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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.
Submitted December 23, 2005
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
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| Copyright © 2006 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
-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.
