Antitumor Vaccination with Synthetic mRNA: Strategies for In Vitro and In Vivo Preclinical Studies

  • Mustafa Diken
  • Sebastian Kreiter
  • Abderraouf Selmi
  • Özlem Türeci
  • Ugur Sahin
Part of the Methods in Molecular Biology book series (MIMB, volume 969)


Synthetic antigen-encoding mRNA is increasingly exploited as a tool for delivery of genetic information of complete antigens into professional antigen presenting dendritic cells for HLA haplotype-independent antigen-specific vaccination against cancer. Two strategies for mRNA-based antitumor vaccination have emerged into the clinical setting. One is transfection of autologous dendritic cells with synthetic mRNA for adoptive transfer into the patient. The other is direct injection of naked synthetic mRNA. Both methods have proven to be feasible and safe and to elicit antigen-specific immune responses. The design of novel synthetic vaccines employing synthetic mRNA requires further in-depth investigation of its bioavailability and immune pharmacology. This chapter summarizes the state-of-art in this field and describes methods elementary for preclinical studies of mRNA-based antitumor vaccine protocols.

Key words

mRNA vaccination Dendritic cell Cancer immunotherapy Lymph node 



This research was supported by the GO-Bio funding of the German Federal Ministry of Education and Research to Ugur Sahin.


  1. 1.
    Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL (1990) Direct gene-transfer into mouse muscle in vivo. Science 247:1465–1468PubMedCrossRefGoogle Scholar
  2. 2.
    Conry RM, Lobuglio AF, Wright M, Sumerel L, Pike MJ, Feng JN, Benjamin R, Lu D, Curiel DT (1995) Characterization of a messenger-RNA polynucleotide vaccine vector. Cancer Res 55:1397–1400PubMedGoogle Scholar
  3. 3.
    Hoerr I, Obst R, Rammensee HG, Jung G (2000) In vivo application of RNA leads to induction of specific cytotoxic T lymphocytes and antibodies. Eur J Immunol 30:1–7PubMedCrossRefGoogle Scholar
  4. 4.
    Kariko K, Buckstein M, Ni HP, Weissman D (2005) Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23:165–175PubMedCrossRefGoogle Scholar
  5. 5.
    Nair SK, Morse M, Boczkowski D, Cumming RI, Vasovic L, Gilboa E, Lyerly HK (2002) Induction of tumor-specific cytotoxic T lymphocytes in cancer patients by autologous tumor RNA-transfected dendritic cells. Ann Surg 235:540–549PubMedCrossRefGoogle Scholar
  6. 6.
    Granstein RD, Ding WH, Ozawa H (2000) Induction of anti-tumor immunity with epidermal cells pulsed with tumor-derived RNA or intradermal administration of RNA. J Invest Dermatol 114:632–636PubMedCrossRefGoogle Scholar
  7. 7.
    Boczkowski D, Nair SK, Snyder D, Gilboa E (1996) Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo. J Exp Med 184:465–472PubMedCrossRefGoogle Scholar
  8. 8.
    Van Tendeloo VF, Ponsaerts P, Lardon F, Nijs G, Lenjou M, Van Broeckhoven C, Van Bockstaele DR, Berneman ZN (2001) Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood 98:49–56PubMedCrossRefGoogle Scholar
  9. 9.
    Ponsaerts P, Van den Bosch G, Cools N, Van Driessche A, Nijs G, Lenjou M, Lardon F, Van Broeckhoven C, Van Bockstaele DR, Berneman ZN, Van Tendeloo VF (2002) Messenger RNA electroporation of human monocytes, followed by rapid in vitro differentiation, leads to highly stimulatory antigen-loaded mature dendritic cells. J Immunol 169:1669–1675PubMedGoogle Scholar
  10. 10.
    Grover A, Kim GJ, Lizee G, Tschoi M, Wang G, Wunderlich JR, Rosenberg SA, Hwang ST, Hwu P (2006) Intralymphatic dendritic cell vaccination induces tumor antigen-specific, skin-homing T lymphocytes. Clin Cancer Res 12:5801–5808PubMedCrossRefGoogle Scholar
  11. 11.
    Nair S, Boczkowski D, Moeller B, Dewhirst M, Vieweg J, Gilboa E (2003) Synergy between tumor immunotherapy and antiangiogenic therapy. Blood 102:964–971PubMedCrossRefGoogle Scholar
  12. 12.
    Heiser A, Coleman D, Dannull J, Yancey D, Maurice MA, Lallas CD, Dahm P, Niedzwiecki D, Gilboa E, Vieweg J (2002) Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors. J Clin Invest 109:409–417PubMedGoogle Scholar
  13. 13.
    Kreiter S, Diken M, Selmi A, Tureci O, Sahin U (2011) Tumor vaccination using messenger RNA: prospects of a future therapy. Curr Opin Immunol 23:399–406PubMedCrossRefGoogle Scholar
  14. 14.
    Carralot JP, Probst J, Hoerr I, Scheel B, Teufel R, Jung G, Rammensee HG, Pascolo S (2004) Polarization of immunity induced by direct injection of naked sequence-stabilized mRNA vaccines. Cell Mol Life Sci 61:2418–2424PubMedCrossRefGoogle Scholar
  15. 15.
    Holtkamp S, Kreiter S, Selmi A, Simon P, Koslowski M, Huber C, Tureci O, Sahin U (2006) Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood 108:4009–4017PubMedCrossRefGoogle Scholar
  16. 16.
    Stepinski J, Waddell C, Stolarski R, Darzynkiewicz E, Rhoads RE (2001) 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 7:1486–1495PubMedGoogle Scholar
  17. 17.
    Pasquinelli AE, Dahlberg JE, Lund E (1995) Reverse 5′ caps in RNAs made in vitro by phage RNA polymerases. RNA 1:957–967PubMedGoogle Scholar
  18. 18.
    Kuhn AN, Diken M, Kreiter S, Selmi A, Kowalska J, Jemielity J, Darzynkiewicz E, Huber C, Tureci O, Sahin U (2010) Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo. Gene Ther 17:961–971PubMedCrossRefGoogle Scholar
  19. 19.
    Kreiter S, Selmi A, Diken M, Koslowski M, Britten CM, Huber C, Tureci O, Sahin U (2010) Intranodal vaccination with naked antigen-encoding RNA elicits potent prophylactic and therapeutic antitumoral immunity. Cancer Res 70:9031–9040PubMedCrossRefGoogle Scholar
  20. 20.
    Kreiter S, Diken M, Selmi A, Diekmann J, Attig S, Husemann Y, Koslowski M, Huber C, Tureci O, Sahin U (2011) FLT3 ligand enhances the cancer therapeutic potency of naked RNA vaccines. Cancer Res 71:6132–6142PubMedCrossRefGoogle Scholar
  21. 21.
    Weide B, Pascolo S, Scheel B, Derhovanessian E, Pflugfelder A, Eigentler TK, Pawelec G, Hoerr I, Rammensee HG, Garbe C (2009) Direct injection of protamine-protected mRNA: results of a phase 1/2 vaccination trial in metastatic melanoma patients. J Immunother 32:498–507PubMedCrossRefGoogle Scholar
  22. 22.
    Weide B, Carralot JP, Reese A, Scheel B, Eigentler TK, Hoerr I, Rammensee HG, Garbe C, Pascolo S (2008) Results of the first phase I/II clinical vaccination trial with direct injection of mRNA. J Immunother 31:180–188PubMedCrossRefGoogle Scholar
  23. 23.
    Rittig SM, Haentschel M, Weimer KJ, Heine A, Muller MR, Brugger W, Horger MS, Maksimovic O, Stenzl A, Hoerr I, Rammensee HG, Holderried TA, Kanz L, Pascolo S, Brossart P (2011) Intradermal vaccinations with RNA coding for TAA generate CD8+ and CD4+ immune responses and induce clinical benefit in vaccinated patients. Mol Ther 19:990–999PubMedCrossRefGoogle Scholar
  24. 24.
    Diken M, Kreiter S, Selmi A, Britten CM, Huber C, Tureci O, Sahin U (2011) Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation. Gene Ther 18:702–708PubMedCrossRefGoogle Scholar
  25. 25.
    Lutz MB, Kukutsch N, Ogilvie AL, Rossner S, Koch F, Romani N, Schuler G (1999) An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods 223:77–92PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Mustafa Diken
    • 1
  • Sebastian Kreiter
    • 1
  • Abderraouf Selmi
    • 1
    • 2
  • Özlem Türeci
    • 3
  • Ugur Sahin
    • 4
    • 5
    • 6
  1. 1.Translational Oncology and Immunology (TRON) at the Johannes Gutenberg University MainzMainzGermany
  2. 2.Division of Translational and Experimental Oncology, Department of Internal Medicine IIJohannes Gutenberg University Medical CenterMainz Germany
  3. 3.Ganymed PharmaceuticalsMainzGermany
  4. 4.TRON-Translational Oncology at the University Medical Center MainzMainzGermany
  5. 5.III. Medical DepartmentUniversity Medical Center of the Johannes Gutenberg-University MainzMainzGermany
  6. 6.Ribological, Biontech AGMainzGermany

Personalised recommendations