mRNA-based therapeutics — developing a new class of drugs

In vitro transcribed (IVT) mRNA has recently come into focus as a potential new drug class to deliver genetic information. Such synthetic mRNA can be engineered to transiently express proteins by structurally resembling natural mRNA. Advances in addressing the inherent challenges of this drug class, particularly related to controlling the translational efficacy and immunogenicity of the IVTmRNA, provide the basis for a broad range of potential applications. mRNA-based cancer immunotherapies and infectious disease vaccines have entered clinical development. 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. This Review provides a comprehensive overview of the current state of mRNA-based drug technologies and their applications, and discusses the key challenges and opportunities in developing these into a new class of drugs.

[1]  S. Akira,et al.  Species-Specific Recognition of Single-Stranded RNA via Toll-like Receptor 7 and 8 , 2004, Science.

[2]  Yurong Lai,et al.  Mitochondrial Expression of the Human Equilibrative Nucleoside Transporter 1 (hENT1) Results in Enhanced Mitochondrial Toxicity of Antiviral Drugs* , 2004, Journal of Biological Chemistry.

[3]  Z. Berneman,et al.  Clinical-grade manufacturing of autologous mature mRNA-electroporated dendritic cells and safety testing in acute myeloid leukemia patients in a phase I dose-escalation clinical trial. , 2009, Cytotherapy.

[4]  Y. Kaneda,et al.  RNA melanoma vaccine: induction of antitumor immunity by human glycoprotein 100 mRNA immunization. , 1999, Human gene therapy.

[5]  B. Neyns,et al.  Enhancing the T-cell stimulatory capacity of human dendritic cells by co-electroporation with CD40L, CD70 and constitutively active TLR4 encoding mRNA. , 2008, Molecular therapy : the journal of the American Society of Gene Therapy.

[6]  F. Bloom,et al.  Reversal of diabetes insipidus in Brattleboro rats: intrahypothalamic injection of vasopressin mRNA. , 1992, Science.

[7]  E. Paoletti,et al.  Purification of mRNA guanylyltransferase and mRNA (guanine-7-) methyltransferase from vaccinia virions. , 1975, The Journal of biological chemistry.

[8]  H. Goossens,et al.  Induction of complete and molecular remissions in acute myeloid leukemia by Wilms’ tumor 1 antigen-targeted dendritic cell vaccination , 2010, Proceedings of the National Academy of Sciences.

[9]  H. Schild,et al.  Correction: Increased Antigen Presentation Efficiency by Coupling Antigens to MHC Class I Trafficking Signals , 2012, The Journal of Immunology.

[10]  E. Ehrenfeld,et al.  Comparison of replication complexes synthesizing poliovirus RNA. , 1981, Virology.

[11]  P. Fournier,et al.  Intra-pinna anti-tumor vaccination with self-replicating infectious RNA or with DNA encoding a model tumor antigen and a cytokine , 2000, Gene Therapy.

[12]  Xi-rong Guo,et al.  Feeder-Free Derivation of Human Induced Pluripotent Stem Cells with Messenger RNA , 2012, Scientific Reports.

[13]  K. Chua,et al.  DNA vaccines for the prevention and treatment of allergy , 2009, Current opinion in allergy and clinical immunology.

[14]  B. Rehermann,et al.  The liver as an immunological organ , 2006, Hepatology.

[15]  N. Seidah,et al.  Proprotein and prohormone convertases: a family of subtilases generating diverse bioactive polypeptides 1 Published on the World Wide Web on 17 August 1999. 1 , 1999, Brain Research.

[16]  C. Mandl,et al.  RNA-based vaccines. , 2012, Vaccine.

[17]  Shizuo Akira,et al.  Innate Antiviral Responses by Means of TLR7-Mediated Recognition of Single-Stranded RNA , 2004, Science.

[18]  P. Bevilacqua,et al.  Nucleoside modifications modulate activation of the protein kinase PKR in an RNA structure‐specific manner , 2008, RNA.

[19]  C. Heirman,et al.  Preclinical evaluation of TriMix and antigen mRNA-based antitumor therapy. , 2012, Cancer research.

[20]  Jeffrey Wilusz,et al.  The highways and byways of mRNA decay , 2007, Nature Reviews Molecular Cell Biology.

[21]  H. Rammensee,et al.  Toll‐like receptor‐dependent activation of several human blood cell types by protamine‐condensed mRNA , 2005, European journal of immunology.

[22]  E. Gilboa,et al.  Induction of primary carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocytes in vitro using human dendritic cells transfected with RNA , 1998, Nature Biotechnology.

[23]  J. Keith Joung,et al.  High frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells , 2013, Nature Biotechnology.

[24]  H. Rammensee,et al.  In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies , 2000 .

[25]  S. Barash,et al.  Human secretory signal peptide description by hidden Markov model and generation of a strong artificial signal peptide for secreted protein expression. , 2002, Biochemical and biophysical research communications.

[26]  P. Lundin.  Is silence still golden? Mapping the RNAi patent landscape , 2011, Nature Biotechnology.

[27]  N. Schmitz,et al.  Generation of Cytotoxic Responses in Mice and Human Individuals Against Hematological Malignancies Using Survivin-RNA-Transfected Dendritic Cells1 , 2003, Journal of Immunology.

[28]  T. Hocking,et al.  Heritable Targeted Gene Disruption in Zebrafish Using Designed Zinc Finger Nucleases , 2008, Nature Biotechnology.

[29]  G. Acsadi,et al.  Direct gene transfer into mouse muscle in vivo. , 1990, Science.

[30]  S. Akira,et al.  Nucleic acid agonists for Toll‐like receptor 7 are defined by the presence of uridine ribonucleotides , 2006, European journal of immunology.

[31]  D. Gallie.  The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. , 1991, Genes & development.

[32]  Philip C. Bevilacqua,et al.  Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation , 2010, Nucleic acids research.

[33]  C. Pichon,et al.  Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition , 2013, BMC Biotechnology.

[34]  W. Keller,et al.  Tailing and 3'-end labeling of RNA with yeast poly(A) polymerase and various nucleotides. , 1998, RNA.

[35]  Elo Leung,et al.  Targeted Genome Editing Across Species Using ZFNs and TALENs , 2011, Science.

[36]  H. Rammensee,et al.  Results of the First Phase I/II Clinical Vaccination Trial With Direct Injection of mRNA , 2008, Journal of immunotherapy.

[37]  Burkhard Ludewig,et al.  Ribose 2′-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5 , 2011, Nature Immunology.

[38]  J. Castle,et al.  Exploiting the mutanome for tumor vaccination. , 2012, Cancer research.

[39]  Lianfeng Zhang,et al.  Generating Rats with Conditional Alleles Using Crispr/cas9 Dear Editor, Generating Conditional Knockout Rats Using Crispr/cas9 2 Generating Conditional Knockout Rats Using Crispr/cas9 4 , 2022 .

[40]  Eli Gilboa,et al.  Induction of cytotoxic T cell responses and tumor immunity against unrelated tumors using telomerase reverse transcriptase RNA transfected dendritic cells. , 2000, Nature Medicine.

[41]  Wenzhong Li,et al.  Non-viral gene delivery methods. , 2013, Current pharmaceutical biotechnology.

[42]  A. J. Shatkin,et al.  5′-Terminal 7-methylguanosine in eukaryotic mRNA is required for translation , 1975, Nature.

[43]  S. Krämer,et al.  Particle size and activation threshold: a new dimension of danger signaling. , 2010, Blood.

[44]  Gunnar Kvalheim,et al.  Transiently redirected T cells for adoptive transfer. , 2011, Cytotherapy.

[45]  Hiroki Kato,et al.  Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. , 2008, Molecular therapy : the journal of the American Society of Gene Therapy.

[46]  Ö. Türeci,et al.  Simultaneous ex vivo quantification of antigen-specific CD4+ and CD8+ T cell responses using in vitro transcribed RNA , 2007, Cancer Immunology, Immunotherapy.

[47]  Yuhua Wang,et al.  Systemic delivery of modified mRNA encoding herpes simplex virus 1 thymidine kinase for targeted cancer gene therapy. , 2013, Molecular therapy : the journal of the American Society of Gene Therapy.

[48]  S. Brenner,et al.  An Unstable Intermediate Carrying Information from Genes to Ribosomes for Protein Synthesis , 1961, Nature.

[49]  Kris Thielemans,et al.  Characterization of CD8+ T-Cell Responses in the Peripheral Blood and Skin Injection Sites of Melanoma Patients Treated with mRNA Electroporated Autologous Dendritic Cells (TriMixDC-MEL) , 2013, BioMed research international.

[50]  Osamu Takeuchi,et al.  Recognition of 5' triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus. , 2009, Immunity.

[51]  Elo Leung,et al.  Knockout rats generated by embryo microinjection of TALENs , 2011, Nature Biotechnology.

[52]  C. Meijer,et al.  Dendritic cells transfected with interleukin-12 and tumor-associated antigen messenger RNA induce high avidity cytotoxic T cells , 2007, Gene Therapy.

[53]  D. Krainc,et al.  Human iPSC-based modeling of late-onset disease via progerin-induced aging. , 2013, Cell stem cell.

[54]  A. Muotri,et al.  Efficient generation of human iPSCs by a synthetic self-replicative RNA. , 2013, Cell stem cell.

[55]  U. Şahin,et al.  Intranodal vaccination with naked antigen-encoding RNA elicits potent prophylactic and therapeutic antitumoral immunity. , 2010, Cancer research.

[56]  H. Rammensee,et al.  Intradermal vaccinations with RNA coding for TAA generate CD8+ and CD4+ immune responses and induce clinical benefit in vaccinated patients. , 2011, Molecular therapy : the journal of the American Society of Gene Therapy.

[57]  T. Geng,et al.  Transfection of cells using flow-through electroporation based on constant voltage , 2011, Nature Protocols.

[58]  Jianglin Fan,et al.  Effective gene targeting in rabbits using RNA-guided Cas9 nucleases. , 2014, Journal of molecular cell biology.

[59]  R. Weiss,et al.  Immunize and disappear-safety-optimized mRNA vaccination with a panel of 29 allergens. , 2009, The Journal of allergy and clinical immunology.

[60]  S. Weissman,et al.  Chimeric receptor mRNA transfection as a tool to generate antineoplastic lymphocytes. , 2008, Human gene therapy.

[61]  Lei Wang,et al.  Generation of Gene-Modified Cynomolgus Monkey via Cas9/RNA-Mediated Gene Targeting in One-Cell Embryos , 2014, Cell.

[62]  K. G. Rajeev,et al.  Rational design of cationic lipids for siRNA delivery , 2010, Nature Biotechnology.

[63]  Yi Zhang,et al.  Erythropoietin gene therapy leads to autoimmune anemia in macaques. , 2004, Blood.

[64]  S. Bhattacharya,et al.  Site Specific Mutation of the Zic2 Locus by Microinjection of TALEN mRNA in Mouse CD1, C3H and C57BL/6J Oocytes , 2013, PloS one.

[65]  M. Yanik,et al.  Innate Immune Suppression Enables Frequent Transfection with RNA Encoding Reprogramming Proteins , 2010, PloS one.

[66]  S. Akira,et al.  Activation of MDA5 Requires Higher-Order RNA Structures Generated during Virus Infection , 2009, Journal of Virology.

[67]  Alan Villalobos,et al.  Engineering genes for predictable protein expression. , 2012, Protein expression and purification.

[68]  Vidya Balagopal,et al.  Ways and means of eukaryotic mRNA decay. , 2012, Biochimica et biophysica acta.

[69]  E. Gilboa,et al.  Immunotherapy with Autologous, Human Dendritic Cells Transfected with Carcinoembryonic Antigen mRNA , 2003, Cancer investigation.

[70]  J. Rose,et al.  Novel infectious particles generated by expression of the vesicular stomatitis virus glycoprotein from a self-replicating RNA , 1994, Cell.

[71]  T. Dubensky,et al.  An Alphavirus Replicon Particle Chimera Derived from Venezuelan Equine Encephalitis and Sindbis Viruses Is a Potent Gene-Based Vaccine Delivery Vector , 2003, Journal of Virology.

[72]  S. Mizutani,et al.  In vitro synthesis of an infectious RNA from cDNA clones of human rhinovirus type 14 , 1985, Journal of virology.

[73]  D. Weissman,et al.  Nucleoside modifications in RNA limit activation of 2′-5′-oligoadenylate synthetase and increase resistance to cleavage by RNase L , 2011, Nucleic acids research.

[74]  J. Castle,et al.  Targeting the tumor mutanome for personalized vaccination therapy , 2012, Oncoimmunology.

[75]  Shizuo Akira,et al.  The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses , 2004, Nature Immunology.

[76]  V. Hornung,et al.  Immunostimulatory RNA oligonucleotides trigger an antigen-specific cytotoxic T-cell and IgG2a response. , 2007, Blood.

[77]  Andrzej Dziembowski,et al.  Novel endoribonucleases as central players in various pathways of eukaryotic RNA metabolism. , 2010, RNA.

[78]  B. Bettencourt,et al.  Safety and efficacy of RNAi therapy for transthyretin amyloidosis. , 2013, The New England journal of medicine.

[79]  Donna Niedzwiecki,et al.  Telomerase mRNA-Transfected Dendritic Cells Stimulate Antigen-Specific CD8+ and CD4+ T Cell Responses in Patients with Metastatic Prostate Cancer 1 , 2005, The Journal of Immunology.

[80]  S. Yao,et al.  Cloning of a human nucleoside transporter implicated in the Cellular uptake of adenosine and chemotherapeutic drugs , 1997, Nature Medicine.

[81]  K. Kawakami,et al.  Transposon-mediated BAC transgenesis in zebrafish and mice , 2009, BMC Genomics.

[82]  S. Fukushima,et al.  In Vivo Messenger RNA Introduction into the Central Nervous System Using Polyplex Nanomicelle , 2013, PloS one.

[83]  A. Thess,et al.  Highly potent mRNA based cancer vaccines represent an attractive platform for combination therapies supporting an improved therapeutic effect , 2012, The journal of gene medicine.

[84]  Thomas Krucker,et al.  Nonviral delivery of self-amplifying RNA vaccines , 2012, Proceedings of the National Academy of Sciences.

[85]  H. Rammensee,et al.  Direct Injection of Protamine-protected mRNA: Results of a Phase 1/2 Vaccination Trial in Metastatic Melanoma Patients , 2009, Journal of immunotherapy.

[86]  C. Broeckhoven,et al.  Nonviral transfection of distinct types of human dendritic cells: high-efficiency gene transfer by electroporation into hematopoietic progenitor- but not monocyte-derived dendritic cells , 1998, Gene Therapy.

[87]  D. Weissman,et al.  Increased Erythropoiesis in Mice Injected With Submicrogram Quantities of Pseudouridine-containing mRNA Encoding Erythropoietin , 2012, Molecular therapy : the journal of the American Society of Gene Therapy.

[88]  Ignacio Anegon,et al.  Knockout Rats via Embryo Microinjection of Zinc-Finger Nucleases , 2009, Science.

[89]  J. Connolly,et al.  B Cell TLR7 Expression Drives Anti-RNA Autoantibody Production and Exacerbates Disease in Systemic Lupus Erythematosus–Prone Mice , 2012, The Journal of Immunology.

[90]  E. Gilboa,et al.  Optimization of the sequence of antigen loading and CD40-ligand-induced maturation of dendritic cells. , 1998, Cancer research.

[91]  Jeffry D. Sander,et al.  Efficient In Vivo Genome Editing Using RNA-Guided Nucleases , 2013, Nature Biotechnology.

[92]  Nicol N. Schraudolph,et al.  A Role for Codon Order in Translation Dynamics , 2010, Cell.

[93]  S. Grupp,et al.  Regimen-specific effects of RNA-modified chimeric antigen receptor T cells in mice with advanced leukemia. , 2013, Human gene therapy.

[94]  M. Aoki,et al.  Autocrine-paracrine effects of overexpression of hepatocyte growth factor gene on growth of endothelial cells. , 1996, Biochemical and biophysical research communications.

[95]  Derrick J. Rossi,et al.  Reprogramming human fibroblasts to pluripotency using modified mRNA , 2013, Nature Protocols.

[96]  R. Löbenberg,et al.  Targeted delivery of nanoparticles for the treatment of lung diseases. , 2008, Advanced drug delivery reviews.

[97]  L. Bossi,et al.  The influence of codon context on genetic code translation , 1980, Nature.

[98]  I. Verma,et al.  Cationic liposome-mediated RNA transfection. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[99]  Hideyuki Okano,et al.  Steps Toward Safe Cell Therapy Using Induced Pluripotent Stem Cells , 2013, Circulation research.

[100]  B. Neyns,et al.  Dendritic cells loaded with mRNA encoding full-length tumor antigens prime CD4+ and CD8+ T cells in melanoma patients. , 2012, Molecular therapy : the journal of the American Society of Gene Therapy.

[101]  K. P. Murphy,et al.  Janeway's immunobiology , 2007 .

[102]  A. van Hoof,et al.  Messenger RNA regulation: to translate or to degrade , 2008, The EMBO journal.

[103]  D. Weissman,et al.  Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA , 2011, Nucleic acids research.

[104]  D. Melton,et al.  Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. , 1984, Nucleic acids research.

[105]  J. A. Bernal.  RNA-Based Tools for Nuclear Reprogramming and Lineage-Conversion: Towards Clinical Applications , 2013, Journal of Cardiovascular Translational Research.

[106]  K. Zdanowski,et al.  Eukaryotic translation initiation is controlled by cooperativity effects within ternary complexes of 4E‐BP1, eIF4E, and the mRNA 5′ cap , 2013, FEBS letters.

[107]  E. Gilboa,et al.  The feasibility and safety of immunotherapy with dendritic cells loaded with CEA mRNA following neoadjuvant chemoradiotherapy and resection of pancreatic cancer , 2002, International journal of gastrointestinal cancer.

[108]  H. Conjeevaram,et al.  Hepatic failure and lactic acidosis due to fialuridine (FIAU), an investigational nucleoside analogue for chronic hepatitis B. , 1995, The New England journal of medicine.

[109]  S. Yoon,et al.  Adoptive immunotherapy using human peripheral blood lymphocytes transferred with RNA encoding Her-2/neu-specific chimeric immune receptor in ovarian cancer xenograft model , 2009, Cancer Gene Therapy.

[110]  J. Ross,et al.  Half-lives of beta and gamma globin messenger RNAs and of protein synthetic capacity in cultured human reticulocytes. , 1985, Blood.

[111]  D. Campana,et al.  Synthetic messenger RNA as a tool for gene therapy. , 2006, Human gene therapy.

[112]  D. Tollervey,et al.  The Many Pathways of RNA Degradation , 2009, Cell.

[113]  Michael J. Davis,et al.  Sensing prokaryotic mRNA signifies microbial viability and promotes immunity , 2011, Nature.

[114]  G. Rhodes,et al.  Self-replicating Semliki Forest virus RNA as recombinant vaccine. , 1994, Vaccine.

[115]  Ö. Türeci,et al.  Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation , 2011, Gene Therapy.

[116]  E. Neumann,et al.  Gene transfer into mouse lyoma cells by electroporation in high electric fields. , 1982, The EMBO journal.

[117]  T. Schlake,et al.  Protein expression from exogenous mRNA: Uptake by receptor-mediated endocytosis and trafficking via the lysosomal pathway , 2011, RNA biology.

[118]  D. Weissman,et al.  HIV Gag mRNA Transfection of Dendritic Cells (DC) Delivers Encoded Antigen to MHC Class I and II Molecules, Causes DC Maturation, and Induces a Potent Human In Vitro Primary Immune Response1 , 2000, The Journal of Immunology.

[119]  R. Rhoads,et al.  Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells. , 2007, RNA.

[120]  Y. Lai,et al.  Identification of the Mitochondrial Targeting Signal of the Human Equilibrative Nucleoside Transporter 1 (hENT1) , 2006, Journal of Biological Chemistry.

[121]  D. Givol,et al.  Reprogramming of human fibroblasts to pluripotent stem cells using mRNA of four transcription factors. , 2010, Biochemical and biophysical research communications.

[122]  U. Şahin,et al.  mRNA as a versatile tool for exogenous protein expression. , 2012, Current gene therapy.

[123]  K Nakayama,et al.  Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. , 1997, The Biochemical journal.

[124]  E. Gilboa,et al.  Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo , 1996, The Journal of experimental medicine.

[125]  J. Wilusz,et al.  Lsm proteins bind and stabilize RNAs containing 5′ poly(A) tracts , 2007, Nature Structural &Molecular Biology.

[126]  Elmar Wahle,et al.  Poly(A) Tail Shortening by a Mammalian Poly(A)-specific 3′-Exoribonuclease* , 1997, The Journal of Biological Chemistry.

[127]  Chow H Lee,et al.  Endoribonucleases – enzymes gaining spotlight in mRNA metabolism , 2010, The FEBS journal.

[128]  R. Vale,et al.  Circularization of mRNA by eukaryotic translation initiation factors. , 1998, Molecular cell.

[129]  Ö. Türeci,et al.  Tumor vaccination using messenger RNA: prospects of a future therapy. , 2011, Current opinion in immunology.

[130]  K. Jacobson,et al.  Cochleate lipid cylinders: formation by fusion of unilamellar lipid vesicles. , 1975, Biochimica et biophysica acta.

[131]  C. Heirman,et al.  mRNA , 2013, Human Vaccines & Immunotherapeutics.

[132]  M. Pfreundschuh,et al.  Human neoplasms elicit multiple specific immune responses in the autologous host. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[133]  N. Seidah,et al.  Precursor Convertases: An Evolutionary Ancient, Cell‐Specific, Combinatorial Mechanism Yielding Diverse Bioactive Peptides and Proteins , 1998, Annals of the New York Academy of Sciences.

[134]  J. Slater,et al.  The latex allergen Hev b 5 transcript is widely distributed after subcutaneous injection in BALB/c mice of its DNA vaccine. , 1998, The Journal of allergy and clinical immunology.

[135]  C. Figdor,et al.  Vaccination with mRNA-Electroporated Dendritic Cells Induces Robust Tumor Antigen-Specific CD4+ and CD8+ T Cells Responses in Stage III and IV Melanoma Patients , 2012, Clinical Cancer Research.

[136]  U. Şahin,et al.  Antitumor vaccination with synthetic mRNA: strategies for in vitro and in vivo preclinical studies. , 2013, Methods in molecular biology.

[137]  Wolfgang Wurst,et al.  Generation of targeted mouse mutants by embryo microinjection of TALEN mRNA , 2013, Nature Protocols.

[138]  E. Gilboa,et al.  Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors. , 2002, The Journal of clinical investigation.

[139]  Rudolf Jaenisch,et al.  One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering , 2013, Cell.

[140]  D. G. Gibson,et al.  Rapidly produced SAM® vaccine against H7N9 influenza is immunogenic in mice , 2013, Emerging Microbes & Infections.

[141]  Patrick Mayeux,et al.  Pure red-cell aplasia and antierythropoietin antibodies in patients treated with recombinant erythropoietin. , 2002, The New England journal of medicine.

[142]  P. Roberts,et al.  Essential role for the dsRNA-dependent protein kinase PKR in innate immunity to viral infection. , 2000, Immunity.

[143]  R. Rhoads,et al.  Synthesis and properties of mRNAs containing the novel "anti-reverse" cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl (3'-deoxy)GpppG. , 2001, RNA.

[144]  N. Yang,et al.  Gene gun delivery of mRNA in situ results in efficient transgene expression and genetic immunization. , 1996, Gene therapy.

[145]  F. Altmann,et al.  Comprehensive glyco‐proteomic analysis of human α1‐antitrypsin and its charge isoforms , 2006, Proteomics.

[146]  C. Pichon,et al.  mRNA transfection of dendritic cells: synergistic effect of ARCA mRNA capping with Poly(A) chains in cis and in trans for a high protein expression level. , 2006, Biochemical and biophysical research communications.

[147]  Aristóbolo Mendes Silva,et al.  Intranasal vaccination with messenger RNA as a new approach in gene therapy: Use against tuberculosis , 2010, BMC biotechnology.

[148]  Bernard Verrier,et al.  Efficient stimulation of HIV-1-specific T cells using dendritic cells electroporated with mRNA encoding autologous HIV-1 Gag and Env proteins. , 2006, Blood.

[149]  M. Hilleman,et al.  Inducers of interferon and host resistance. 3. Double-stranded RNA from reovirus type 3 virions (reo 3-RNA). , 1967, Proceedings of the National Academy of Sciences of the United States of America.

[150]  U. Şahin,et al.  Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. , 2006, Blood.

[151]  H. Goossens,et al.  mRNA-based dendritic cell vaccination induces potent antiviral T-cell responses in HIV-1-infected patients , 2012, AIDS.

[152]  E. Levine,et al.  Fialuridine and its metabolites inhibit DNA polymerase gamma at sites of multiple adjacent analog incorporation, decrease mtDNA abundance, and cause mitochondrial structural defects in cultured hepatoblasts. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[153]  J. Wilusz.  RNA stability: is it the endo' the world as we know it? , 2009, Nature Structural &Molecular Biology.

[154]  Corey M. Carlson,et al.  Mammalian germ-line transgenesis by transposition , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[155]  Ö. Türeci,et al.  FLT3 ligand enhances the cancer therapeutic potency of naked RNA vaccines. , 2011, Cancer research.

[156]  H. Schellekens,et al.  Antibodies against Erythropoietin and Other Protein‐Based Therapeutics: An Overview , 2005, Annals of the New York Academy of Sciences.

[157]  M. Kiledjian,et al.  Regulation of mRNA decapping , 2010, Wiley interdisciplinary reviews. RNA.

[158]  R. Valenta,et al.  From allergen genes to allergy vaccines. , 2010, Annual review of immunology.

[159]  D. Kufe,et al.  Induction of Antitumor Immunity by Vaccination of Dendritic Cells Transfected with MUC1 RNA1 , 2000, The Journal of Immunology.

[160]  G. Vanham,et al.  Type I IFN counteracts the induction of antigen-specific immune responses by lipid-based delivery of mRNA vaccines. , 2013, Molecular therapy : the journal of the American Society of Gene Therapy.

[161]  M. Fotin‐Mleczek,et al.  Messenger RNA-based Vaccines With Dual Activity Induce Balanced TLR-7 Dependent Adaptive Immune Responses and Provide Antitumor Activity , 2011, Journal of immunotherapy.

[162]  H. Rammensee,et al.  The regulatory landscape for actively personalized cancer immunotherapies , 2013, Nature Biotechnology.

[163]  R. Granstein,et al.  Induction of anti-tumor immunity with epidermal cells pulsed with tumor-derived RNA or intradermal administration of RNA. , 2000, The Journal of investigative dermatology.

[164]  J. Lötvall,et al.  Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells , 2007, Nature Cell Biology.

[165]  C. Fathman,et al.  A short pulse of IL-4 delivered by DCs electroporated with modified mRNA can both prevent and treat autoimmune diabetes in NOD mice. , 2010, Molecular therapy : the journal of the American Society of Gene Therapy.

[166]  Lippincott-Schwartz,et al.  Supporting Online Material Materials and Methods Som Text Figs. S1 to S8 Table S1 Movies S1 to S3 a " Silent " Polymorphism in the Mdr1 Gene Changes Substrate Specificity Corrected 30 November 2007; See Last Page , 2022 .

[167]  E. G. Westaway,et al.  Subgenomic replicons of the flavivirus Kunjin: construction and applications , 1997, Journal of virology.

[168]  P. Eilers,et al.  A phase I/IIa immunotherapy trial of HIV-1-infected patients with Tat, Rev and Nef expressing dendritic cells followed by treatment interruption. , 2012, Clinical immunology.

[169]  D. Harrich,et al.  Kunjin Virus Replicon Vaccine Vectors Induce Protective CD8+ T-Cell Immunity , 2002, Journal of Virology.

[170]  K. Miura,et al.  A blocked structure at the 5′ terminus of mRNA from cytoplasmic polyhedrosis virus , 1975, Nature.

[171]  P. Liljeström,et al.  A New Generation of Animal Cell Expression Vectors Based on the Semliki Forest Virus Replicon , 1991, Bio/Technology.

[172]  K. Dyer,et al.  The RNase a superfamily: Generation of diversity and innate host defense , 2006, Molecular Diversity.

[173]  M. Bonneville,et al.  +1 Frameshifting as a Novel Mechanism to Generate a Cryptic Cytotoxic T Lymphocyte Epitope Derived from Human Interleukin 10 , 2002, The Journal of experimental medicine.

[174]  C. Mandl,et al.  In vitro-synthesized infectious RNA as an attenuated live vaccine in a flavivirus model , 1998, Nature Medicine.

[175]  R. Hewson.  RNA viruses: emerging vectors for vaccination and gene therapy. , 2000, Molecular medicine today.

[176]  K. Lundstrom.  Alphaviruses in Gene Therapy , 2009, Viruses.

[177]  B. Levine,et al.  T Cells Expressing Chimeric Antigen Receptors Can Cause Anaphylaxis in Humans , 2013, Cancer Immunology Research.

[178]  R. Rhoads,et al.  Novel "anti-reverse" cap analogs with superior translational properties. , 2003, RNA.

[179]  J. Sampson,et al.  Selective modification of antigen-specific T cells by RNA electroporation. , 2008, Human gene therapy.

[180]  C. Mandl,et al.  RNA: the new revolution in nucleic acid vaccines. , 2013, Seminars in immunology.

[181]  Gunther Hartmann,et al.  5'-Triphosphate RNA Is the Ligand for RIG-I , 2006, Science.

[182]  Y. Uematsu,et al.  A chimeric alphavirus RNA replicon gene-based vaccine for human parainfluenza virus type 3 induces protective immunity against intranasal virus challenge. , 2007, Vaccine.

[183]  James E. DiCarlo,et al.  RNA-Guided Human Genome Engineering via Cas9 , 2013, Science.

[184]  N. Schmitz,et al.  Induction of cytotoxic T-cell responses against the oncofetal antigen-immature laminin receptor for the treatment of hematologic malignancies. , 2003, Blood.

[185]  K. Kawakami,et al.  A simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection. , 2010, Genomics.

[186]  Jun Zhang,et al.  Generation of gene-modified mice via Cas9/RNA-mediated gene targeting , 2013, Cell Research.

[187]  Ö. Türeci,et al.  Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo , 2010, Gene Therapy.

[188]  W. Lewis.  Defective mitochondrial DNA replication and NRTIs: pathophysiological implications in AIDS cardiomyopathy. , 2003, American journal of physiology. Heart and circulatory physiology.

[189]  M. Czech,et al.  RNAi-based therapeutic strategies for metabolic disease , 2011, Nature Reviews Endocrinology.

[190]  Viswanath Devanarayan,et al.  Recommendations on risk-based strategies for detection and characterization of antibodies against biotechnology products. , 2008, Journal of immunological methods.

[191]  J. H. Strauss,et al.  Production of infectious RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants , 1987, Journal of virology.

[192]  Tao Wang,et al.  Design of multifunctional non-viral gene vectors to overcome physiological barriers: dilemmas and strategies. , 2012, International journal of pharmaceutics.

[193]  D. Curiel,et al.  Characterization of a messenger RNA polynucleotide vaccine vector. , 1995, Cancer research.

[194]  G. Ciliberto,et al.  Gene electro‐transfer of an improved erythropoietin plasmid in mice and non‐human primates , 2005, The journal of gene medicine.

[195]  V. M. Pain,et al.  A Reevaluation of the Cap-binding Protein, eIF4E, as a Rate-limiting Factor for Initiation of Translation in Reticulocyte Lysate (*) , 1996, The Journal of Biological Chemistry.

[196]  M. Idzko,et al.  Modified Foxp3 mRNA protects against asthma through an IL-10-dependent mechanism. , 2013, The Journal of clinical investigation.

[197]  A. Thess,et al.  A development that may evolve into a revolution in medicine: mRNA as the basis for novel, nucleotide-based vaccines and drugs , 2014, Therapeutic advances in vaccines.

[198]  F. Marincola,et al.  Cancer therapy using a self-replicating RNA vaccine , 1999, Nature Medicine.

[199]  Timothy B. Stockwell,et al.  Synthetic Generation of Influenza Vaccine Viruses for Rapid Response to Pandemics , 2013, Science Translational Medicine.

[200]  R. Flavell,et al.  Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3 , 2001, Nature.

[201]  E. Gilboa,et al.  Induction of Human Dendritic Cell Maturation Using Transfection with RNA Encoding a Dominant Positive Toll-Like Receptor 41 , 2004, The Journal of Immunology.

[202]  G. Lenzen,et al.  Induction of virus‐specific cytotoxic T lymphocytes in vivo by liposome‐entrapped mRNA , 1993, European journal of immunology.

[203]  E. Raz,et al.  Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[204]  B. Levine,et al.  Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor. , 2010, Cancer research.

[205]  B. Neyns,et al.  Intravenous and intradermal TriMix-dendritic cell therapy results in a broad T-cell response and durable tumor response in a chemorefractory stage IV-M1c melanoma patient , 2012, Cancer Immunology, Immunotherapy.

[206]  N. Shastri,et al.  Presentation of out-of-frame peptide/MHC class I complexes by a novel translation initiation mechanism. , 1999, Immunity.

[207]  G. J. Dimitriadis.  Translation of rabbit globin mRNA introduced by liposomes into mouse lymphocytes , 1978, Nature.

[208]  Alexander Meissner,et al.  Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. , 2010, Cell stem cell.

[209]  C. Y. Chen,et al.  AU-rich elements: characterization and importance in mRNA degradation. , 1995, Trends in biochemical sciences.

[210]  J. Rosenecker,et al.  Expression of therapeutic proteins after delivery of chemically modified mRNA in mice , 2011, Nature Biotechnology.

[211]  Ralf Dahm,et al.  Friedrich Miescher and the discovery of DNA. , 2005, Developmental biology.

[212]  R. Behringer,et al.  Insertional Mutagenesis by a Hybrid PiggyBac and Sleeping Beauty Transposon in the Rat , 2012, Genetics.

[213]  Ronald A. Li,et al.  Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction , 2013, Nature Biotechnology.

[214]  M. F. Mallette,et al.  The use of basic proteins to increase the infectivity of enterovirus ribonucleic acid , 1961 .

[215]  S. Govindarajan,et al.  Codon bias and heterologous protein expression. , 2004, Trends in biotechnology.

[216]  S. Aamdal,et al.  Phase I/II trial of melanoma therapy with dendritic cells transfected with autologous tumor-mRNA , 2006, Cancer Gene Therapy.

[217]  D. Baltimore,et al.  Cloned poliovirus complementary DNA is infectious in mammalian cells. , 1981, Science.

[218]  Vladimir Torchilin,et al.  Tumor delivery of macromolecular drugs based on the EPR effect. , 2011, Advanced drug delivery reviews.

[219]  Shizuo Akira,et al.  Shared and Unique Functions of the DExD/H-Box Helicases RIG-I, MDA5, and LGP2 in Antiviral Innate Immunity1 , 2005, The Journal of Immunology.

[220]  M. Debenedette,et al.  Cytokine Maturation Followed by CD40L mRNA Electroporation Results in a Clinically Relevant Dendritic Cell Product Capable of Inducing a Potent Proinflammatory CTL Response , 2008, Journal of immunotherapy.

[221]  J. Gurdon,et al.  Use of Frog Eggs and Oocytes for the Study of Messenger RNA and its Translation in Living Cells , 1971, Nature.

[222]  K. Karikó,et al.  Overexpression of urokinase receptor in mammalian cells following administration of the in vitro transcribed encoding mRNA , 1999, Gene Therapy.

[223]  R. E. Lockard,et al.  The synthesis of mouse hemoglobin beta-chains in a rabbit reticulocyte cell-free system programmed with mouse reticulocyte 9S RNA. , 1969, Biochemical and biophysical research communications.

[224]  Samira Kiani,et al.  Genetic engineering of human ES and iPS cells using TALE nucleases , 2011, Nature Biotechnology.

[225]  R. Rhoads,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. , 2008, RNA.

[226]  R. A. Cox,et al.  Foreign nucleic acids as the stimulus to make interferon. , 1963, Lancet.

[227]  D. Largaespada,et al.  RNA as a source of transposase for Sleeping Beauty-mediated gene insertion and expression in somatic cells and tissues. , 2006, Molecular therapy : the journal of the American Society of Gene Therapy.

[228]  B. Neyns,et al.  A phase IB study on intravenous synthetic mRNA electroporated dendritic cell immunotherapy in pretreated advanced melanoma patients. , 2013, Annals of oncology : official journal of the European Society for Medical Oncology.

[229]  B. Neyns,et al.  Therapeutic Vaccination With an Autologous mRNA Electroporated Dendritic Cell Vaccine in Patients With Advanced Melanoma , 2011, Journal of immunotherapy.

[230]  T. Thalhamer,et al.  Prophylactic mRNA Vaccination against Allergy Confers Long-Term Memory Responses and Persistent Protection in Mice , 2015, Journal of immunology research.

[231]  P. Chomez,et al.  A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. , 2007, Journal of immunology.

[232]  S. Pascolo.  Vaccination with messenger RNA (mRNA). , 2008, Handbook of experimental pharmacology.

[233]  T. Schlake,et al.  Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection , 2012, Nature Biotechnology.

[234]  N. Shastri,et al.  Constitutive Display of Cryptic Translation Products by MHC Class I Molecules , 2003, Science.

[235]  Adam James Waite,et al.  An improved zinc-finger nuclease architecture for highly specific genome editing , 2007, Nature Biotechnology.

[236]  H. Rammensee,et al.  Polarization of immunity induced by direct injection of naked sequence-stabilized mRNA vaccines , 2004, Cellular and Molecular Life Sciences CMLS.

[237]  B. Neyns,et al.  Single-Step Antigen Loading and Activation of Dendritic Cells by mRNA Electroporation for the Purpose of Therapeutic Vaccination in Melanoma Patients , 2009, Clinical Cancer Research.

[238]  Houping Ni,et al.  Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. , 2005, Immunity.

[239]  Mehmet Fatih Yanik,et al.  mRNA-engineered mesenchymal stem cells for targeted delivery of interleukin-10 to sites of inflammation. , 2013, Blood.

[240]  T. Tagawa,et al.  Bax mRNA therapy using cationic liposomes for human malignant melanoma , 2008, Journal of Gene Medicine.

[241]  Clay W Scott,et al.  Human induced pluripotent stem cells and their use in drug discovery for toxicity testing. , 2013, Toxicology letters.

[242]  S. Yamanaka,et al.  Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors , 2006, Cell.

[243]  J. Keene,et al.  Autoimmune epitopes in messenger RNA. , 2002, RNA.

[244]  N. Sang,et al.  Engineering Factor Viii for Hemophilia Gene Therapy. , 2011, Journal of genetic syndromes & gene therapy.

[245]  C. Rice,et al.  Sindbis virus: an efficient, broad host range vector for gene expression in animal cells. , 1989, Science.

[246]  S. Grupp,et al.  Treatment of advanced leukemia in mice with mRNA engineered T cells. , 2011, Human gene therapy.

[247]  W. Rottbauer,et al.  Successful Use of mRNA-Nucleofection for Overexpression of Interleukin-10 in Murine Monocytes/Macrophages for Anti-inflammatory Therapy in a Murine Model of Autoimmune Myocarditis , 2012, Journal of the American Heart Association.

[248]  H. Schild,et al.  Increased Antigen Presentation Efficiency by Coupling Antigens to MHC Class I Trafficking Signals1 , 2008, The Journal of Immunology.

[249]  L. Silbart,et al.  Modulating gene expression using DNA vaccines with different 3'-UTRs influences antibody titer, seroconversion and cytokine profiles. , 2003, Vaccine.

[250]  M. Murphy,et al.  Self-replicative RNA vaccines elicit protection against influenza A virus, respiratory syncytial virus, and a tickborne encephalitis virus. , 2001, The Journal of infectious diseases.

[251]  Ö. Türeci,et al.  mTOR Inhibition Improves Antitumor Effects of Vaccination with Antigen-Encoding RNA , 2013, Cancer Immunology Research.

[252]  J. Routy,et al.  Immunologic activity and safety of autologous HIV RNA-electroporated dendritic cells in HIV-1 infected patients receiving antiretroviral therapy. , 2010, Clinical immunology.