NIH Public Access
`Author Manuscript
`Mol Ther. Author manuscript; available in PMC 2009 November 10.
`Published in final edited form as:
`Mol Ther. 2008 November ; 16(11): 1833–1840. doi:10.1038/mt.2008.200.
`
`Incorporation of Pseudouridine Into mRNA Yields Superior
`Nonimmunogenic Vector With Increased Translational Capacity
`and Biological Stability
`
`Katalin Karikó1, Hiromi Muramatsu1, Frank A Welsh1, János Ludwig2, Hiroki Kato3, Shizuo
`Akira3, and Drew Weissman4
`1Department of Neurosurgery, University of Pennsylvania, Philadelphia, Pennsylvania, USA
`2Laboratory of RNA Molecular Biology, The Rockefeller University, New York, New York, USA
`3Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka,
`Japan
`4Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
`
`Abstract
`In vitro–transcribed mRNAs encoding physiologically important proteins have considerable
`potential for therapeutic applications. However, in its present form, mRNA is unfeasible for clinical
`use because of its labile and immunogenic nature. Here, we investigated whether incorporation of
`naturally modified nucleotides into transcripts would confer enhanced biological properties to
`mRNA. We found that mRNAs containing pseudouridines have a higher translational capacity than
`unmodified mRNAs when tested in mammalian cells and lysates or administered intravenously into
`mice at 0.015–0.15 mg/kg doses. The delivered mRNA and the encoded protein could be detected
`in the spleen at 1, 4, and 24 hours after the injection, where both products were at significantly higher
`levels when pseudouridine-containing mRNA was administered. Even at higher doses, only the
`unmodified mRNA was immunogenic, inducing high serum levels of interferon-(cid:302) (IFN-(cid:302)). These
`findings indicate that nucleoside modification is an effective approach to enhance stability and
`translational capacity of mRNA while diminishing its immunogenicity in vivo. Improved properties
`conferred by pseudouridine make such mRNA a promising tool for both gene replacement and
`vaccination.
`
`INTRODUCTION
`In vitro–synthesized mRNA seems an ideal nonviral gene replacement tool with many inherent
`advantages, including rapid protein production and efficient transduction of primary cells
`(reviewed in ref. 1). mRNA-based therapy also avoids deleterious side effects (e.g., integration
`into chromosomes) that limit clinical application of most virus- and DNA-based vectors.2 The
`first in vivo gene transfer therapy using mRNA was reported in 1990 (ref. 3). Subsequently,
`however, mRNA has rarely been used for introducing genetic material into animals or even
`into cultured cells. The use of mRNA has been mostly limited to vaccination in which antigen-
`encoding transcripts were administered in vivo or delivered to dendritic cells (DCs) ex vivo in
`
`© The American Society of Gene Therapy
`Correspondence: Katalin Karikó, Department of Neurosurgery, University of Pennsylvania, Room 371 Stemmler Hall, 36th & Hamilton
`Walk, Philadelphia, Pennsylvania 19104-6087, USA. kariko@mail.med.upenn.edu.
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`order to induce cellular and humoral immune responses.4,5 However, RNA is unsuitable for
`gene replacement because of its high immunogenicity and low effectiveness.
`
`Recent studies have demonstrated that RNA activates cells of the innate immune system by
`stimulating Toll-like receptors (TLRs), specifically TLR3, TLR7, and TLR8.6–8 However,
`when naturally occurring modified nucleosides, for example, pseudouridine ((cid:524)), 5-
`methylcytidine (m5C), N6-methyladenosine (m6A), 5-methyluridine (m5U), or 2-thiouridine
`(s2U) were incorporated into the transcript, most of the TLRs were no longer activated.9 The
`immunostimulatory potential of such RNAs as measured by proinflammatory cytokine release
`and induction of co-stimulatory molecules was greatly diminished.9 Hartmann et al. also
`reported that modified nucleosides, including s2U and (cid:524), abrogated 5(cid:397)-triphosphate RNA-
`mediated activation of another RNA-responsive immune sensor, retinoic acid-inducible protein
`I (RIG-I).10 If any of the in vitro transcripts containing nucleoside modifications would remain
`translatable and also avoid immune activation in vivo, such an mRNA could be developed into
`a new therapeutic tool for both gene replacement and vaccination. Accordingly, in this report,
`we tested the nucleoside-modified mRNAs for their translational potentials and in vivo immune
`characteristics.
`
`Our results reveal that incorporating pseudouridine, a naturally occurring modified nucleoside,
`into mRNA not only suppresses RNA-mediated immune activation in vitro and in vivo, but
`also enhances the translational capacity of the RNA. These characteristics and the ease of
`generating such an RNA by in vitro transcription make (cid:524)-containing mRNA a unique tool for
`expression of any protein in vitro and in vivo.
`
`RESULTS
`In vitro transcription and translation of nucleoside-modified mRNAs
`In nature, RNA is synthesized from adenosine triphosphate, uridine triphosphate, cytidine
`triphosphate, and guanosine triphosphate. After transcription, selected nucleosides become
`modified. Due to technical limitations, at present, the simplest method to generate RNA with
`modified nucleosides is to perform in vitro transcription reactions in which one of the four
`basic nucleotide triphosphates is replaced with a corresponding modified nucleotide
`triphosphate.9 In these transcripts, one particular nucleotide is substituted with the modified
`nucleotide at every position. In a series of transcription reactions using reporter protein–
`encoding plasmids and RNA polymerases, we obtained full-length transcripts containing (cid:524),
`m5U, s2U, m6A, or m5C (Figure 1a). Structures of these nucleosides are shown in
`Supplementary Figure S1. Under similar transcriptional conditions, we were unable to obtain
`full-length mRNA containing other naturally occurring nucleosides such as N1-
`methylguanosine, N1-methyladenosine, N7-methylguanosine, 2(cid:397)-O-methyluridine, and 2(cid:397)-O-
`methylcytidine.
`
`First, a set of nucleoside-modified mRNAs encoding firefly luciferase (TEVlucA50) and
`Renilla luciferase (capRen) (Supplementary Figure S2) was tested to determine whether they
`were translated in rabbit reticulocyte lysates. By measuring the enzyme activity of the encoded
`proteins, we were surprised to find that (cid:524)-containing mRNA translated more efficiently than
`its unmodified counterpart, resulting in twice as much protein from both TEVlucA50 and
`capRen templates (Figure 1b). The translational yield of mRNAs with m5U or m5C was similar
`to the unmodified transcripts, while mRNAs containing s2U translated poorly. The presence
`of m6A in mRNAs completely abolished their translatability. The (cid:524)-mediated translational
`enhancement was not observed in all translational systems. In wheat germ extracts, ~50% less
`protein was produced from the (cid:524)-containing mRNAs compared with unmodified mRNAs,
`while in bacterial cell lysates, mRNAs with (cid:524)-modifications were not translated (Figure 1b).
`Analyses of radiolabeled translational products generated in reticulocyte lysates confirmed that
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`lack of luciferase activities in samples programmed with s2U- and m6A-containing mRNAs
`were indeed due to lack of translation rather than loss of enzyme activity as a result of amino
`acid misincorporation caused by the modified nucleosides (Figure 1c). Although (cid:524) has not
`been found in any mRNA,11 its ability to form hydrogen bonds with adenosine is similar to
`that of uridine (Figure 1d), a structural characteristic that should allow efficient translation of
`such an mRNA.
`
`Superior translation of pseudouridine-containing mRNAs in cultured cells
`To determine whether the nucleoside-modified mRNAs are translated in cultured cells, sets of
`Renilla luciferase–encoding mRNAs were lipofectin complexed and delivered into 293 cells.
`mRNA containing (cid:524) translated ~10 times as much as unmodified mRNA, while the presence
`of m5C led to a fourfold enhancement (Figure 2a). mRNAs with s2U or m6A constituents, as
`in the cell-free system, were poorly or not at all translated. The translational capacity of mRNA
`in which only 5% of the adenosines were substituted with m6A, a composition closer to that
`which occurs in nature,11 was equivalent to that of the unmodified transcript. Similar results
`were obtained when sets of mRNAs with nucleoside modifications were tested in primary cells,
`including mouse DCs, embryonic fibroblasts and splenocytes, and human DCs (Figure 2b and
`data not shown). Control and (cid:524)-containing mRNAs delivered by polyethyleneimine (PEI) gave
`results consistent with those obtained with lipofectin (Supplementary Figure S3).
`
`In all of cell types, the (cid:524) modification conferred the highest translational yield to RNA and,
`because it was one of the nucleoside modifications that abolished RNA-mediated activation
`of primary DCs,9 as well as interferon (IFN) induction by RIG-I,10 subsequent studies focused
`on (cid:524)-modified mRNAs. To determine whether superior translation of (cid:524)-containing transcripts
`was dependent on structural elements known to enhance translation of the mRNA, a set of
`firefly luciferase–encoding mRNAs were synthesized with a unique 5(cid:397)-untranslated sequence
`(TEV, a cap-independent translational enhancer12), cap and/or poly(A) tail, or no additional
`sequences. As expected, these structural elements enhanced translational efficiency of the
`mRNA and presence of (cid:524) in the transcripts further increased the amount of protein made from
`all tested constructs (Figure 2c), demonstrating that (cid:524)-mediated translational enhancement is
`independent of cap and poly(A) tail. The most efficiently translated mRNAs
`(capTEVlucA50) were extended with longer poly(A) tails, which is known to enhance
`translation,12 and analyzed in time-course studies. mRNA with (cid:524) modification was translated
`to a greater extent than unmodified RNA at every tested time point and, as the incubation time
`increased, the difference between levels of luciferase made from the two types of mRNAs also
`increased (Figure 2d). Considering the short functional half-life of luciferase in mammalian
`cells and the fact that at 8–50 hours after transfection, cells programmed with (cid:524)-containing
`mRNA had unproportionally higher luciferase level, it is likely that (cid:524)-modification stabilizes
`the mRNA.
`
`Routinely, (cid:533)-galactosidase (lacZ)- and green fluorescent protein (GFP)–encoding constructs
`are used to visualize viral- or plasmid vector–mediated gene expression. Accordingly, we
`generated lacZ and GFP mRNAs (Supplementary Figure S2) with or without (cid:524) to use this
`detection system. To enhance translation, a subset of the RNAs was also extended with poly
`(A) tail. Analyses of 293 cells at 24 hours following RNA delivery demonstrated that the
`presence of (cid:524) in the mRNA significantly improved translational capacity of all tested RNA
`(caplacZ, caplacZ-An, and capGFP-An) as compared to the corresponding control, uridine-
`containing transcripts (Figure 2e and f). Not only did more cells express the encoded proteins
`when (cid:524)-modified mRNAs were used, but also the cells exhibited more intense staining,
`suggesting higher cellular levels of those proteins (Figure 2e and f and Supplementary Figure
`S4). The level of GFP staining in both number of positive cell and intensity was proportional
`to the amounts of delivered capGFP-An RNA added to cells cultured (Figure 2f). Transfection
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`of GFP mRNA containing (cid:524) modification resulted in higher levels of GFP expression in
`Chinese hamster ovary (CHO) cells as well as in cultured primary neurons from rat brain cortex
`(Figure 2g).
`
`Superior translation of (cid:524)-containing mRNA is independent of RIG-I
`RIG-I, a cytoplasmic sensor of pathogenic RNA, is ubiquitously present in all cells.13 It directs
`type I IFN secretion when exposed to RNA containing 5(cid:397)-triphoshate; however, replacement
`of uridine with (cid:524) in RNA blocks such an activation.10 The RNA samples we have synthesized
`and analyzed, including a significant fraction of those transcribed in the presence of cap analog,
`contained a 5(cid:397)-triphoshate. This raises the possibility that the observed translational differences
`between U- and (cid:524)-containing RNA (Figure 2) were caused by type I IFN, secreted when RIG-
`I was exposed to uridine- but not to (cid:524)-containing RNA. To address the role of RIG-I and type
`I IFN in the differential translation of the two RNA types, mouse embryonic fibroblasts (MEFs)
`derived from RIGI((cid:237)/(cid:237)) and wild-type mice were treated with U- or (cid:524)-containing mRNAs,
`and then translation and IFN induction were measured. Transcripts containing either 5(cid:397)-
`triphosphates (ppplucA50) or cap (capTEVluc-An) were tested. (cid:524)-containing mRNA was
`translated at increased levels compared to U-containing mRNA independent from the presence
`of RIG-I (Figure 3). As expected, the level of IFN-(cid:533) secreted by wild-type MEFs greatly
`diminished when the triphosphates were replaced with cap at the 5(cid:397)-ends of U-containing RNAs
`(Figure 3a and b).
`
`Most important, the U-containing mRNA did not induce IFN-(cid:533) from the RIG-I((cid:237)/(cid:237)) MEFs but
`was still translated less efficiently than (cid:524)-containing mRNA (Figure 3), making unlikely that
`translational differences were mediated by RIG-I-induced IFN actions.
`
`Efficient translation of (cid:524)-containing mrnA in mice
`For in vivo studies, we used firefly luciferase–encoding mRNA because, unlike other reporter
`enzymes (e.g., (cid:533)-galactosidase, Renilla luciferase), no endogenous mammalian enzyme
`interferes with its detection. The transcripts, unmodified and (cid:524)-modified capTEVlucA50, were
`extended with ~200-nt long poly(A) tails to increase their translatability (Figure 4a). mRNAs
`were lipofectin complexed and injected into mice through their caudal veins. Intravenous
`administration of 0.3-(cid:541)g capTEVluc-An containing (cid:524) resulted in high luciferase activity in the
`spleen, but not in the lung, liver, heart, kidney, or brain (Figure 4b), thus in subsequent studies
`only spleens were examined. Before analysis, spleens were bisected with one-half used for
`luciferase enzyme measurements and the other half for RNA isolation and subsequent northern
`blotting. First, the efficacies of (cid:524)-modified and unmodified capTEVluc-An mRNAs at 0.015
`mg/kg (0.3 (cid:541)g/animal) dose were compared in time-course experiments. Luciferase activity
`was readily detectable at 1 hour, peaked at 4 hours, and declined by 24 hours after mRNA
`administration (Figure 4c). At 1 and 4 hours, significantly more (up to 12-fold) luciferase
`activity was detected in animals that received the (cid:524)-modified mRNA compared to those
`injected with the unmodified mRNA. By 24 hours, the only samples with detectable levels of
`luciferase (fourfold above background) were from animals injected with (cid:524)-containing mRNA.
`The overall expression patterns were similar when mRNAs encoding Renilla luciferase
`(capRen with or without (cid:524) modifications) were injected into the animals or when isolated
`mouse splenocytes were exposed to mRNAs in culture (Supplementary Figure S5). Northern
`analyses of splenic RNA revealed that the administered mRNAs, in their intact and partially
`degraded forms, were readily detectable at 15 minutes, 1 hour, and 4 hours postinjection (Figure
`4d and Supplementary Figure S6). Because pilot studies demonstrated that the hybridization
`properties of (cid:524)-modified and unmodified RNAs are the same, we conclude that although equal
`amounts of the delivered mRNA reached the spleens, more (cid:524)-containing mRNA than control
`mRNA remained at 1 and 4 hours (Figure 4d and Supplementary Figure S6). By 24 hours,
`unmodified capTEVluc-An was below the level of detection, but trace amounts of (cid:524)-containing
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`capTEVluc-An, though partially degraded, were detectable (Figure 4d). These results suggest
`that presence of (cid:524) in RNA likely increases its biological stability. By altering the mRNA-lipid
`formulation, the delivery of greater amounts of mRNAs resulted in proportionally greater
`production of luciferase in cultured cells (Supplementary Figure S7). To determine whether
`protein production from the encoding mRNAs increases similarly in vivo, mRNAs were
`administered at 0.015–0.150 mg/kg doses, corresponding to delivery of 0.3–3.0 (cid:541)g capTEVluc-
`An per animal. Six hours later, the mice were killed and their spleens were analyzed. We found
`that luciferase levels were proportional to the amount of injected RNAs (Figure 4e). At these
`doses of RNA, luciferase levels were 12- to 78-fold higher when (cid:524)-modified capTEVluc-An
`was injected compared to the corresponding unmodified capTEVluc-An (Figure 4e). Again,
`no luciferase activity could be detected in any of the other organs even in animals injected with
`the highest dose of mRNA. For comparison, 3.0 (cid:541)g of pCMVluc plasmid was injected in a
`similar manner, but no luciferase activity could be detected in any organ when analyzed 24
`hours postadministration (Figure 4e and data not shown).
`
`(cid:524)-Modified mRNA is nonimmunogenic in mice
`We recently demonstrated that unmodified RNA, in contrast to (cid:524)-modified RNA, activated
`human DCs to secrete IFN-(cid:302) and tumor necrosis factor-(cid:302) (TNF-(cid:302)).9 In order to demonstrate
`whether (cid:524)-modified RNA activated the innate immune system after in vivo systemic delivery,
`serum samples were collected from animals at 6 hours postinjection and tested for
`proinflammatory cytokines. We found high IFN-(cid:302) levels only in those animals that were
`injected with 3.0 (cid:541)g of unmodified capTEVluc-An, but not with the (cid:524)-modified capTEVluc-
`An (Figure 4f). Although TNF-(cid:302) protein was below the detection level in the collected serum
`samples, northern blot analysis demonstrated the highest amount of TNF-(cid:302) mRNA in the spleen
`of animals that were administered unmodified mRNA at 4 hours postinjection (Figure 4d).
`
`DISCUSSION
`Progress in understanding the molecular details of many human diseases and instant access to
`human gene collections brings the possibility of using genes for treatment closer to reality.
`However, safe and effective gene therapy, which would take advantage of all these recent
`achievements, is still lacking. Unlike viral- and plasmid-based vectors, in vitro–transcribed
`mRNAs have never been vigorously tested for in vivo gene replacement, only for therapeutic
`vaccination (reviewed in ref. 14), because they were generally considered labile, inefficient,
`and immunogenic. Our most significant finding, however, demonstrates that in vitro–
`transcribed mRNAs containing pseudouridines possess superior translational capacities,
`increased biological stability, and no immunogenicity. The potential therapeutic advantages
`of using mRNA, especially (cid:524)-containing mRNA, over plasmid and viral vectors for delivering
`genetic material are numerous: (i) improved safety, because RNA is inherently incapable of
`integrating into the genome, thus preventing deleterious side effects that have stalled other
`vectors;2 (ii) lack of inflammatory response to (cid:524)-containing mRNA, thereby avoiding
`devastating systemic inflammation that can be fatal with a viral vector;15 (iii) efficient
`transduction of primary cells, unlike DNA, RNA does not require cell proliferation for
`expression of the encoded protein; (iv) rapid protein expression, mRNAs are translated within
`minutes following entry into the cytoplasm, whereas plasmids require time-consuming nuclear
`import and transcription; (v) virtually no size limit for the encoded proteins, because long
`mRNAs (we have generated 12-kb long mRNAs) can be easily obtained, unlike viral vectors
`that possess limited packaging capacities; (vi) the extent and duration of the encoded protein
`expression can be closely controlled because mRNAs have shorter half lives and, unlike other
`vectors, do not replicate; and (vii) manufacturing of mRNA is much simpler than producing
`viral or plasmid vectors, one DNA template can be transcribed many (~100) times and, by
`immobilizing the template, the process is adaptable for large scale continuous production in
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`bioreactors.16 Overall, these beneficial characteristics make (cid:524)-containing mRNA an excellent
`tool not only for in vivo expression of therapeutic proteins but also for vaccination. In the latter
`case, coadministration of adjuvant (e.g., immunostimulatory oligo-DNA, lipopolysaccha-ride)
`would also be required.
`
`Our most surprising result is that mRNAs with (cid:524) modification have a higher translational
`capacity than those without modification in all tested mammalian systems. Although further
`studies are needed to understand the reason for this difference fully, using RIG-I((cid:237)/(cid:237)) MEFs,
`we excluded a role for RIG-I and type I IFN in this phenomenon (Figure 3). It is possible that
`protein synthesis might be inhibited by RNA-dependent protein kinase activated by structural
`motifs present in mRNA-containing uridine17 but not (cid:524) modification. Consistent with this
`interpretation, attenuated translation of nonmodified mRNAs as compared to (cid:524)-modified
`mRNAs was observed in mammalian cells and lysates that contain RNA-dependent protein
`kinase but not observed in wheat germ extracts (Figures 1b and 2), which have no RNA-
`dependent protein kinase.18
`
`A likely contributing factor to the enhanced translation observed with (cid:524) modification is an
`increase in biological stability of the mRNAs (Figure 4d). Indeed, higher resistance to
`hydrolysis by phosphodiesterases from snake venom and spleen has been reported when uridine
`was replaced with (cid:524) in dinucleotide substrates.19 Previous studies have also demonstrated that
`(cid:524) stabilizes RNA secondary structures by promoting base stacking,20 which could slow
`degradation. However, stability of mRNAs containing either uridines or pseudouridines was
`the same when tested by in vitro assays using human skin–associated RNases21 (data not
`shown). Enhanced translation might be another factor that improves stability by protecting the
`RNA with high ribosome occupancy.
`
`From a potential adverse event standpoint, it is important to note that (cid:524), a natural constituent
`of RNA, likely lacks toxicity. In fact, (cid:524) is the most common modification found in RNA and
`natural pathways for metabolism of (cid:524)-modified mRNA and subsequent disposal into the urine
`exist.22
`
`In previous studies, we demonstrated that bacterial RNA and in vitro transcripts without
`modified nucleoside constituents stimulated human TLR3, TLR7, and TLR8, while
`mammalian RNA and in vitro transcripts containing nucleoside modifications including (cid:524)
`were minimally or nonstimulatory.9 We also observed high levels of TNF-(cid:302) and IFN-(cid:302)
`secretion by human primary DCs when exposed to RNAs that lacked nucleoside modification,
`but not when (cid:524)-modified RNAs were used.9 Because mammalian RNA, unlike bacterial RNA,
`abundantly possesses modified nucleosides, these results collectively demonstrate that
`nucleoside modifications suppress RNA immunogenicity. This study extends these findings
`to in vivo conditions by showing induction of IFN-(cid:302) secretion and an increase of TNF-(cid:302) mRNA
`in mice following administration of unmodified, but not (cid:524) modified, mRNA (Figure 4d and
`f).
`
`In summary, we demonstrate that mRNA containing (cid:524) is a new and effective vector to deliver
`genetic material for protein expression into both cultured cells and animals. The presence of
`(cid:524) in mRNA improved its translational capacity and overall stability. It also diminished its
`immunogenicity in vivo. Administering (cid:524)-containing mRNA at very low doses (0.015 mg/kg)
`resulted in robust expression of the encoded protein. We identified spleen as the target organ
`where the intravenously delivered, lipofectin-complexed mRNA and its encoded protein
`accumulate. These collective findings are important steps in developing the therapeutic
`potential of mRNA, such as using modified mRNA as an alternative to conventional
`vaccination and as a means for expressing clinically beneficial proteins in vivo safely and
`effectively.
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`MATERIALS AND METHODS
`mRNA synthesis and characterization
`For templates, reporter plasmids encoding firefly luciferase (pTEVluc, pTEVlucA50, pLuc,
`and pLucA50)12 (Daniel Gallie, University of California at Riverside, CA), Renilla luciferase
`(pSVren),23 bacterial (cid:533)-galactosidase (placZ) (Jay Hecker, University of Pennsylvania, PA),
`and enhanced GFP (peGFP) were used. Plasmid peGFP was generated by inserting coding
`sequences from pEGFP-N3 (Clontech, Mountain View, CA) into the EcoRV site of pT7TS
`(Paul Krieg, University of Texas at Austin, TX) containing the corresponding 5(cid:397)- and 3(cid:397)-
`untranslated region sequences of Xenopus (cid:533)-globin mRNA and a stretch of dA30dC30. First,
`plasmids were linearized with BamHI (pTEVluc, pLuc), NdeI (pTEVlucA50, pLucA50), SspI
`(pSVren), or XbaI/SalI (placZ, peGFP) to generate templates. Transcriptions were performed
`at 37 °C for 3 hours using T7 or SP6 RNA polymerases and nucleotide triphosphates at 7.5
`mmol/l final concentration (MEGAscript kits; Ambion, Austin, TX). To obtain mRNAs with
`modified nucleosides, the transcription reaction was assembled with the replacement of one
`nucleotide triphosphate with the corresponding triphosphate derivative of the following
`modified nucleosides: 5-methylcytidine (m5C), 5-methyluridine (m5U), 2-thiouridine (s2U),
`N6-methyladenosine (m6A), pseudouridine ((cid:524)), N1-methylguanosine (m1G), N1-
`methyladenosine (m1A), 2(cid:397)-O-methyluridine (Um), 2(cid:397)-O-methylcytidine (Cm) (TriLink, San
`Diego, CA), or N7-methylguanosine (m7G) (Sigma, St. Louis, MO). Where noted, only 5%
`of the adenosine triphosphate was replaced with N6-methyladenosine triphosphates in the
`reaction (5% m6A mRNA). Capped mRNAs were generated by supplementing the
`transcription reactions with 6 mmol/l 3(cid:397)-O-Me-m7GpppG, a nonreversible cap analog, (New
`England Biolabs, Beverly, MA) and lowering the concentration of guanosine triphosphate
`(3.75 mmol/l). Selected mRNAs were also poly(A) tailed in a reaction of ~1.5 (cid:541)g/(cid:541)l RNA, 5
`mmol/l adenosine triphosphate, and 60 U/(cid:541)l yeast poly(A) polymerase (USB, Cleveland, OH)
`according to the manufacturer's instructions and incubated at 30 °C for 3 hours. The length of
`poly(A) tails were estimated to be ~200-nt long and is indicated with An. Purification of the
`transcripts were performed by Turbo DNase (Ambion, Austin, TX) digestion followed by LiCl
`precipitation and 75% ethanol wash. The concentrations of RNA reconstituted in water were
`determined by measuring the optical density at 260 nm. Efficient incorporation of modified
`nucleotides to the transcripts was demonstrated by HPLC analyses.9 All RNA samples were
`analyzed by denaturing agarose gel electrophoresis for quality assurance. Each RNA type was
`synthesized in 4–10 independently performed transcription experiments and all experiments
`were performed with at least two different batches of mRNA. Schematic representations of all
`mRNAs used in the study are shown in Supplementary Figure S2.
`
`In vitro translation assay
`Aliquots (9 (cid:541)l) of rabbit reticulocyte lysates, wheat germ extracts, or Escherichia coli S30
`extracts (Promega, Madison, WI) were programmed with 1-(cid:541)l (0.5 (cid:541)g) TEVlucA50 or capRen
`mRNAs and incubated for 60 minutes at 30, 25, or 37 °C, respectively. Using cell culture lysis
`reagent (Promega, Madison, WI), the translation was stopped and aliquots were analyzed for
`enzyme activities using firefly and Renilla luciferase assay systems (Promega, Madison, WI)
`and a LUMAT LB 950 luminometer (Berthold/EG&G; Wallac, Gaithersburg, MD) at 10-
`second measuring time. Using recombinant firefly luciferase (Promega, Madison, WI), a
`standard curve was determined to be linear up to 3.6 × 107 relative light units measured activity,
`corresponding to 1.57-ng enzyme. All measurements were performed in the linear range of the
`standard curve. In selected experiments, the capRen-programmed rabbit reticulocyte lysates
`were also supplemented with 35S-methionine (PerkinElmer, Waltham, MA). At the end of
`incubation, 1-(cid:541)l aliquots were removed to analyze the labeled Renilla luciferase by 15%
`polyacrylamide gel electrophoresis. The gel containing the labeled samples was treated with
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`NIH-PA Author Manuscript
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`NIH-PA Author Manuscript
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`CUREVAC EX2033
`Page 7
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`Karikó et al.
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`Cells
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`1 mol/l Na salicylate, dried, and a fluorogram was generated by exposure to BioMax MS film
`(Kodak, Rochester, NY).
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`Page 8
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`Human embryonic kidney 293 cells and dihydrofolate reductase((cid:237)/(cid:237)) CHO cells were obtained
`from the American Type Culture Collection (Manassas, VA). Splenocytes were isolated from
`mouse (C57Bl/6) spleens using a cell strainer (70 (cid:541)m; BD Discovery Labware, Bedford, MA)
`and ACK lysis buffer (Sigma, St. Louis, MO). Embryonic fibroblasts from wild-type and RIG-
`I((cid:237)/(cid:237)) mice,24 293 cells, and splenocytes were cultured in Dulbecco's modified Eagle's
`medium (DMEM) supplemented with glutamine (Life Technologies, Gaithersburg, MD) and
`10% fetal calf serum (HyClone, Logan, UT). CHO cells were cultured in Iscove's modified
`Dulbecco's medium supplemented with hypoxanthine (0.1 mmol/l), thymidine (0.016 mmol/
`l), and 10% fetal calf serum. Murine DCs were generated from bone marrow cells of femurs
`and tibia of 6- to 9-week-old BALB/c mice as described.25 The cells were cultured in RPMI
`(Life Technologies, Gaithersburg, MD) containing glutamine, 10% fetal calf serum, and 50
`ng/ml muGM-CSF (R&D, Minneapolis, MN). Cells were maintained with fresh medium
`containing 50 ng/ml muGM-CSF every 2 days and used on day 7. Primary cortical culture was
`prepared from brain neocortices of E19 rat embryos as previously described,26 cultured in
`DMEM supplemented with 10% fetal calf serum (HyClone), 10% Ham's F12 with glutamine
`(Whittaker Bioproducts, Walkersville, MD), and 50 U/ml penicillin-streptomycin (Sigma, St.
`Louis, MO).
`
`Assembling the RNA complex
`Lipofectin (Invitrogen, Carlsbad, CA) and mRNA were complexed in phosphate buffer that
`has been shown to enhance transfection in vitro and in vivo.27,28 To assemble a 50-(cid:541)l complex
`of RNA-lipofectin, first 0.4 (cid:541)l potassium phosphate buffer (0.4 mol/l, pH 6.2) containing 10
`(cid:541)g/(cid:541)l bovine serum albumin (Sigma, St. Louis, MO) was added to 6.7 (cid:541)l DMEM, then 0.8 (cid:541)l
`lipofectin was mixed in and the sample was incubated for 10 minutes. In a separate tube, 0.25–
`3.0 (cid:541)g of RNA was added to DMEM to a final volume of 3.3 (cid:541)l. Diluted RNA was added to
`the lipofectin mix and incubated for 10 minutes. Finally, the RNA-lipofectin complex was
`further diluted by adding 38.8 (cid:541)l DMEM. Fifty microliter of such a complex was used to
`transfect cells present in 1 well of a 96-well plate. RNA was complexed with PEI (jetPEI;
`Polyplus Transfection, Illkirch, France) according to the manufacturer's instructions. Cells
`were exposed to 50 (cid:541)l NaCl (150 mmol/l) containing 0.25 (cid