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Blood, 15 December 2006, Vol. 108, No. 13, pp. 4009-4017.
Prepublished online as a Blood First Edition Paper on August 29, 2006; DOI 10.1182/blood-2006-04-015024.


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Submitted December 23, 2005
Accepted July 25, 2006

Modification of antigen encoding RNA increases stability, translational efficacy and T-cell stimulatory capacity of dendritic cells

Silke Holtkamp, Sebastian Kreiter, Abderraouf Selmi, Petra Simon, Michael Koslowski, Christoph Huber, Ozlem Tureci, and Ugur Sahin*

JohannesGutenberg-University, Mainz, Germany

* Corresponding author; email: sahin{at}uni-mainz.de.

Adoptive transfer of dendritic cells (DCs) transfected with in vitro transcribed RNA encoding tumor-associated antigens has recently entered clinical testing as a promising approach for cancer immunotherapy. However, pharmacokinetic exploration of RNA as potential drug compound as a key aspect of clinical development is still pending. Investigating the impact of different structural modifications of RNA molecules on the kinetics of the encoded protein in DCs, we have identified components located 3’ of the coding region, which contribute to a higher transcript stability and translational efficiency. Using quantitative RT-PCR and eGFP variants to measure transcript amounts and protein yield, we show that (i) a poly(A) tail of 120 nucleotides length as compared to a shorter one, (ii) an unmasked poly(A) tail with a free 3’ end rather than one extended with unrelated nucleotides and (iii) two sequential {beta} -globin 3’ untranslated regions cloned head- to-tail between the coding region and the poly(A) tail, independently enhance RNA stability and translational efficiency. Consecutively, the density of antigen- specific peptide/MHC complexes on the transfected cells as well as their potency to stimulate and expand antigen- specific CD4+ as well as CD8+ T-cells are increased as well. In summary, our data provides a strategy for optimization of RNA transfected DC vaccines and a basis for defining release criteria for such vaccine preparations.


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S. Kreiter, A. Selmi, M. Diken, M. Sebastian, P. Osterloh, H. Schild, C. Huber, O. Tureci, and U. Sahin
Increased Antigen Presentation Efficiency by Coupling Antigens to MHC Class I Trafficking Signals
J. Immunol., January 1, 2008; 180(1): 309 - 318.
[Abstract] [Full Text] [PDF]



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