mRNA-based therapeutics: powerful and versatile tools to combat diseases

Abstract

The therapeutic use of messenger RNA (mRNA) has fueled great hope to combat a wide range of incurable diseases. Recent rapid advances in biotechnology and molecular medicine have enabled the production of almost any functional protein/peptide in the human body by introducing mRNA as a vaccine or therapeutic agent. This represents a rising precision medicine field with great promise for preventing and treating many intractable or genetic diseases. In addition, in vitro transcribed mRNA has achieved programmed production, which is more effective, faster in design and production, as well as more flexible and cost-effective than conventional approaches that may offer. Based on these extraordinary advantages, mRNA vaccines have the characteristics of the swiftest response to large-scale outbreaks of infectious diseases, such as the currently devastating pandemic COVID-19. It has always been the scientists' desire to improve the stability, immunogenicity, translation efficiency, and delivery system to achieve efficient and safe delivery of mRNA. Excitingly, these scientific dreams have gradually been realized with the rapid, amazing achievements of molecular biology, RNA technology, vaccinology, and nanotechnology. In this review, we comprehensively describe mRNA-based therapeutics, including their principles, manufacture, application, effects, and shortcomings. We also highlight the importance of mRNA optimization and delivery systems in successful mRNA therapeutics and discuss the key challenges and opportunities in developing these tools into powerful and versatile tools to combat many genetic, infectious, cancer, and other refractory diseases.

Fig. 1

Key discoveries and advances in mRNA-based therapeutics. The development of mRNA-based therapeutics can be divided into three stages. Phase 1, mRNA discovery, in vitro synthesis and nucleic acid delivery system construction (1961–1990), including discovery mRNA and using protamine for RNA delivery, in vitro translation of isolated mRNA, mRNA cap was discovered, Liposome-entrapped mRNA delivery, Cap analog commercialized, T7 RNA polymerases commercialized, Cationic lipid-mediated mRNA delivery, Naked mRNA is translated in vivo by direct injection. Phase2 (1990-2019), accumulated knowledge with the continuous attempts and diverse applications, especially protein replacement therapies and vaccination approaches for cancer and infectious diseases, including using mRNAs for cancer immunotherapy, mRNA-based company founded and 3′-UTR regulates mRNA localization, antitumor T cell response induced by mRNA, first clinical trial with mRNA using ex vivo transfected DCs, mRNA-based immunotherapy for human cancer, preclinical study with intranodally injected DC-targeted mRNA, protective mRNAs vaccination in influenza and respiratory syncytial virus, CRISPR–Cas9 mRNA for gene editing, personalized mRNA cancer vaccine for clinical trials. Phase 3, mRNA-based therapeutics, as a disruptive therapeutic technology, is becoming powerful and versatile tools for therapy diseases (2019 to present), including clinical trials of mRNA vaccines for cancer and infectious disease, mRNA-1273, and BNT162b emergency use for SARS-CoV-2 pandemic

Fig. 2

mRNA drugs production pipeline. The encoding of peptide/protein is designed and inserted into a plasmid DNA construct. Plasmid DNA is transcribed into mRNA by bacteriophage polymerases in vitro, and mRNA transcripts are purified by high-performance liquid chromatography (HPLC) or nanoprecipitation to remove contaminants and reactants. Subsequently, purified mRNA is entrapped in various vehicles. The interactions between vehicles and mRNA can be divided into three types: (a) electrostatic adsorption with phosphate ions of the ribonucleotides; (b) complementary paired hydrogen bonding with bases of the ribonucleotides; and (c) coordination with the phosphate ions. Thus, vehicles for mRNA delivery consist of the following categories: cationic compounds, such as cationic lipids, ionizable lipids, and cationic polymers. Nucleoside-based lipids, e.g., DNCA, or nucleoside-based amphiphilic polymers, e.g., Chol(+)-oligoRNA. Metal-based compounds provide vacant orbitals to coordinate with phosphate ions. Furthermore, the efficacy, pharmacology, and safety of mRNA drugs were evaluated in vaccinated mice and primates. Finally, the scale-up manufacturing of mRNA therapeutics is conducted and followed by clinical trials

Fig. 3

In vitro transcribed (IVT) mRNA and translation initiation.IVT mRNA preparation includes several steps, plasmid cloning, plasmid linearization, in vitro transcription, 5′ capping, and the poly(A) tail adding. Transcription, capping and the tail adding can combine into one, two or three steps that depend on the design of synthesis routes. After entering into the cell, mRNA translation can be initiated in an eIF4F-dependent manner to recruit a preinitiation complex (PIC). The 43S PIC is formed by 40S ribosomal subunit, the eukaryotic translation initiation factors (eIF, including eIF1, eIF1A, eIF3, eIF5) and the ternary complex, including a trimeric complex comprising eIF2 that contains α-, β-, and γ-subunits, initiating methionyl tRNA (tRNAiMet), and GTP. eIF4F is a complex composed of eIF4A, eIF4E and eIF4G. eIF4E binds to mRNA cap. eIF4G interacts with eIF3 and poly(A)-binding protein (PABP) that binds to the 3′ poly(A) tail. These interactions result in mRNA circularization and 48S PIC assembly. The 48S PIC ribosomal subunit scans and finds the start codon with the help of eIF4A helicase to resolve secondary mRNA structure in the 5′ UTR. Then, eIFs are released and 60S ribosomal subunit joins to initiate translation elongation by forming 80S ribosome

Fig. 4

Mechanisms of mRNA decay. Degradation of messenger mRNA plays an essential role in regulating sustained mRNA expression. mRNA is generally degraded in the following three pathways: ① Deadenylation-dependent mRNA decay: The poly(A) tail is removed by deadenylase activity (such as CCR4, CAF1 or PARN). The LSM1-7 complex associates with the 3′-end of the mRNA transcript to induce decapping by the Dcp1–Dcp2 complex and is then degraded by exoribonuclease XRN1. Alternatively, deadenylated mRNA can be degraded by exosomes. ② Endonuclease-mediated mRNA decay: The mRNA is cleaved into two fragments, and then the fragments are degraded by XRN1 and exosomes. ③ Deadenylation-independent pathways require recruitment of the decapping machinery. RPS28B interacts with the enhancer of decapping-3 (Edc3) to engage the decapping enzyme. Subsequently, the mRNA is degraded by XRN1

Fig. 5

Commercialization and commonly used Cap. The 5′ cap of mRNA is critical to improve mRNA stability and promote translation efficiency. Modification of the 5′-5′ phosphate bridge can increase the resistance to DcpS and Dcp1/Dcp2, but the translation efficiency may not necessarily increase (such as the introduction of methylene groups on the phosphate bridge). The modification of ribose nucleosides also plays essential functions in mRNA translation by recruiting translation initiation factors, such as the methylation modification on the N7 position of the guanosine cap and the ribose-2′O position of the first nucleotide (Cap 1), increasing the affinity for eIF4E and thereby improving translation efficiency^,

Fig. 6

Positively charged lipids in mRNA-loaded lipid nanoparticles. The most widely used carrier of mRNA preparations is LNPs. Positively charged lipids play a vital role in LNPs because LNPs encapsulate mRNA through electrostatic adsorption between lipids and mRNA. These lipids can be classified into cationic lipids and ionizable lipids according to the generation of a positive charge. Furthermore, ionizable lipids can be divided into single-charged lipids and multicharged lipids. Here, we listed the representative lipids used in LNPs, including DOTMA, DOTAP, DSTAP, DMTAP, DDA, DOBAQ, DC-Chol,^, DLin-MC3-DMA, SM-102, A6, ALC-0315, and Lipid 5. Multicharged lipids in LNPs include C12-200, 5A2-SC8, cKK-E12, G0-C14, OF-2, 306Oi10, OF-Deg-Lin, 92-O17S, OF-C4-Deg-Lin, A18-Iso5-2DC18, TT3, BAMEA-O16B, FTT5, Vc-Lipid, C14-4, Lipid 14, 4A3-Cit, and ssPalmO-Phe

Fig. 7

Strategies and potential application of mRNA-based therapeutics. mRNA drugs have yielded numerous inspiring treatments for refractory or previously incurable diseases, including infectious diseases, genetic diseases, cancers, and cardiovascular diseases. In particular, the mRNA vaccine has shown a strong advantage in the prevention of SARS-CoV-2 infection and may also be a potential approach against the infection of other viruses and pathogenic microorganisms, including malaria, respiratory syncytial virus, and HIV

Fig. 8

mRNA drugs elicit immunity using disease-specific targeting antigen strategies. mRNA drugs mainly go through the following three aspects from synthesis to initiate immune protection, including mRNA synthesis, intracellular processing, and initiating immune protection. Briefly, IVT mRNA drugs are encapsulated into carriers (such as nanoparticles) and are endocytosed by antigen-presenting cells (①-②); mRNA is released into the cytoplasm after escaping from endosomes and then translated into antigenic proteins by ribosomes (③). Subsequently, endogenous antigens are degraded into polypeptides by the proteasome and are presented by MHC I and activate cytotoxic T cells (CD8⁺ T cells) (④-⑥). In addition, 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 (⑦-⑨). Finally, helper T cells (CD4⁺ T cells) stimulate B cells to produce neutralizing antibodies against pathogens

Fig. 9

SARS-CoV-2 mRNA antigen immunogenicity and vaccine design. Full-length S-protein or RBD as a vaccine immunogen has been widely confirmed to induce high-affinity neutralizing antibodies. SARS-CoV-2 S protein is intrinsically metastable and can be stabilized in a prefusion conformation by structure-based design.^, Prefusion-stabilized SARS-CoV-2 Spike immunogen induces potent humoral and cellular immune responses.^, The RBD peptide is one of the most promising targets to design candidate vaccines. However, RBD has a low molecular weight, which leads to its weak immunogenicity, and can be further improved by forming multimers. Multimerization of RBD protein using humanized IgG Fc, T4 trimerization (FD) or Ferritin have been shown to induce higher neutralizing antibody compared to monomeric antigens, which will provide us with new ideas for designing powerful mRNA vaccines