Optimization of RNA cancer vaccines using 3' UTR sequences selected for stabilization of RNA
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Abstract
In the last decades, in vitro-transcribed (IVT-)messenger (m)RNAs encoding tumor-specific antigens have emerged as a powerful new tool to deliver genetic information into human immature dendritic cells (hiDCs) to induce a specific antitumor T cell response for therapeutic cancer vaccination. However, the short half-life of mRNA is still a major challenge. This makes the pharmacologically effective dosing of IVT-mRNA difficult, resulting in a time-restricted protein expression and, thus, a limited immune response. The stability of mRNAs is mainly regulated by the 3' untranslated region (3' UTR) as exemplified by the well-characterized ß-globin 3' UTR that is responsible for the high stability of globin mRNA in erythrocytes. Stability is controlled by binding of regulatory factors to respective sequence elements in the 3' UTR. While the ß-globin 3' UTR also stabilizes synthetic mRNAs to some degree in other cell types, such as hiDCs, the expression of the specific regulatory factors for RNA-stability is cell-type specific. Therefore, the identification of hiDC-specific stabilizing RNA-sequence elements could further improve intracellular pharmacokinetics of mRNA cancer therapeutics.
In this work, a novel in vitro selection process within hiDCs was developed to find naturally occurring RNA sequence elements, which stabilize the mRNA when used as 3' UTR in preclinical and clinical studies. Instead of using a chemically synthesized library for the selection process, a self-made RNA-library was built with hiDC-specific RNA sequences cloned as 3' UTR downstream of a suitable reporter gene. Additionally, hiDCs were used as a selective environment. This ensured the selection of cell-type specific RNA sequences as the cell's inner regulatory factors [RNA-binding proteins (RBPs) and microRNAs (miRNAs)] and degradation machineries would determine the survival of the transfected RNAs. The stringency of the selection was increased with each selection round by extending the time frame, in which the transfected RNA-pool was left in the cells before purification, amplification and preparation for the next selection round. The selected 3' UTR-sequences were analyzed and characterized afterwards. New single RNA-elements were identified and analyzed individually as single 3' UTR or in combination as double 3' UTR regarding their stabilizing effect on mRNA. Differences in the stabilizing effect of the analyzed 3' UTRs were rationalized in silico regarding binding of RBPs and miRNAs. Results revealed less binding of miRNAs, particularly in two combinations, which also proved to be superior compared to 2hBg, the in-house gold standard consisting of two copies of the human ß-globin 3' UTR. These combinations enhanced not only stability, but also translational efficiency of the synthetic mRNA in hiDCs yielding more protein over time. With these new potent 3' UTRs, RNA cancer immunotherapy can be further improved regarding antitumor efficacy due to a prolonged lifespan of the synthetic mRNA and an increased protein amount, thus, ultimately providing better patient care. Moreover, with this generally applicable method, cell-type specific 3' UTRs can be selected for other therapeutic fields like stem cell research or protein-replacement therapies to ensure optimal pharmacokinetics of synthetic mRNAs.