Karik K., Muramatsu H., Ludwig J., Weissman D. liposome-entrapped mRNA encoding the nucleoprotein of the influenza virus.9 By 2017, the first proof-of-concept phase 1 human trial of an mRNA vaccine against an infectious disease, the rabies virus, demonstrated the ability of this platform to induce an immune response to the encoded antigen of interest.10 These early successes supported mRNA technology as a highly versatile and viable contender in ongoing efforts toward innovative vaccine development. EXPRESSION SYSTEM/MECHANISM mRNA is a single stranded RNA, and an intermediate transporter of the genetic code, that plays a key role between translation of protein-encoding deoxyribonucleic acid (DNA) to protein production by ribosomes in the cytoplasm.2 , 11 Functional synthetic mRNA is the result of in vitro transcription of a linearized plasma DNA template by a DNA-dependent RNA polymerase, such as the T7, T3, or Sp6 bacteriophage RNA polymerase, in the presence of nucleoside triphosphates.11, 12, 13, 14 The product is a functional synthetic mRNA that resembles mature mRNA found in eukaryotic cells, and the template DNA is subsequently degraded by DNases.2 , 4 , 11 There are 2 types of mRNA utilized in the current vaccines under development: non-replicating mRNA and self-amplifying mRNA (SAM). Conventional non-replicating mRNA contains a 5 cap, an open reading frame containing the target protein sequence, 5 and 3 untranslated regions (UTRs), and a poly(A) tail.2 , 4 SAM contains these essential elements, but it also encodes viral replication machinery which enables CALCR intracellular RNA amplification thereby increasing Gastrofensin AN 5 free base protein expression compared with non-replicating RNA.2 , 15 The process of in vitro transcription results in many byproducts, including contaminating double-stranded RNA (dsRNA), that can provoke an undesirable innate immune response and hasten the degradation of the newly made RNA.11 By acting as pathogen-associated molecular patterns (PAMPs), these byproducts are detected by the immune system and can incite production of type I interferon which can lead to degradation of both the cellular and ribosomal RNA.2 , 16 Additionally, the intrinsically immunogenic nature of RNA itself can provoke such an immune response and thus the process of purification of the mRNA becomes an important step.3 Purification also plays a role in optimizing the Gastrofensin AN 5 free base of expression of mRNA once it Gastrofensin AN 5 free base enters the cell.3 Several methods of removing the Gastrofensin AN 5 free base contaminating dsRNA have been described, including fast protein liquid chromatography, high-performance liquid chromatography, and nucleoside modification.17 Utilization of these techniques facilitates maximal production of the desired protein and avoidance of adverse activation of innate immune responses. Gastrofensin AN 5 free base The post-transcription process provides the essential modifications that impart structural stability and enhanced expression of the mRNA code. The 5 cap and poly(A) tail are essential for efficient protein translation and stabilization of the mRNA molecule in the cytoplasm.11 , 18 , 19 The 5 and 3 UTRs have important structural roles as they provide the mRNA molecule with stability and regulate translation.2 , 18 The 5 and 3 UTRs also prolong the half-life and enhance expression of the mRNA.20 The post-transcription cytoplasmic mRNA then encounters the cell’s translation machinery and the protein of interest is produced.11 This protein then undergoes additional post-translational modifications, including proper folding, to make a fully functional protein. 11 Other methods of increasing protein translation and expression have been described.2 Sequence optimization and modification of codon usage by substituting rare codons for synonymous codons can increase transfer ribonucleic acid (tRNA) in the cytoplasm.2 , 4 , 21 Another method described involves increasing the guanine and cytosine sequence content of the genetic code, which leads to increased steady-state mRNA levels resulting in increased gene expression.2 , 15 , 22 DELIVERY Once delivered at the site of injection, the mRNA in the vaccine must avoid degradation by nucleases and cross the cell’s plasma membrane to reach the translation machinery in the cytoplasm in order to effect an antigen-specific immune response.15 , 23 Fig?1 summarizes the mechanism of nonreplicating mRNA vaccines against COVID-19. Multiple studies have demonstrated the ability of.