical trials against various infectious diseases and cancers. Recent technological advancements have alleviated some issues that hinder mRNA vaccine development, such as low efficiency that exist in both gene translation and in vivo deliveries. mRNA immunogenicity can also be greatly adjusted as a result of upgraded technologies.

mRNA, an intermediate hereditary substance in the central dogma, was first discovered in 1961. However, the concept of mRNA-based drugs was not conceived until 1989, when Malone et al. demonstrated that mRNA could be successfully transfected and expressed in various of eukaryotic cells under the package of a cationic lipid (N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTMA)). In 1990, in vitro-transcribed mRNA was sufficiently expressed in mouse skeletal muscle cells through direct injection, which became the first successful attempt on mRNA in vivo expression and thus proved the feasibility of mRNA vaccine development. Since then, mRNA structure researches and other related technologies have been rapidly developed. Under this condition, several development restrictions stemmed from mRNA instability, high innate immunogenicity, and inefficient in vivo

delivery have been mitigated, and now mRNA vaccines have been widely studied in different kinds of diseases.

Fig. 1 Timeline of some key discoveries and advances in the development of mRNA-based drug technology.

mRNA vaccines have demonstrated many specific advantages that conventional vaccines do not have.

• mRNA can theoretically meet all genetic information requirements to encode and express all kinds of proteins.
• Vaccine developing efficiency can be optimized by modifying mRNA sequence, which is a more convenient way compared to other kinds of vaccine modification.
• Most of the mRNA vaccine production and purification processes are quite similar despite different encoded antigens, so it is potential to be retained or even standardized to develop other similar mRNA vaccines.
• mRNA vaccines can save both time and economic costs. Second of all, mRNA has self-adjuvanting properties which activate strong and long-lasting adaptive immune responses through tumor necrosis factor-α (TNF-α), interferon-α (IFN-α) and other cytokines secretion by immune cells.

mRNA In Vitro Synthesis and Modification

To date, in vitro transcription technology of mRNA has been mature, and the most popular method is using T3, T7, or SP6 RNA polymerase and linear DNA (linearized plasmid DNA or synthetic DNA prepared by PCR) for mRNA synthesis. There are some basic structural elements of mature mRNA in the eukaryocyte that are required to keep mRNA functional, including five-prime cap (5′ cap), five-prime untranslated region (5′ UTR), open reading frame (ORF) region, three-prime untranslated region (3′ UTR), and poly (A) tail structure. Keeping mRNA structure intact is beneficial for mRNA stability and expression capability. Modifying the mRNA sequence based on its complete structure can further optimize the efficiency of an mRNA vaccine. However, the initial product of mRNA in vitro transcription is the mixture of targeted mRNA, untargeted RNA, nucleotides, oligodeoxynucleotides, and proteins. To purify the mRNA, precipitation and extraction techniques are used to remove common impurities and chromatographic techniques are generally used to separate the target mRNA from other mRNA impurities in this system.

The in vitro synthesis and modification of mRNA consists mainly of:

• Five-prime cap (5′ cap) and modification.
• Optimization of untranslated regions (UTRs).
• Codon optimization of open reading frame (ORF).
• Poly (A) tail and mRNA stability.

Immunogenic Regulation of mRNA

Based on its self-adjuvanting effect, mRNA can exhibit some properties similar to the mRNA virus when it works as the vector of exogenous genes. In this case, mRNA can be recognized by antigen-presenting cells (APCs), which subsequently activates pattern recognition receptors (PRRs) such as Toll-like receptor 3 (TLR3), TLR7, and TLR8. The double-stranded RNA (dsRNA) can combine with some Retinoic-acid-inducible gene I (RIG-I) -like receptors (RLRs) in the cytoplasm, such as RIG-I and melanoma differentiation-associated 5 (MDA5), which promotes APCs maturation, pro-inflammatory cytokines secretion, and type I interferon (IFN) secretion. Eventually this leads to strong antigen-specific humoral and cellular immune responses. However, subunit vaccines composed of peptide or protein antigens are generally unable to activate PRRs, so it is necessary to add adjuvants which can initiate and support adaptive immune responses, achieving the final result of carrying out the body's immune response of subunit vaccines. Therefore, mRNA's strong adaptive immune response and self-adjuvanting property can provide a huge advantage shown in mRNA vaccines. Single-stranded RNA (ssRNA) can trigger the DCs' antiviral activation state through TLR7 and TLR8 recognition during mRNA in vivo transmission. The dsRNA contaminants can also trigger immune activation via TLR3 recognition. However, excessive immune response stimulated by mRNA in the cytoplasm would stimulate cells to secrete large amounts of type I IFN and other interferons which can inhibit the translation of mRNA and eventually lead to translational stagnation, RNA degradation, CD8 (cluster of differentiation 8) T cells activation reduction, and ultimately immune response termination. This could leave negative effects on some mRNA applications such as vaccines and protein replacement therapies. Self-adjuvanting properties of mRNA have both advantages and disadvantages in mRNA vaccine applications, therefore, it is necessary to form mRNA immunogenic regulations according to different medical demands, which in return would effectively improve the application efficacy of mRNA vaccines.

Fig. 2 mRNA in vitro transcription and innate immunity activation.

mRNA Delivery System

mRNA needs to enter the host cytoplasm to express specific antigens to remain functional; however, the mRNA molecule is not small enough to pass through cell membrane by free diffusion. Additionally, mRNA and cell membrane are both negatively charged, which increases the difficulty of mRNA delivery. Furthermore, mRNA can be easily degraded by extracellular ribonucleases which exist in skin and blood. Therefore, delivering mRNA into enough numbers of cells with sufficiently high translation levels is one of the most difficult application problems of mRNA vaccines, as it demands highly specific and efficient mRNA delivery systems. A variety of mRNA delivery methods and mRNA delivery vehicles have been developed and applied currently.

• Naked mRNA delivery system
1. direct injection of naked mRNA
   Early study has demonstrated that naked mRNA in vivo injection can provoke the immunotherapy response in mice. At present, administration strategies of mRNA generally include subcutaneous injection, intradermal injection, intranodular injection, intramuscular injection, intravenous injection, intratumoral injection, etc., which are essential methods that help stimulate antigen presentation and initiate immune responses.
2. Physical delivery of naked mRNA
   The efficiency of naked mRNA antigen presentation can be improved with the assistance of common physical methods including electroporation, gene gun, microneedles, etc. Electroporation can increase mRNA delivery efficiency without the demand of other mode receptors, which can reduce unnecessary immunoreactions.
• Ex Vivo loading of DCs delivery system
  DCs are one of the most potent APCs of immune system. They can present processed antigens to CD4, CD8 T cell via the major histocompatibility complex (MHC), which triggers cellular immunity. Meanwhile, DCs can also present intact antigens to B cells, triggering humoral immunity.
• Protamine-formulated delivery system
  Protamine is an alkali cationic protein with resin-like structure. Combining mRNA with protamine in different mass ratios can yield electrostatic protamine-mRNA complex particles with different diameters. This tight conjugate form can effectively protect mRNA from being degraded by serum RNases, and the complex can cause a strong immune-reaction of immune cells such as DCs, monocytes, B cells, natural killer cells, and neutrophils.
• Cationic lipid-based delivery system
  As a commonly used gene carrier, cationic liposomes can also combine with negatively charged nucleic acids to form electrostatic complexes, improving mRNA delivery efficiency. The cationic lipid-mRNA complex and other preparations together can form an 80-200 nm nanoparticle called lipid nanoparticles (LNP), which can be transfected into the cytoplasm by endocytosis.
• Polymer-based delivery system
  Currently, cationic polymers have been widely used as mRNA delivery vectors. Commonly used polymer delivery materials include polyethylenimine (PEI), poly (beta-amino esters) (PBAEs), etc.

Applications of mRNA as a Drug Platform

Immunotherapy, especially vaccines against infectious diseases and cancers, is the core field of the mRNA drug platform. mRNA vaccines are generally categorized into two major types according to their construction and replication abilities: self-amplifying mRNA (SAM) vaccines and non-replicating mRNA vaccines.

• mRNA vaccines against infectious diseases
  Vaccines against infectious pathogens has always been the most effective way to prevent and limit infectious diseases, a classic example of which is the complete eradication of the smallpox virus. Unfortunately, traditional strategies of vaccines, such as non-live freeze-dried vaccines and live attenuated vaccines, underperform against some chronic or recurrent pathogenic infections with a long duration of disease such as AIDS and tuberculosis (TB). Traditional vaccines' lack of adequate speed, owing to relatively slow process of development, would not be able to address outbreaks of virulent pathogens such as Zaire ebolavirus, Zika virus (ZIKV) and coronavirus.

Fig. 3 Examples of mRNA vaccine clinical trials for infectious diseases.

• mRNA cancer vaccines
  As our knowledge of tumor-specific antigens gradually deepens, people are now discussing more about the possibility of developing cancer vaccines. Tumor-associated antigens (antigens preferentially expressed in cancerous cells and usually relevant to dysregulation and abnormal behaviors of cancerous cells) and tumor-specific neo-epitopes (small peptides derived from tumor-specific somatic mutation that are exposed to the surface of cancer cells and can be recognized by T cells) are now the core targets of mRNA cancer vaccines. Considering the diversity and uncertainty of the cancerogenesis, cancer vaccines that are mainly therapeutic are now aimed to stimulate cellular immunity, which would potentially act as an effective cancer treatment. There have been some clinical trials of hopeful candidates in progress.

Fig. 4 Examples of mRNA vaccine clinical trials for infectious diseases.

Discussion

In conclusion, the mRNA vaccine is a versatile and powerful platform. From increasing productive capacity of mRNA and various carrier materials, to screening potential carrier molecules and adjuvants, to improving the composition and construction of vaccines, to arranging a corresponding route for administration, to optimizing the core encoding mRNA sequence and to demonstrating immune mechanisms of delivery and induction, the field of mRNA vaccines is still far from maturity, but its potential to be the preferred vaccine pattern has been fully shown.