Section Overview
The potential for synthetic mRNA to recapitulate the function of mature mRNA has elevated its potential for a spectrum of biopharmaceutical applications. These therapeutics include in vivo delivery of mRNA for protein replacement, stem cell induction, or cancer immunotherapies. Recent significant pharmaceutical uses include novel approaches for vaccines against infectious disease, most notably the mRNA-based vaccines that protect against SARS-CoV-2.
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This technical article focuses on pDNA and mRNA preparation for lab-scale applications. For a complete overview of our products and solutions suitable for GMP manufacturing, visit our mRNA Manufacturing and Formulation page.
Messenger RNA (mRNA)-based vaccines are derived from the central dogma that mRNA encodes for proteins. This vaccine modality is straightforward, inducing an immunological response by delivering a genetic element that codes for all (or part) of an antigenic protein in a “translation-ready” molecule. This process is facilitated when the mRNA encoding the target antigen is taken up by antigen-presenting cells and translated into the target protein. This in turn induces an immune response, closely mimicking the natural process by which viruses infect cells. Generally, mRNA vaccines are produced byin vitrosynthesis through an enzymatic process. The development and manufacture of mRNA vaccines are relatively simple compared to more traditional modalities and can be accomplished in a shorter time period. These attributes make them suitable for accelerated development and scaleup, and are critical for emergent public health scenarios. Future innovations in mRNA vaccine design, formulation, and enhanced thermostability (beyond cold-chain distribution) would further reduce production costs and enhance global accessibility of mRNA as a modality. For mRNA-based vaccine design,in vitrotranscription of a plasmid DNA (pDNA) template is typically used to produce functional synthetic mRNA. The plasmid vector may contain the following elements: an upstream promoter exclusively recognized by T7, SP6, or T3 RNA polymerase, all of which are derived from bacteriophages; 5' UTR; cDNA; 3' UTR; a downstream poly A-tail; and a unique cleavage site downstream of the poly A-tail. Preparation of the pDNA template often involves PCR amplification and gel purification of your target DNA, followed by restriction digestion (of both insert and plasmid backbone) and ligation using enzymes such as T4 DNA Ligase. Selection of proper transformation reagents (BL21 competent cells, SOC broth, and LB-agar plates with selection antibiotics) and purification techniques (GenElute™ Plasmid Miniprep Kit) are all considerations as researchers optimize conditions for template generation.HowmRNA Vaccines Induce Immunity
Advantages ofmRNA as a Vaccine Modality
Plasmid DNA (pDNA) Preparation formRNA Synthesis
Figure 1.Plasmid generation for in vitro transcription (IVT). Following gene synthesis, cloning of the target DNA uses a plasmid vector (pDNA). The pDNA is expanded in bacterial culture, then purified using nucleic acid purification methods, such as silica-based membranes in spin columns.
Bacteriophage RNA polymerase is normally used to transcribe linearized plasmid DNA. The pDNA is first linearized with the selected unique restriction site enzyme. After digestion, the linearized pDNA may be purified using methods such as the phenol-chloroform protocol or theGenElute™ PCR Clean-Up Kit. For large scale purification, tangential flow filtration (TFF) is often advisable as it can be easily scaled up. Following linearization,in vitrotranscription and capping is performed in a mixed solution of recombinant RNA polymerase (T7, SP6 or T3) and nucleoside triphosphates (ATP, CTP, GTP, UTP), plus a cap analog such as CleanCap®Reagent or ARCA (Anti-Reverse Cap Analog). A modified nucleoside such as N1-Methylpseudouridine-5'-Triphosphate (N1-Methylpseudo-UTP, 1-Methylpseudo-UTP) can be used instead of GTP to suppress the innate immune system and is used in the current mRNA vaccines. The efficiency of capping mRNA with ARCA is ~70-80% on average because it competes with GTP, while the yield of mRNA is reduced by about one-fourth compared to standard mRNA synthesis. In comparison, the CleanCap®reagent has been shown to work at 94% capping efficiency without affecting the yield of mRNA production. Capping may also be achieved post-transcriptionally, without a cap analog, by employing the vaccinia virus-encoded capping complex (Vaccinia capping enzyme, 2'-O-Methyltransferase, GTP, and S-adenosyl methionine (SAM)). Capping efficiency will depend on the secondary structure of the mRNA of interest. Finally, the length of the poly A tail of the template pDNA (up to 150 bases) can be extended by use of poly A enzyme when necessary.mRNA IN VITRO TRANSCRIPTION AND CAPPING
Plasmid Preparation Reagents
Figure 2.mRNA synthesis is completed by linearization of pDNA, in vitro transcription (IVT) of mRNA using cell-free methods, and capping of the mRNA using cap analog or virus-encoded capping complex.
