Viral and Synthetic RNA Vector Technologies and Applications
Authors: Juliane W Schott, Michael Morgan, Melanie Galla, Axel Schambach
Journal: Molecular Therapy (2016)

Use of RNA is an increasingly popular method to transiently deliver genetic information for cell manipulation in basic research and clinical therapy. In these settings, viral and nonviral RNA platforms are employed for delivery of small interfering RNA and protein-coding mRNA. Technological advances allowing RNA modification for increased stability, improved translation and reduced immunogenicity have led to increased use of nonviral synthetic RNA, which is delivered in naked form or upon formulation. Alternatively, highly efficient viral entry pathways are exploited to transfer genes of interest as RNA incorporated into viral particles. Current viral RNA transfer technologies are derived from Retroviruses, nonsegmented negative-strand RNA viruses or positive-stranded Alpha- and Flaviviruses. In retroviral particles, the genes of interest can either be incorporated directly into the viral RNA genome or as nonviral RNA. Nonsegmented negative-strand virus-, Alpha- and Flavivirus-derived vectors support prolonged expression windows through replication of viral RNA encoding genes of interest. Mixed technologies combining viral and nonviral components are also available. RNA transfer is ideal for all settings that do not require permanent transgene expression and excludes potentially detrimental DNA integration into the target cell genome. Thus, RNA-based technologies are successfully applied for reprogramming, transdifferentiation, gene editing, vaccination, tumor therapy, and gene therapy. Use of RNA is an increasingly popular method to transiently deliver genetic information for cell manipulation in basic research and clinical therapy. In these settings, viral and nonviral RNA platforms are employed for delivery of small interfering RNA and protein-coding mRNA. Technological advances allowing RNA modification for increased stability, improved translation and reduced immunogenicity have led to increased use of nonviral synthetic RNA, which is delivered in naked form or upon formulation. Alternatively, highly efficient viral entry pathways are exploited to transfer genes of interest as RNA incorporated into viral particles. Current viral RNA transfer technologies are derived from Retroviruses, nonsegmented negative-strand RNA viruses or positive-stranded Alpha- and Flaviviruses. In retroviral particles, the genes of interest can either be incorporated directly into the viral RNA genome or as nonviral RNA. Nonsegmented negative-strand virus-, Alpha- and Flavivirus-derived vectors support prolonged expression windows through replication of viral RNA encoding genes of interest. Mixed technologies combining viral and nonviral components are also available. RNA transfer is ideal for all settings that do not require permanent transgene expression and excludes potentially detrimental DNA integration into the target cell genome. Thus, RNA-based technologies are successfully applied for reprogramming, transdifferentiation, gene editing, vaccination, tumor therapy, and gene therapy. Ribonucleic acid (RNA) is an important macromolecule critical for evolution and cellular life, and has been widely exploited for biotechnological applications. The RNA world hypothesis, in which RNA is considered as the primordial macromolecule that gave rise to all extant life, is well accepted.1Crick FH The origin of the genetic code.J Mol Biol. 1968; 38: 367-379Crossref PubMed Scopus (1512) Google Scholar,2Orgel LE Evolution of the genetic apparatus.J Mol Biol. 1968; 38: 381-393Crossref PubMed Google Scholar,3Gilbert W. Origin of life: The RNA world.Nature. 1986; 319: 618Crossref Scopus (0) Google Scholar,4Cech TR The RNA worlds in context.Cold Spring Harb Perspect Biol. 2012; 4: a006742Crossref Scopus (0) Google Scholar RNA is chemically suitable to store genetic information and to act as a catalytic molecule. Although the more stable biomolecule deoxyribonucleic acid (DNA) has replaced RNA for most cellular data storage systems and proteins have become the main effectors in biocatalysis, RNA still has a central role in several physiological processes.4Cech TR The RNA worlds in context.Cold Spring Harb Perspect Biol. 2012; 4: a006742Crossref Scopus (0) Google Scholar RNA bridges the information flow from DNA into protein through messenger RNA (mRNA), and there are diverse classes of noncoding RNA that are involved in lagging strand DNA replication, post-transcriptional gene regulation and the catalysis of chemical reactions (e.g., splicing and protein synthesis). In a cell-extrinsic manner, RNA acts as a messenger involved in cellular crosstalk and communication.5Bang C Thum T Exosomes: new players in cell-cell communication.Int J Biochem Cell Biol. 2012; 44: 2060-2064Crossref PubMed Scopus (0) Google Scholar Furthermore, specific RNA modifications serve to discriminate "self" from invading pathogens lacking these modifications, which constitutes an important aspect in cell-intrinsic innate immunity.6Karikó K Buckstein M Ni H Weissman D Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA.Immunity. 2005; 23: 165-175Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar Due to the limited half-life and versatile functions of RNA, the naturally evolved principle of information transfer through RNA is increasingly exploited for biotechnological purposes, with expression of selected proteins as the most common application. RNA administration offers several advantages over DNA- and protein-based technologies. When compared with protein manufacture, RNA technologies are easier to design and produce, and are more flexible. For example, almost every protein can be encoded by RNA and transferred to target cells for proper post-translational modification. Compared with DNA transfer, RNA administration has clear safety advantages, particularly as protein expression from mRNA is naturally cytosolic, alleviating the need to enter the nucleus and thus excluding potential insertional mutagenesis risks. This property can also contribute to higher transfer efficiencies, as crossing the nuclear envelope provides a major limitation to the success of DNA-based methods.7Hecker JG Nonviral, cationic lipid-mediated delivery of mRNA.Methods Mol Biol. 2013; 969: 73-88Crossref PubMed Scopus (0) Google Scholar,8Lechardeur D Lukacs GL Nucleocytoplasmic transport of plasmid DNA: a perilous journey from the cytoplasm to the nucleus.Hum Gene Ther. 2006; 17: 882-889Crossref PubMed Scopus (0) Google Scholar However, RNA molecules are inherently fragile as a result of sensitivity to both extracellular and cytosolic ribonucleases (RNases),9Tabachnick J Freed R Demonstration of nucleases on mammalian skin surface and in saline extracts of hair.Nature. 1961; 190: 921-922Crossref PubMed Scopus (0) Google Scholar,10Blank A Dekker CA Ribonucleases of human serum, urine, cerebrospinal fluid, and leukocytes. Activity staining following electrophoresis in sodium dodecyl sulfate-polyacrylamide gels.Biochemistry. 1981; 20: 2261-2267Crossref PubMed Google Scholar,11Sorrentino S Human extracellular ribonucleases: multiplicity, molecular diversity and catalytic properties of the major RNase types.Cell Mol Life Sci. 1998; 54: 785-794Crossref PubMed Scopus (0) Google Scholar,12Fritz DT Bergman N Kilpatrick WJ Wilusz CJ Wilusz J Messenger RNA decay in mammalian cells: the exonuclease perspective.Cell Biochem Biophys. 2004; 41: 265-278Crossref PubMed Scopus (21) Google Scholar temporally restricting gene expression. Thus, RNA-based technologies are especially suitable in settings requiring transient rather than permanent expression of a selected gene. Current fields of RNA application include: reprogramming and lineage conversion, vaccination and immunization, gene editing, as well as tumor and gene therapy. Several RNA applications were successfully used in clinically relevant in vivo models. Diverse options regarding RNA origin and delivery mode were developed (summarized in Figure 1), including in vitro synthesized nonviral RNA and viral RNA transfer through viral particles. In addition to these conventional technologies, there are mixed systems comprising both viral and nonviral components. Moreover, self-replicating RNA derived from replication-competent RNA viruses allows prolonged protein expression. This review provides information on the basic components and technological requirements of current RNA technologies, summarizes previous applications of the individual systems, and also discusses advantages and limitations of the different technologies to facilitate selection of an appropriate system for a given application. Transfer of genes of interest (GOI) using nonviral RNA facilitates RNA design as no viral regulatory elements are required for expression and no motifs are necessary for packaging into viral particles. An exception to the latter is the MS2 system as described below. Basically, two alternative systems are available for the delivery of nonviral RNA. Synthetic RNA can be generated in vitro, purified and subsequently introduced into target cells. This approach provides maximum safety, with no expression or cotransfer of potentially immunogenic or harmful viral components. However, naked RNA may be degraded by extracellular ribonucleases prior to reaching the cytosol of target cells,11Sorrentino S Human extracellular ribonucleases: multiplicity, molecular diversity and catalytic properties of the major RNase types.Cell Mol Life Sci. 1998; 54: 785-794Crossref PubMed Scopus (0) Google Scholar thus requiring protection through formulation with synthetic vehicles for many applications. Furthermore, distinct cell types may be partially or completely refractory to physicochemical delivery modes, lowering overall efficiency. Thus, viral transfer of nonviral RNA has recently emerged as an attractive alternative.13Hamann MV Stanke N Müllers E Stirnnagel K Hütter S Artegiani B et al.Efficient transient genetic manipulation in vitro and in vivo by prototype foamy virus-mediated nonviral RNA transfer.Mol Ther. 2014; 22: 1460-1471Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar,14Prel A Caval V Gayon R Ravassard P Duthoit C Payen E et al.Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles.Mol Ther Methods Clin Dev. 2015; 2: 15039Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar Protected within the core of retroviral particles, nonviral RNA was successfully delivered to target cells. This approach also offers the possibility for cell targeting conferred by specific interaction of viral envelope glycoproteins with their cognate cellular receptors. However, the transferred RNA population is less pure and defined as compared with nonviral transfer of synthetic RNA and may also include diverse cellular RNA. Initial evidence for the potential of biomedical synthetic mRNA (syn-mRNA) application was demonstrated in 1990. Injection of naked mRNA into murine skeletal muscle allowed local mRNA uptake and drove expression of different proteins.15Wolff JA Malone RW Williams P Chong W Acsadi G Jani A et al.Direct gene transfer into mouse muscle in vivo.Science. 1990; 247: 1465-1468Crossref PubMed Google Scholar However, concerns about mRNA stability during storage and after administration as well as the difficult manufacturing process initially hampered widespread use of syn-mRNA as therapeutic molecules.16Quabius ES Krupp G Synthetic mRNAs for manipulating cellular phenotypes: an overview.N Biotechnol. 2015; 32: 229-235Crossref PubMed Scopus (0) Google Scholar Following step-wise resolution of these obstacles together with the development of efficient and widely applicable in vitro RNA transfection methods,17Malone RW Felgner PL Verma IM Cationic liposome-mediated RNA transfection.Proc Natl Acad Sci USA. 1989; 86: 6077-6081Crossref PubMed Google Scholar mRNA technology has emerged as an attractive and promising platform for transient gene delivery (Figure 1a).18Sahin U Karikó K Türeci Ö mRNA-based therapeutics–developing a new class of drugs.Nat Rev Drug Discov. 2014; 13: 759-780Crossref PubMed Scopus (80) Google Scholar Syn-mRNA is now commercially available and can even be produced under good manufacturing practice (GMP) conditions (syn-mRNA production details can be found in refs. 16Quabius ES Krupp G Synthetic mRNAs for manipulating cellular phenotypes: an overview.N Biotechnol. 2015; 32: 229-235Crossref PubMed Scopus (0) Google Scholar,19Schlake T Thess A Fotin-Mleczek M Kallen KJ Developing mRNA-vaccine technologies.RNA Biol. 2012; 9: 1319-1330Crossref PubMed Scopus (34) Google Scholar). Functional syn-mRNA can be synthesized through in vitro transcription (IVT) from a cDNA template with bacteriophage RNA polymerases (e.g., SP6 or T7).20Yisraeli JK Melton DA Synthesis of long, capped transcripts in vitro by SP6 and T7 RNA polymerases.Methods Enzymol. 1989; 180: 42-50Crossref PubMed Scopus (0) Google Scholar For eukaryotic mRNA, a 5′ cap and a long 3′ poly(A) tail are the minimal design requirements for efficient translation and these act synergistically.21Gallie DR The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency.Genes Dev. 1991; 5: 2108-2116Crossref PubMed Google Scholar Although enzymatic RNA tailing of syn-mRNA is possible with recombinant poly(A) polymerase,22Lingner J Keller W 3′-end labeling of RNA with recombinant yeast poly(A) polymerase.Nucleic Acids Res. 1993; 21: 2917-2920Crossref PubMed Google Scholar,23Martin G Keller W Tailing and 3′-end labeling of RNA with yeast poly(A) polymerase and various nucleotides.RNA. 1998; 4: 226-230PubMed Google Scholar templated poly(A) addition creates poly(A) tails of defined length and is therefore preferred.18Sahin U Karikó K Türeci Ö mRNA-based therapeutics–developing a new class of drugs.Nat Rev Drug Discov. 2014; 13: 759-780Crossref PubMed Scopus (80) Google Scholar This is accomplished through usage of matrices harboring poly[d(A/T)] sequences for IVT. Capping is essential for eukaryotic RNA stability, efficient translation, and splicing. For syn-mRNA, capping can be achieved both co- and post-transcriptionally. The Vaccinia virus-derived enzymatic capping complex is employed to add a natural cap (m7GpppG) at the 5′ end of uncapped RNA transcripts.24Martin SA Paoletti E Moss B Purification of mRNA guanylyltransferase and mRNA (guanine-7-) methyltransferase from vaccinia virions.J Biol Chem. 1975; 250: 9322-9329PubMed Google Scholar,25Ensinger MJ Martin SA Paoletti E Moss B Modification of the 5′-terminus of mRNA by soluble guanylyl and methyl transferases from vaccinia virus.Proc Natl Acad Sci USA. 1975; 72: 2525-2529Crossref PubMed Google Scholar,26Martin SA Moss B Modification of RNA by mRNA guanylyltransferase and mRNA (guanine-7-)methyltransferase from vaccinia virions.J Biol Chem. 1975; 250: 9330-9335Abstract Full Text PDF PubMed Google Scholar Cotranscriptional capping is achieved by the simple inclusion of excess cap analogues in the IVT reaction to compete with the standard guanosine triphosphate. As natural cap analogues can also be incorporated in the reverse orientation, resulting in a biologically inactive configuration in about one-third to one-half of generated molecules,27Pasquinelli AE Dahlberg JE Lund E Reverse 5′ caps in RNAs made in vitro by phage RNA polymerases.RNA. 1995; 1: 957-967PubMed Google Scholar so-called anti-reverse cap analogs were designed to only allow nucleotide addition at the nonmethylated guanosine, yielding exclusively correctly capped transcripts with drastically improved translation efficiency.28Stepinski J Waddell C Stolarski R Darzynkiewicz E Rhoads RE Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl(3′-O-methyl)GpppG and 7-methyl (3′-deoxy)GpppG.RNA. 2001; 7: 1486-1495PubMed Google Scholar,29Jemielity J Fowler T Zuberek J Stepinski J Lewdorowicz M Niedzwiecka A et al.Novel "anti-reverse" cap analogs with superior translational properties.RNA. 2003; 9: 1108-1122Crossref PubMed Scopus (0) Google Scholar Further cap modifications resulted in increased stability and improved translational profiles, e.g., using phosphorothioate cap analogs conferred resistance to pyrophosphatase-mediated RNA decapping and stabilized the complex between the eukaryotic translation initiation factor eIF4E and the cap.30Grudzien-Nogalska E Jemielity J Kowalska J Darzynkiewicz E Rhoads RE Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells.RNA. 2007; 13: 1745-1755Crossref PubMed Scopus (0) Google Scholar,31Kowalska J Lewdorowicz M Zuberek J Grudzien-Nogalska E Bojarska E Stepinski J et al.Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS.RNA. 2008; 14: 1119-1131Crossref PubMed Scopus (0) Google Scholar,32Kuhn AN Diken M Kreiter S Selmi A Kowalska J Jemielity J et al.Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo.Gene Ther. 2010; 17: 961-971Crossref PubMed Scopus (0) Google Scholar In addition to the 5′-cap and 3′-poly(A) tail, 5′- and 3′- untranslated regions (UTR) can be included to improve mRNA stability and translation efficiency.19Schlake T Thess A Fotin-Mleczek M Kallen KJ Developing mRNA-vaccine technologies.RNA Biol. 2012; 9: 1319-1330Crossref PubMed Scopus (34) Google Scholar,33Tan X Wan Y Enhanced protein expression by internal ribosomal entry site-driven mRNA translation as a novel approach for in vitro loading of dendritic cells with antigens.Hum Immunol. 2008; 69: 32-40Crossref PubMed Scopus (0) Google Scholar,34Yakubov E Rechavi G Rozenblatt S Givol D Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors.Biochem Biophys Res Commun. 2010; 394: 189-193Crossref PubMed Scopus (123) Google Scholar In some cases, internal ribosomal entry sites (IRES) can replace the 5′ cap. Finally, a purification step to separate the desired transcript from remaining nucleotides, oligonucleotides, protein as well as shorter abortive and longer transcripts was reported to improve mRNA translation.35Pascolo S Messenger RNA-based vaccines.Expert Opin Biol Ther. 2004; 4: 1285-1294Crossref PubMed Scopus (70) Google Scholar,36Karikó K Muramatsu H Ludwig J Weissman D Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA.Nucleic Acids Res. 2011; 39: e142Crossref PubMed Scopus (65) Google Scholar Despite incorporation of these structures to mimic eukaryotic mRNA, syn-mRNA has strong immunostimulatory properties because it lacks additional characteristics inherent to eukaryotic mRNA, most prominently specific nucleotide modifications.6Karikó K Buckstein M Ni H Weissman D Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA.Immunity. 2005; 23: 165-175Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar Therefore, syn-mRNA strongly activates pattern recognition receptors, in particular Toll-like receptors 3, 7, and 8 as well as retinoic acid-inducible gene I-like receptors.37Karikó K Ni H Capodici J Lamphier M Weissman D mRNA is an endogenous ligand for Toll-like receptor 3.J Biol Chem. 2004; 279: 12542-12550Crossref PubMed Scopus (563) Google Scholar,38Heil F Hemmi H Hochrein H Ampenberger F Kirschning C Akira S et al.Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8.Science. 2004; 303: 1526-1529Crossref PubMed Scopus (2153) Google Scholar,39Diebold SS Kaisho T Hemmi H Akira S Reis e Sousa C Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA.Science. 2004; 303: 1529-1531Crossref PubMed Scopus (1859) Google Scholar,40Schott JW Galla M Godinho T Baum C Schambach A Viral and non-viral approaches for transient delivery of mRNA and proteins.Curr Gene Ther. 2011; 11: 382-398Crossref PubMed Google Scholar Immunogenicity constitutes a major impediment leading to reduced protein expression efficiencies. However, incorporation of modified nucleosides (e.g., m5C, m6A, m5U, s2U, or pseudouridine) present in native RNA was identified as an efficient means to avoid recognition of syn-mRNA, to reduce signaling by pattern recognition receptors and to block induction of the translation repressor protein kinase R.6Karikó K Buckstein M Ni H Weissman D Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA.Immunity. 2005; 23: 165-175Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar,41Karikó K Muramatsu H Welsh FA Ludwig J Kato H Akira S et al.Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability.Mol Ther. 2008; 16: 1833-1840Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar,42Nallagatla SR Bevilacqua PC Nucleoside modifications modulate activation of the protein kinase PKR in an RNA structure-specific manner.RNA. 2008; 14: 1201-1213Crossref PubMed Scopus (60) Google Scholar,43Pardi N Muramatsu H Weissman D Karikó K In vitro transcription of long RNA containing modified nucleosides.Methods Mol Biol. 2013; 969: 29-42Crossref PubMed Scopus (0) Google Scholar 5′ terminal triphosphates are also recognized by protein kinase R44Nallagatla SR Hwang J Toroney R Zheng X Cameron CE Bevilacqua PC 5′-triphosphate-dependent activation of PKR by RNAs with short stem-loops.Science. 2007; 318: 1455-1458Crossref PubMed Scopus (137) Google Scholar and retinoic acid-inducible gene I45Hornung V Ellegast J Kim S Brzózka K Jung A Kato H et al.5′-Triphosphate RNA is the ligand for RIG-I.Science. 2006; 314: 994-997Crossref PubMed Scopus (1246) Google Scholar,46Pichlmair A Schulz O Tan CP Näslund TI Liljeström P Weber F et al.RIG-I-mediated antiviral responses to single-stranded RNA bearing 5′-phosphates.Science. 2006; 314: 997-1001Crossref PubMed Scopus (1108) Google Scholar as an immunogenic motif. As inclusion of cap analogues still generates ~20% of uncapped transcripts, phosphatase treatment of IVT products is frequently accomplished to remove any remaining 5′ triphosphates.16Quabius ES Krupp G Synthetic mRNAs for manipulating cellular phenotypes: an overview.N Biotechnol. 2015; 32: 229-235Crossref PubMed Scopus (0) Google Scholar The combined effects of nucleoside modification, phosphatase treatment, and additional inhibition of residual interferon type-I (IFN-I) responses by the Vaccinia virus decoy receptor B18R have enabled successful mRNA-based reprogramming by rescuing initially low reprogramming factor (RF) expression levels.47Warren L Manos PD Ahfeldt T Loh YH Li H Lau F et al.Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA.Cell Stem Cell. 2010; 7: 618-630Abstract Full Text Full Text PDF PubMed Scopus (1166) Google Scholar Reprogramming was achieved although the generation of sufficiently long RF expression windows required repeated mRNA administration, which triggers innate immune responses even more potently than single mRNA application. In another study, siRNA-mediated knockdown of cellular/intrinsic innate immune factors enabled expression of active RF.48Angel M Yanik MF Innate immune suppression enables frequent transfection with RNA encoding reprogramming proteins.PLoS One. 2010; 5: e11756Crossref PubMed Scopus (50) Google Scholar In a more recent approach, induced pluripotent stem cells (iPSC) were generated using nonmodified mRNA for RF expression.49Poleganov MA Eminli S Beissert T Herz S Moon JI Goldmann J et al.Efficient reprogramming of human fibroblasts and blood-derived endothelial progenitor cells using nonmodified RNA for reprogramming and immune evasion.Hum Gene Ther. 2015; 26: 751-766Crossref PubMed Google Scholar Coadministration of mRNA molecules to supply B18R and two additional Vaccinia virus-derived immune evasion factors (protein kinase R inhibitors E3 and K3) supported this reprogramming strategy. Importantly, iPSC generation via RNA-based reprogramming was faster and produced iPSC clones whose molecular signatures more closely resembled embryonic stem cells when compared with standard protocols utilizing integrating retroviral vectors.50Rosa A Brivanlou AH Synthetic mRNAs: powerful tools for reprogramming and differentiation of human cells.Cell Stem Cell. 2010; 7: 549-550Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar Beyond reprogramming and other cell-based in vitro systems,19Schlake T Thess A Fotin-Mleczek M Kallen KJ Developing mRNA-vaccine technologies.RNA Biol. 2012; 9: 1319-1330Crossref PubMed Scopus (34) Google Scholar,51Zou X Sorenson BS Ross KF Herzberg MC Augmentation of epithelial resistance to invading bacteria by using mRNA transfections.Infect Immun. 2013; 81: 3975-3983Crossref PubMed Scopus (0) Google Scholar,52Boros G Miko E Muramatsu H Weissman D Emri E Rózsa D et al.Transfection of pseudouridine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage in human keratinocytes: a new approach with future therapeutic potential.J Photochem Photobiol B. 2013; 129: 93-99Crossref PubMed Google Scholar in vivo delivery of syn-mRNA was also demonstrated. Since the initial proof-of-principle of reporter protein expression in injected mouse muscle,15Wolff JA Malone RW Williams P Chong W Acsadi G Jani A et al.Direct gene transfer into mouse muscle in vivo.Science. 1990; 247: 1465-1468Crossref PubMed Google Scholar mRNA technology has advanced to express proteins with clear therapeutic benefit in disease models, including lethal congenital surfactant B protein deficiency,53Kormann MS Hasenpusch G Aneja MK Nica G Flemmer AW Herber-Jonat S et al.Expression of therapeutic proteins after delivery of chemically modified mRNA in mice.Nat Biotechnol. 2011; 29: 154-157Crossref PubMed Scopus (168) Google Scholar therapy to increase erythropoiesis via injection of erythropoietin mRNA,53Kormann MS Hasenpusch G Aneja MK Nica G Flemmer AW Herber-Jonat S et al.Expression of therapeutic proteins after delivery of chemically modified mRNA in mice.Nat Biotechnol. 2011; 29: 154-157Crossref PubMed Scopus (168) Google Scholar,54Karikó K Muramatsu H Keller JM Weissman D Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin.Mol Ther. 2012; 20: 948-953Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar and treatment of myocardial infarction through intramyocardially injected mRNA encoding human vascular endothelial growth factor-A.55Zangi L Lui KO von Gise A Ma Q Ebina W Ptaszek LM et al.Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction.Nat Biotechnol. 2013; 31: 898-907Crossref PubMed Scopus (135) Google Scholar Of note, most reports using syn-mRNA employed nucleoside modification to improve efficacy, with pseudouridine incorporation as the most frequently used strategy. While detrimental to efficient protein expression, syn-mRNA immunogenicity has contributed to the success of vaccination approaches by acting as a suitable adjuvant. Compared with peptide vaccines or use of attenuated viruses, syn-mRNA displays the following advantages: (i) fully defined sequence, (ii) no replication within target cells, (iii) no genomic integration, (iv) high flexibility with respect to antigens that can be expressed, also including complex antigens, (v) access to major histocompatibility complex class I compartment, and (vi) self-adjuvanticity. Especially the latter contributes to high efficiencies, with RNA matrices used for antigen expression simultaneously costimulating innate immunity. Syn-mRNA stability and adjuvanticity can be improved by complexation or chemical modification.19Schlake T Thess A Fotin-Mleczek M Kallen KJ Developing mRNA-vaccine technologies.RNA Biol. 2012; 9: 1319-1330Crossref PubMed Scopus (34) Google Scholar,56Scheel B Braedel S Probst J Carralot JP Wagner H Schild H et al.Immunostimulating capacities of stabilized RNA molecules.Eur J Immunol. 2004; 34: 537-547Crossref PubMed Scopus (0) Google Scholar RNA-complexed particle size seems to affect the cytokine pattern induced by syn-mRNA, triggering either antiviral or antimicrobial/antifungal adaptive immunity.57Rettig L Haen SP Bittermann AG von Boehmer L Curioni A Krämer SD et al.Particle size and activation threshold: a new dimension of danger signaling.Blood. 2010; 115: 4533-4541Crossref PubMed Scopus (48) Google Scholar For optimal performance, a combined approach was developed employing both naked mRNA, which is mainly responsible for antigen production, and protamine-condensed mRNA, which provides strong immuno-costimulation.58Fotin-Mleczek M Duchardt KM Lorenz C Pfeiffer R Ojkić-Zrna S Probst J et al.Messenger RNA-based vaccines with dual activity induce balanced TLR-7 dependent adaptive immune responses and provide antitumor activity.J Immunother. 2011; 34: 1-15Crossref PubMed Scopus (86) Google Scholar RNA-based vaccination was successfully used in prophylactic and therapeutic tumor immunization in animal models and clinical trials.59Boczkowski D Nair SK Snyder D Gilboa E Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo.J Exp Med. 1996; 184: 465-472Crossref PubMed Google Scholar,60Nair SK Heiser A Boczkowski D Majumdar A Naoe M Lebkowski JS et al.Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse t