IVT mRNA can be manufactured at relatively low costs and within a few hours, and the production and purification processes are robust, enabling the generation of mRNA ranging from a few hundred to more than 10,000 nucleotides in length. Science 303, 15291531 (2004). Nanoparticles are incredibly small structures that have been developed for many uses. With the exception of the 5 cap, this DNA template encodes all the structural elements of a functional mRNA. Opin. Gene Ther. The robustness and ease of the production process facilitates the implementation of high-throughput approaches for drug discovery and iterative drug optimization. Ying, H. et al. The other more commonly used approach is to add a synthetic cap analogue into the in vitro transcription reaction and perform capping and in vitro transcription in a single step. Zeis, M. et al. Van Driessche, A. et al. The mRNA is fragile and it delivers the instructions to your cells to make antibodies against SARS-CoV-2. Feeder-free derivation of human induced pluripotent stem cells with messenger RNA. Cells may be modified ex vivo for subsequent administration to humans, or may be altered in vivo by gene therapy given directly to the subject. In preclinical studies, interferon-, IL-6, tumour necrosis factor- and interferon--induced protein 10 (IP-10; also known as CXCL10) were determined as key cytokines that are upregulated by systemic IVT mRNA delivery. Sci. Wang, W. et al. Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells. Drug Deliv. Hence, the IVT mRNA is single-stranded, has a 5 cap and a 3 poly(A) tail. An improved zinc-finger nuclease architecture for highly specific genome editing. 209) and laid the foundation for genetic engineering of self-amplifying viral mRNA replicons. Lorenz, C. et al. Okumura, K. et al. Qiu, P. et al. Gene Ther. A limitation of enzymatic polyadenylation is that each RNA preparation consists of a mixture of RNA species differing in the length of the poly(A) tail. CAS J. Biol. J. Immunol. J. Immunother. About this content. Epub 2021 Mar 1. Safety monitoring has to consider that adverse effects may only occur after prolonged treatment with nucleoside analogues. This review will introduce the history of RNA therapy, and explain the basic concepts of RNA therapy and RNA-based drugs on the market or clinical trials. IVT mRNA containing modified nucleosides (2-thiouridine and 5-methylcytidine) and encoding surfactant protein B (SPB) was tested in a mouse model of congenital lethal lung disease caused by the lack of the SPB protein. J. Virol. The discovery that modified nucleosides can reduce the immune-stimulatory activity of RNA was pivotal in advancing this field of application75. This approach was further optimized, resulting in a pharmaceutical-grade two-component vaccine combining a naked antigen-encoding mRNA plus a protamine-complexed mRNA adjuvant (developed by CureVac). Van Tendeloo, V. F. et al. Khromykh, A. The machinery of the transfected cell is utilized for in vivo translation of the message to the corresponding protein, which is the pharmacologically active product. mRNA Vaccines: What They Are & How They Work - Cleveland Clinic B. et al. HHS Vulnerability Disclosure, Help Particle size and activation threshold: a new dimension of danger signaling. Immunother. Cells that are transfected with IVT mRNA need to have the required convertase or endoprotease to process the encoded precursors to functional products. Curr. RNA therapeutics - Wikipedia Cisco, R. M. et al. Mol. Meanwhile, emerging novel approaches include in vivo delivery of IVT mRNA to replace or supplement proteins, IVT mRNA-based generation of pluripotent stem cells and genome engineering using IVT mRNA-encoded designer nucleases. An increasing number of patent applications are from academic groups featuring findings in preclinical studies and from industry for the use of IVT mRNA to replace defined disease targets. The production of mRNA by in vitro transcription is a well-defined procedure. Such mRNA-binding proteins are ubiquitously expressed in all human cells and tissues, and play a critical role in numerous biological processes. J. The mRNA is synthesized in a cell-free system by in vitro transcription from a DNA template, such as a linearized plasmid or a PCR product. USA 99, 44954499 (2002). Med. Science 314, 994997 (2006). This is because the makeup and delivery mechanism now provides scientists a basic template to target any virus. FEBS J. Safety considerations regarding the encoded protein. Cancer therapy using a self-replicating RNA vaccine. Different types of vaccines work in different ways to offer protection. Nature. and transmitted securely. 16, 18331840 (2008). Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells. B. 58, 29652968 (1998). https://doi.org/10.1038/nrd4278. 29, 695696 (2011). The https:// ensures that you are connecting to the Commun. Is gene therapy available to treat my disorder? Korner, C. G. & Wahle, E. Poly(A) tail shortening by a mammalian poly(A)-specific 3-exoribonuclease. Dis. An alphavirus replicon particle chimera derived from venezuelan equine encephalitis and sindbis viruses is a potent gene-based vaccine delivery vector. Not every cell has the capability to glycosylate each protein properly, particularly if highly complex glycosylation is required149. Calderhead, D. M. et al. 32, 498507 (2009). Niu, Y. et al. Nature Immunol. Teens 12-17 years old can only get a Pfizer booster. 5-triphosphate RNA is the ligand for RIG-I. The mRNA vaccine technology and its success against COVID-19 have opened doors to explore its uses against a variety of diseases. Smull, C. E., Mallette, M. F. & Ludwig, E. H. The use of basic proteins to increase the infectivity of enterovirus ribonucleic acid. Vaccin. Lai, Y., Tse, C.-M. & Unadkat, J. D. Mitochondrial expression of the human equilibrative nucleoside transporter 1 (hENT1) results in enhanced mitochondrial toxicity of antiviral drugs. Martinon, F. et al. The highest currently visible patenting activity stems from the small group of biotechnology companies specialized in mRNA-based therapeutics. Similar to recombinant DNA-based approaches (reviewed in Ref. These responses protected the mice against the induction of allergen-specific IgE and inhibited lung inflammation mediated by allergen exposure147,148. Li, W. M., Barnes, T. & Lee, C. H. Endoribonucleases enzymes gaining spotlight in mRNA metabolism. Knockout rats generated by embryo microinjection of TALENs. Bernal, J. Recently, a phosphorothioate-containing ARCA cap analogue was developed39. This impairs its use for tissues with non-fenestrated capillaries and restricts the number of transfectable cells in tissues with endothelial fenestration to those undergoing mitosis at the time of exposure. Mol. Furuichi, Y. Eur. These spike proteins are ideal Trends Biotechnol. Biochem. The type of immune response appears to depend on a range of factors, including the characteristics of the type and size of particles in which the IVT mRNA is incorporated62,63,64. The body recognizes that spike protein and remembers that it has seen it before, so that next time when there is an infection, it attacks the full virus. Blood 102, 44164423 (2003). Moreover, professional antigen-presenting cells, which are the targets for mRNA-based vaccine delivery, are constitutively equipped with a specialized mechanism for mRNA uptake. The translated protein can then act in the cell in which it has been generated. 70, 90539061 (2010). Federal government websites often end in .gov or .mil. This type of treatment combines the technologies of gene therapy and cell therapy. Genomics 95, 306311 (2010). IRF, interferon regulatory factor; ISRE7, interferon-stimulated response element; MAVS, mitochondrial antiviral signalling protein; MDA5, melanoma differentiation-associated protein 5; MYD88, myeloid differentiation primary response protein 88; MX1, myxovirus (influenza) resistance 1; NF-B, nuclear factor-B; TRIF, Toll-IL-1 receptor domain-containing adapter protein inducing IFN. Eur. Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Two mRNA vaccines have been tested in large-scale clinical trials that included elderly and medically at-risk individuals; 30% of participants in these trials were from racially and ethnically diverse backgrounds. Diebold, S. S. et al. These may be either intrinsic to the natural protein sequence or recombinantly engineered to direct the protein to the desired cell compartment within the host cell. Messenger RNA (mRNA) Vaccines for COVID-19: How Do They Work? Thomsen, L. B. et al. Gene Ther. J. Immunol. mRNA vaccine: a potential therapeutic strategy - PMC mRNA-based therapeutics: powerful and versatile tools to combat Messenger RNA (mRNA) is a type of single-stranded ribonucleic acid that is transcribed from a strand of DNA, which carries the coding information for protein synthesis to be further transcribed. contracts here, Sign up for Nature Briefing: Translational Research. The relative secretory signal strengths differ153; thus, exchanging the natural signal peptides could lead to increased protein secretion. mRNA therapeutics in cancer immunotherapy. Nat Rev Drug Discov 13, 759780 (2014). RNA 14, 11191131 (2008). Gene Ther. Nature Cell Biol. 70, 90319040 (2010). The reprogramming and direct differentiation of cells with IVT mRNA profits from its high in vitro transfection efficiency, transient expression with no genomic integration and the ability to transfer complex mixtures. In vitro transfection strategies. Seidah, N. G. & Chrtien, M. Proprotein and prohormone convertases: a family of subtilases generating diverse bioactive polypeptides. Step 5: the translated protein product undergoes post-translational modification, the nature of which depends on the properties of the host cell. After your body builds antibodies against the spike protein, it gives you immunity against the real SARS-CoV-2 virus infection. Zhou, W. Z. et al. (Individuals who get an mRNA vaccine are not exposed to the virus, nor can they become infected with the virus by the vaccine.) Suster, M. L., Sumiyama, K. & Kawakami, K. Transposon-mediated BAC transgenesis in zebrafish and mice. Efficient genome editing in zebrafish using a CRISPR-Cas system. 31, 898907 (2013). Induction of virus-specific cytotoxic T lymphocytes in vivo by liposome-entrapped mRNA. Google Scholar. A. et al. CAR T cell therapy (or chimeric antigen receptor T cell therapy) is an example of cell-based gene therapy. The recent progress in identifying RNA sensors in cells and the structural elements within mRNA that are involved in immune recognition provides opportunities to augment immune activation by IVT mRNA, or alternatively, to create 'de-immunized' mRNA as needed. J. Immunother. Benteyn, D. et al. The spike protein your cells use to build antibodies may linger in your body for a few weeks. The role of mRNA is to carry protein information from the DNA in a cell's nucleus to the cell's cytoplasm (watery interior), where the protein-making machinery reads the mRNA sequence and translates each three-base codon into its corresponding amino acid in a growing protein chain. It was shown that the injected DNA persisted for 2 weeks and could spread from the injection site to immune and non-immune tissues throughout the body, thus posing the potential risk of inducing severe anaphylactic side effects146. 25, 152159 (2013). This could induce an immune response to fight off a viral attack. Transl. 23, 17191722 (1993). 83, 3746 (2012). CAR, chimeric antigen receptor; Cas9, CRISPR-associated protein 9; CRISPR, clustered regularly interspaced short palindromic repeat; DC, dendritic cell; dsRNA, double-stranded RNA; iPSC, induced pluripotent stem cell; RSV, respiratory syncytial virus; ssRNA, singlestranded RNA; TALEN, transcription activator-like effector nuclease; TLR, Toll-like receptor. mRNA therapeutics in cancer immunotherapy - PMC - National Center for Nature 274, 923924 (1978). The plasmid is linearized with a restriction enzyme that cleaves the DNA downstream from sequences encoding the poly(A) tail. Biophys. 9, 265274 (2013). J. Immunol. Activation of MDA5 requires higher-order RNA structures generated during virus infection. Morse, M. A. et al. The protein that the person makes in response to the vaccine can cause an immune response without a person ever having been exposed to the virus that causes COVID-19. Cancer Immunol. Mol. Chem. 2nd International mRNA Health Conference; Boston, November 11-12, 2014, FierceBiotech (Industry Voices: mRNA-Based Therapies Blueprints for Therapeutics; 25 Jul 2013), Zone in with Zon (What's trending in nucleic acid research): Modified mRNA Mania; 2 Dec 2013, Sahin, U., Karik, K. & Treci, . mRNA-based therapeutics developing a new class of drugs. 142, 252268 (2012). B. AU-rich elements: characterization and importance in mRNA degradation. J. Immunol. J. Since most of the immune-mediated adverse effects against a therapeutic protein product are mediated by humoral mechanisms, circulating antibodies to the therapeutic protein product have been the main criterion for defining an immune response. Sci. Moreover, immune stimulation is unwanted. Your Genome from the Wellcome Genome campus explains the first gene therapy trial to treat a condition called severe combined immunodeficiency (SCID). It's not the entire virus, as it's impossible to make a whole virus out of the mRNA. Koren, E. et al. Biochim. Steps toward safe cell therapy using induced pluripotent stem cells. This means that the question of consistent dosing across tissues and patients can become a significant roadblock for the clinical development of in vivo administered IVT mRNA. Muthukrishnan, S., Both, G. W., Furuichi, Y. As the number of precedents is limited and the diversity of mRNA-based applications is broad, one cannot predict for each individual investigational mRNA drug how the United States, the European Union and European national competent authorities may view in vitro transcribed (IVT) mRNA from a regulatory perspective. Induction of antitumor immunity by vaccination of dendritic cells transfected with MUC1 RNA. It cannot change your DNA in any way. Therefore, no general exhaustive risk assessment can be provided; instead, risks have to be evaluated with due diligence on a case-by-case basis. Immunol. Schlee, M. et al. Roberts, A. Three Phase I/II clinical trials using IVT mRNA encoding combinations of different viral proteins showed that the vaccines are safe and that antigen-specific CD8+ and CD4+ T cell responses can be induced115,134,135. In 1995, it was demonstrated that intramuscular injection of naked RNA encoding carcinoembryonic antigen elicited antigen-specific antibody responses4. The replicon RNA can be produced easily by in vitro transcription from cDNA templates. One such technique uses special structures called nanoparticles as vectors to deliver the genetic material or gene-editing components into cells. Curr. However, given the unique immunological properties of this organ, it may even be de-risking to use the liver as a depot organ for protein expression of the first-generation of mRNA-based therapeutics for protein replacement, as its capacity to induce antigen-specific tolerance may counteract immunogenecity196. -, Dimitriadis, G. J. If an altered gene causes a necessary protein to be faulty or missing, gene transfer therapy can introduce a normal copy of the gene to recover the function of the protein. 3) (Left to right) Javier Zayas Photography / Getty Images. Optimization of the sequence of antigen loading and CD40-ligand-induced maturation of dendritic cells. 180, 309318 (2008). RNA-based vaccines. The vector can be injected or given intravenously (by IV) directly into a specific tissue in the body, where it is taken up by individual cells. Am. mRNA nanomedicine-based gene therapy may offer opportunities for cancer treatment. Kreiter, S. et al. USA 86, 60776081 (1989). Balagopal, V., Fluch, L. & Nissan, T. Ways and means of eukaryotic mRNA decay. Gene Ther. Kreiter, S. et al. Nucleoside reverse transcriptase inhibitors used for treating HIV-infected patients caused severe clinical toxicities (for example, myopathy, polyneuropathy, lactic acidosis, liver steatosis, pancreatitis and lipodystrophy) including fatal complications due to mitochondrial dysfunction199. Vaccination with mRNA-electroporated dendritic cells induces robust tumor antigen-specific CD4+ and CD8+ T cells responses in stage III and IV melanoma patients. The synthetic mRNA in the vaccine acts like any other mRNA that your cells make. First-in-human testing of the intranodal injection of naked IVT mRNA encoding cancer antigens (developed by BioNTech) has recently begun in patients with melanoma (ClinicalTrials.gov identifier: NCT01684241). 1, 841845 (1982). What does an mRNA vaccine prevent? Rolls, M. M. et al. Nature Biotech. Other key challenges related to the complex pharmacology of mRNA, and to its delivery in particular, concern applications in which precise targeting of a particular cell type and organ is required. Res. a | A linearized DNA plasmid template with the antigen-coding sequence is used for in vitro transcription. Kuhn, A. N. et al. The 3-UTR of the eukaryotic elongation factor 1 (EEF1A1) mRNA46 and a 5-UTR element present in many orthopoxvirus mRNAs, for example, inhibited both decapping and 35 exonucleolytic degradation47 (reviewed in Ref. CAS 3, 8993 (1997). A further impact on ease and costs of manufacturing might result from progress in the field of chemical synthesis of functional mRNAs. Overall, these create a pro-inflammatory microenvironment poised for inducing specific immune responses. Another type of post-translational modification is proteolytic processing. Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation. Systemic delivery of IVT mRNA to other tissues, however, is hindered by non-fenestrated, non-permeable vascular endothelia, intercellular junctions and dense extracellular fibril networks that limit the accessibility of target cells93 (Fig. Sign up for the Nature Briefing: Translational Research newsletter top stories in biotechnology, drug discovery and pharma. Transl. In experiments with mice and macaques, pseudouridine-modified IVT mRNA purified by high-performance liquid chromatography and encoding erythropoietin was administered, and therapeutically relevant levels of erythropoietin were detected26. Please enable it to take advantage of the complete set of features! As IVT mRNA is produced in cell-free systems, process development and manufacturing of clinical-grade material can be easily standardized. B. et al. PubMed mRNA breaks down right after it delivers instructions to your cells. Ma, Y. et al. Given the diversity of proteins that may be potential candidates for the IVT mRNA approach and are currently being explored as such, it is difficult to predict which of these will be the first to be advanced into clinical development. 19, 990999 (2011). Int. Beck JD, Reidenbach D, Salomon N, Sahin U, Treci , Vormehr M, Kranz LM. Warren, L. et al. 72, 16611671 (2012). Type I IFN counteracts the induction of antigen-specific immune responses by lipid-based delivery of mRNA vaccines. Slater, J. E. et al. Gene therapy: What is it and how does it work? | Live Science Moreover, exogenous antigens that are taken up by dendritic cells can also be processed and loaded onto MHC class I molecules by a mechanism that is known as cross-priming232. Aerosol delivery of SPB mRNA into the lung twice a week protected mice from respiratory failure and prolonged their average lifespan25. Koido, S. et al. Protein-replacement therapies. Unauthorized use of these marks is strictly prohibited. In case of a real viral infection, your immune system is now prepared to destroy it and prevent serious illness. Invest. Britten, C. M. et al. J. Gene Med. Tumor vaccination using messenger RNA: prospects of a future therapy. MHC class I molecules are expressed by almost all cells. Microbes Infect. Purif. Invest. Nature 286, 123127 (1980). Mol. The protein enables a virus to enter your body, attach itself to a cell, replicate, and spread. The immune-activating property of IVT mRNA is an important feature to be considered from a safety perspective, particularly for systemically administered IVT mRNA. If the treatment is successful, the new gene delivered by the vector will make a functioning protein or the editing molecules will correct a DNA error and restore protein function.