Download Beta‐S‐ARCA(D1) Cap Analog Increases Protein Expression

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beta‐S‐ARCA(D1) Cap Analog
RNA Tools Beta‐S‐ARCA(D1) Cap Analog Increases Protein Expression from Synthetic Messenger RNA
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Andreas N. Kuhn , Mustafa Diken , Joanna Kowalska2, Jacek Jemielity2, Edward Darzynkiewicz2, and Ugur Sahin1
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BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany, and Division of Biophysics, University of Warsaw, Warsaw, Poland Synthetic messenger (m)RNA is increasingly used as a platform technology to express proteins of interest in cell culture and even in vivo. The 5’ cap of the mRNA is an important feature that affects mRNA stability as well as translational efficiency and thus ultimately the total amount of protein expressed from the mRNA. Capping of in vitro transcribed mRNA is commonly achieved by adding a corresponding dinucleotide cap analog to the reaction. Here we demonstrate that a modified cap analog called beta‐S‐ARCA(D1) enhances protein expression from synthetic mRNA compared to conventionally capped mRNA in immature dendritic cells both in cell culture as well as in mice. Importantly, this leads to an enhanced immune response in vivo, as has been shown using capped mRNA encoding the model epitope SIINFEKL from chicken ovalbumin. Together, the results demonstrate superiority of beta‐S‐ARCA(D1)‐capped synthetic mRNA. Introduction Synthetic messenger (m)RNA, which can be easily produced in large amounts and with high purity by in vitro transcription, can be used as a tool for the delivery of genetic information of a 1
protein of interest into cells in vitro and in vivo. To reach high levels of protein expression, considerable effort has been put in the improvement of delivered RNA with respect to its stability and 2
translational efficiency. A critical element for this is the 5’‐cap structure of the mRNA. The natural cap structure in eukaryotes consists of a guanine nucleotide that is connected to the first 5’‐
nucleotide of the transcribed RNA by a 5’‐5’ triphosphate bridge 7
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and is further methylated at the N7 position (m GpppG; Fig. 1). During capping of mRNA by in vitro transcription in the presence 7
of a cap dinucleotide, it was found that the plain m GpppG cap analog can be incorporated in both orientations, where only the 7
correctly orientated derivative, i.e. with the m G at the outward 4
position, takes part in the translation process. This has been addressed by designing modified cap analogs with a substitution 7
of the 3'‐ or 2'‐hydroxyl group of the m G with O‐CH3 and is 5,6
therefore referred to as 'anti reverse cap analog' (ARCA). Further modifications of the cap structure led to the synthetic cap 7,2’‐O
GppSpG). Beta‐S‐ARCA contains analog beta‐S‐ARCA(D1) (m2
additionally a phosphorothioate modification in the beta‐
phosphate group (Fig. 1). As this introduces a stereogenic center, there are two beta‐S‐ARCA diastereomers, called beta‐S‐ARCA(D1) and (D2). Initial characterization demonstrated that beta‐S‐ARCAs confer resistance against decapping by human Dcp2 and at the 7,8
same time higher affinity for eIF4E. Here, we summarize our results testing synthetic mRNAs capped 9
with different cap analogs in dendritic cells in vitro and in vivo. Beta‐S‐ARCA(D1)‐capped mRNA was shown to be superior, which results not only in higher levels of reporter gene expression like luciferase or eGFP, but also confers an increased T cell immune response against an encoded antigen in mice. Thus, it is recommended to use beta‐S‐ARCA(D1) for the capping of synthetic mRNAs to be applied into dendritic cells. D1 and D2), m2
GpppG (ARCA) and the plain structural homolog 7
of the natural cap, m GpppG, were tested. In addition, mRNA that was post‐transcriptionally capped enzymatically after transcription 7
(m GpppG/p.t.) was included for comparison. Human immature dendritic cells (hiDCs) were electroporated with equal amounts of the differentially capped synthetic mRNAs coding for the reporter luciferase or d2eGFP using a Gene‐Pulser‐II apparatus (Bio‐Rad, Munich, Germany) with voltage and capacitance settings of 300 V/150 µF. Cells were diluted in culture medium immediately after electroporation. For in vivo testing, mice were anaesthetized with xylazine/ ketamine, the inguinal lymph node was surgically exposed, and the RNA was injected in a maximal volume of 10 µl. Afterwards the wound was closed. 7,3’‐O
Results To test whether protein expression from synthetic mRNA can be increased in human immature dendritic cells (hiDCs) using modified cap analogs, equal amounts of differentially capped Materials & Methods In vitro transcription of the linearized DNA template (containing a promotor, the respective coding region flanked by 5’‐ and 3’‐UTR 9
sequences, and the poly(A)‐tail) was performed with T7 RNA polymerase (available from multiple commercial sources) in the presence of 7.5 mM ATP, CTP, UTP, a reduced GTP concentration (1.5 mM) and a 4‐fold excess of the particular cap analog (6 mM). 7,2’‐O
GppspG Along with the two diastereomers of beta‐S‐ARCA (m2
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beta‐S‐ARCA(D1) Cap Analog
RNA Tools mRNAs coding for luciferase or d2eGFP were electroporated into these cells. In Figure 1B the activity of the reporter genes is plotted as a function of time after electroporation. mRNA capped with beta‐S‐ARCA(D1) clearly showed the highest and longest lasting activity for both reporters in hiDCs (increased by more than 7
tenfold compared to plain m GpppG, and still more than threefold compared to ARCA or post‐transcriptional capping). This is based on both higher translational efficiency (as indicated by the initial slope of the curves), and improved mRNA stability (leading to a maximum at later time points). The latter could be independently confirmed by direct measurement of the mRNA stability via quantitative RT‐PCR (data not shown). To verify the positive effect of the modified cap analog in vivo, mRNAs encoding luciferase and capped either with ARCA or beta‐
S‐ARCA(D1) were injected into the inguinal lymph node of mice, 10
where these are taken up by resident dendritic cells. As observed in cell culture, protein expression was clearly higher and lasted longer for beta‐S‐ARCA(D1)‐capped mRNA (Fig. 2A). Most importantly, the increase in protein translation conferred an improved antigen‐specific immune response, when mRNA coding for the model epitope SIINFEKL of chicken ovalbumin was applied to mice by intranodal injection. Both in peripheral blood and +
spleen the amount of CD8 T cells recognizing the SIINFEKL epitope was measured by MHC tetramer staining. For beta‐S‐ARCA(D1)‐
capped mRNA, a proportion of 12.5 and 14.0% of SIINFEKL specific CD8+ T cells was measured, respectively, which is about threefold higher compared to ARCA‐capped mRNA (4.8 and 5.1%, Fig. 2B). Discussion & Conclusions Modified cap analogs were evaluated to optimize synthetic mRNA. Our data revealed that mRNAs capped with beta‐S‐ARCA(D1) showed enhanced stability and translational efficiency, which ultimately leads to higher total protein expression from the same amount of mRNA in vitro and in vivo. Importantly, this correlates with higher functionality as evidenced by increased induction of an antigen‐specific T cell immune response. In summary, beta‐S‐
ARCA(D1) is superior compared to commonly used cap analogs, and also to enzymatic capping. References 1. Recombinant messenger RNA technology and its application in cancer immunotherapy, transcript replacement therapies, pluripotent stem cell induction, and beyond. Vallazza et al.,WIREs RNA (2015) 2. Determinants of intracellular RNA pharmacokinetics: Implications for RNA‐based immunotherapeutics. Kuhn et al., RNA Biol. (2011) 3. ‘Cap‐tabolism’. Cougot et al., Trends Biochem. Sci. (2004) 4. Reverse 5’ caps in RNAs made in vitro by phage RNA polymerases. Pasquinelli et al.,RNA J. (1995) 5. Novel “anti‐reverse” cap analogs with superior translational properties. Jemielity et al., RNA J. (2003) 6. Synthesis and properties of mRNAs containing the novel “anti‐reverse” cap analogs 7‐methyl(3 ‘‐O‐methyl)GpppG and 7‐methyl(3 ‘‐
deoxy)GpppG. Stepinski et al., RNA J. (2001) 7. Phosphorothioate cap analogs stabilize mRNA and increase translational efficiency in mammalian cells. Grudzien‐Nogalska et al., RNA J. (2007) 8. Synthesis and characterization of mRNA cap analogs containing phosphorothioate substitutions that bind tightly to eIF4E and are resistant to the decapping pyrophosphatase DcpS. Kowalska et al., RNA J. (2008) 9. Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo. Kuhn et al., Gene Ther. (2010) 10. Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation. Diken et al., Gene Ther. (2011) The Author Andreas N. Kuhn [email protected] BioNTech RNA Pharmaceuticals GmbH, Mainz Germany Andreas N. Kuhn, Ph.D. has worked in the field of RNA biochemistry and molecular biology for more than twenty years. His current focus at BioNTech RNA Pharmaceuticals is to increase the efficacy of RNA immunotherapies and to optimize GMP‐compatible manufacturing processes for RNA. He has co‐authored several publications and patents ranging from basic research on RNA to its application as a diagnostic and therapeutic agent. The Company JPT Peptide Technologies is a DIN ISO 9001:2015 certified and GCLP compliant integrated provider of innovative peptide solutions for: cellular and humoral immune monitoring, seromarker discovery & validation, vaccine target discovery, peptide lead identification & optimization, targeted proteomics, and enzyme profiling.
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