Fig. 1

A Adjusting mRNA Medicine Dosage Pharmacokinetics. a) Crucial structural components of in vitro transcribed (IVT) mRNA and approaches for their alterations. b) Based on the individual or combined use of these elements (such as modification of caps, UTRs, or poly(A) tails), the protein product's expression duration and kinetic profile can be controlled and optimized. eIF4E represents eukaryotic translation initiation factor 4E; IRES refers to the internal ribosome entry site; and ORF denotes open reading frame. Reprinted from [51] with permission from Springer Nature. B Fundamentals of mRNA-based antigen pharmacology. a) A linear DNA plasmid containing the antigen-encoding sequence is employed for in vitro transcription. The transcribed mRNA consists of the cap, 5′ and 3′ UTRs, the open reading frame (ORF), and the poly(A) tail, which influence the mRNA's translational activity and stability once introduced into cells. b) Step 1: A portion of the foreign mRNA avoids degradation by common RNases and is taken up by cell-specific mechanisms (such as macropinocytosis in immature dendritic cells) into endosomal pathways. Step 2: The release of mRNA into the cytoplasm is not entirely understood. Step 3: Host cell protein synthesis machinery translates the mRNA. mRNA translation's rate-limiting step involves eukaryotic eIF4E binding to the cap structure. The formation of circular structures and active translation result from mRNA binding to ribosomes, eIF4E, eIF4G, and poly(A)-binding protein. Step 4: Exonucleases catalyze the termination of translation via mRNA degradation. Decapping enzymes D CP1, DCP2, and DCPS hydrolyze the cap, followed by the digestion of residual mRNA by 5′–3′ exoribonuclease 1 (XRN1). Degradation might be delayed if mRNA is silenced and located within cytoplasmic processing bodies. Alternatively, exosomal endonucleolytic cleavage of mRNA may take place. Various mechanisms control the breakdown of aberrant mRNA (such as mRNA with a premature stop codon). Step 5: The translated protein undergoes post-translational modifications based on the host cell's characteristics. The synthesized protein can then function within the cell it was produced in. Step 6: Alternatively, the protein is secreted and can function through autocrine, paracrine, or endocrine pathways. Step 7: For immunotherapeutic mRNA application, the protein must be broken down into antigenic peptide epitopes. These peptides are loaded onto major MHC molecules, which present the antigens to immune effector cells. Proteasomes degrade cytoplasmic proteins, which are then transported to the endoplasmic reticulum and loaded onto MHC class I molecules for presentation to CD8 + cytotoxic T lymphocytes. Almost all cells express MHC class I molecules. Step 8: In antigen-presenting cells, the protein must be directed to MHC class II loading compartments to obtain T cell assistance for a stronger, lasting immune response. This can be achieved by incorporating routing signal-encoding sequences into the mRNA. Additionally, DCs can process and load exogenous antigens onto MHC class I molecules through a mechanism called cross-priming. Step 9: Antigens derived from the protein can be displayed on the cell surface by both MHC class I and MHC class II molecules, enabling the immune system to recognize and respond to them accordingly. Reprinted from [51] with permission from Springer Nature