Developing mRNA-Vaccine Technologies PDF

RNA Biology ISSN: 1547-6286 (Print) 1555-8584 (Online) Journal homepage: https://www.tandfonline.com/loi/krnb20 Developing mRNA-vaccine technologies Thomas Schlake, Andreas Thess, Mariola Fotin-Mleczek & Karl-Josef Kallen To cite this article: Thomas Schlake, Andreas Thess, Mariola F...

RNA Biology ISSN: 1547-6286 (Print) 1555-8584 (Online) Journal homepage: https://www.tandfonline.com/loi/krnb20 Developing mRNA-vaccine technologies Thomas Schlake, Andreas Thess, Mariola Fotin-Mleczek & Karl-Josef Kallen To cite this article: Thomas Schlake, Andreas Thess, Mariola Fotin-Mleczek & Karl-Josef Kallen (2012) Developing mRNA-vaccine technologies, RNA Biology, 9:11, 1319-1330, DOI: 10.4161/ rna.22269 To link to this article: https://doi.org/10.4161/rna.22269 Published online: 12 Oct 2012. Submit your article to this journal Article views: 79457 View related articles Citing articles: 219 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=krnb20  review REVIEW RNA Biology 9:11, 1319–1330; November 2012; © 2012 Landes Bioscience Developing mRNA-vaccine technologies Thomas Schlake,* Andreas Thess, Mariola Fotin-Mleczek and Karl-Josef Kallen CureVac GmbH; Tübingen, Germany Keywords: mRNA, adjuvant, vaccine, mRNA production, mRNA design, mRNA uptake, formulation, protein expression Abbreviations: ARCA, anti-reverse cap analog; cDNA, complementary DNA; CTL, cytotoxic T cell; dsRNA, double-stranded RNA; EMCV, encephalomyocarditis virus; GMP, good manufacturing practice; HPLC, high performance liquid chromatography; IRES, internal ribosome entry site; mRNA, messenger RNA; MHC, major histocompatibility complex; miRNA, microRNA; ORF, open reading frame; pAPC, professional antigen presenting cell; pDNA, plasmid DNA; PEG, polyethylenglycol; siRNA, ©2012 Landes Bioscience. Do not distribute small interfering RNA; tRNA, transfer RNA; TLR, toll-like receptor; UTR, untranslated region mRNA vaccines combine desirable immunological properties excluded. Finally, this lack of genomic integration in combina- with an outstanding safety profile and the unmet flexibility of tion with mRNA being non-replicative as well as metabolically genetic vaccines. Based on in situ protein expression, mRNA decaying within a few days8 makes mRNA a merely transient vaccines are capable of inducing a balanced immune response carrier of information. comprising both cellular and humoral immunity while not mRNA as the technological basis of therapeutics and vaccines subject to MHC haplotype restriction. In addition, mRNA is is characterized by a great flexibility with respect to production an intrinsically safe vector as it is a minimal and only transient and application. Any protein can be encoded and expressed by carrier of information that does not interact with the genome. mRNA, in principle enabling the development of prophylactic Because any protein can be expressed from mRNA without the and therapeutic vaccines fighting diseases as diverse as infections need to adjust the production process, mRNA vaccines also and cancer as well as protein replacement therapies. Since changes offer maximum flexibility with respect to development. Taken of the encoded protein just alter the sequence of the RNA mol- together, mRNA presents a promising vector that may well become the basis of a game-changing vaccine technology ecule, leaving its physico-chemical characteristics largely unaf- platform. Here, we outline the current knowledge regarding fected, diverse products can be manufactured using the same different aspects that should be considered when developing established production process without any adjustment, saving an mRNA-based vaccine technology. time and reducing cost compared with other vaccine platforms. In terms of efficacy, mRNA-based therapeutics profit from the fact that they do not need to cross the nuclear envelope as opposed to DNA. In contrast to peptides, mRNA vaccines lack Introduction MHC haplotype restriction. In addition, mRNA binds to pattern recognition receptors and mRNA vaccines may be designed to be RNA is considered as notoriously unstable making its therapeu- self-adjuvanting,9 a property which peptide- and protein-based tic use a provocative idea. Despite the sensitivity of the molecule vaccines lack. to the virtually omnipresent ribonucleases (RNases),1 mRNA as All in all, mRNA presents a promising, even if challenging, a therapeutic was first promoted in 1989 after the development class of therapeutic molecules that has the potential to become of a broadly applicable in vitro transfection technique.2 Only a the basis of a “disruptive technology.”10 In the following we are couple of years later, mRNA was advocated as a vaccine platform, casting light on what has to be considered when developing an perhaps being ideal in the sense that it brings together the immu- mRNA-vaccine technology touching important topics such as nological features of live attenuated vaccines such as endogenous mRNA manufacturing and quality, mRNA format and formu- antigen expression and T cell induction with those of killed or lation as well as antigen/protein expression and immunological subunit vaccines like defined composition and safety.3,4 properties of mRNA-vaccines. Particularly compared with DNA as a therapeutic or more specifically as a vaccine, mRNA offers strong safety advantages.5 mRNA Production As the minimal genetic construct, it harbors only the elements directly required for expression of the encoded protein. Moreover, mRNA synthesis. Functional synthetic mRNA may be obtained while recombination between single-stranded RNA molecules by in vitro transcription of a cDNA template, typically plasmid may occur in rare cases,6,7 mRNA does not interact with the DNA (pDNA), using a bacteriophage RNA polymerase.11 Hence, genome. Thus, potentially detrimental genomic integration is the preparation of pDNA is the first step in the production of mRNA. Manufacture of mRNA might thus appear to require *Correspondence to: Thomas Schlake; more effort than manufacture of pDNA. However, unpol- Email: [email protected] ished pDNA contains traces of bacterial genomic DNA and Submitted: 08/15/12; Revised: 09/17/12; Accepted: 09/17/12 three forms of pDNA (supercoiled, relaxed circle or linear) in http://dx.doi.org/10.4161/rna.22269 www.landesbioscience.com RNA Biology 1319 variable proportions. Hence, the reproducible preparation of pure The authors demonstrated that increased protein expression after and invariant pDNA, as required for a vaccine, is demanding. HPLC purification was also due to the removal of contaminat- Remains of bacterial DNA and the heterogeneity of pDNA are ing, e.g., double-stranded, RNA that activates innate immune not a concern, on the other hand, if linearized pDNA is tran- sensors, thereby reducing protein expression. scribed using bacteriophage RNA polymerase,5 because all DNA is removed during further processing steps (see below). mRNA Design Synthetic mRNA contains a protein-encoding open reading frame (ORF) flanked at the minimum by two elements essen- Synthetic mRNA for therapy is in general designed following the tial for the function of mature eukaryotic mRNA: a “cap,” blueprint of eukaryotic mRNA. Cap and poly(A) tail are essential i.e., a 7-methyl-guanosine residue joined to the 5'-end via a elements because they are required for efficient translation.2,15,21 5'-5' triphosphate,12 and a poly(A) tail at the 3'-end.13 Accordingly, Positioned at the very 5'- and 3'-end of mRNA, Cap and poly(A) a pDNA template for in vitro transcription contains at least a tail are also required to stabilize mRNA in the cytosol, where ©2012 Landes Bioscience. Do not distribute bacteriophage promoter, an ORF, optionally a poly[d(A/T)] decay is catalyzed predominantly by exonucleases.22,23 sequence transcribed into poly(A) and a unique restriction site However, to further increase both translation and stability, for linearization of the plasmid to ensure defined termination of mRNA requires 5' and 3' untranslated regions (UTRs) to flank transcription (the cap is not encoded by the template). the ORF.24-27 UTRs have to be carefully chosen because they The linearized pDNA template is transcribed into mRNA in may also impair translation or mRNA stability.28 In particular, a mixture containing recombinant RNA polymerase (T7, T3 or specific cis-acting destabilizing sequences like AU-rich elements29 SP6) and nucleoside triphosphates. It is possible to obtain capped and miRNA binding sites30,31 mostly reside in UTRs, although mRNA by transcription. To this end, a cap analog like the dinu- they may also be found in ORFs.32 Care must be taken to avoid cleotide m7G(5')-ppp-(5')G (called “regular cap analog” in the such destabilizing signals. following) may be included in the reaction.14 If the cap analog is Following these considerations, efforts have been made to in excess of GTP, transcription initiates with the cap analog rather identify beneficial mRNA elements in order to improve transla- than GTP, yielding capped mRNA.15 Alternatively, the cap may tion and stability of synthetic mRNA molecules inside the cell. be added enzymatically post transcription. A poly(A) tail may Improved mRNA formats thus identified will likely also yield bet- also be added post transcription if it is not provided by the pDNA ter mRNA vaccines, as it is widely assumed that the efficacy of template. Following transcription, the pDNA template as well as an mRNA vaccine will rise as protein expression is increased and contaminating bacterial DNA is digested by DNase. prolonged. mRNA purification. At this point, the sample contains the Cap. mRNA may be capped during transcription by includ- desired mRNA transcript within a complex mixture including ing a cap analog in the reaction. However, it has been found that various nucleotides, oligodeoxynucleotides, short abortive tran- the regular cap analog is often incorporated in the reverse orien- scripts from abortive cycling during initiation,16 as well as pro- tation so that the m7G nucleotide does not constitute the cap but tein. These contaminants may be removed from the sample by a is instead the first transcribed nucleotide. As a result, about one combination of precipitation and extraction steps. third of mRNA molecules are not methylated at their cap.33 Such However, the sample includes additional contaminating RNA mRNA lacking methylation of the cap base is not translated.34 species that cannot be separated from the correct transcript by In order to avoid unmethylated cap by reverse orientation, simple means: Shorter than designated transcripts arise from mRNA may be transcribed without cap analog and subsequently premature termination during elongation. Longer than desig- capped using the vaccinia virus capping complex.35 This complex nated transcripts arise from template DNA linearized with an with triphosphatase, guanylyltransferase and (guanine-7-)meth- enzyme that leaves a 3'-overhang17 or from traces of nonlinear- yltransferase activity adds a natural cap to the 5'-triphosphate of ized template DNA. Undesirable transcripts are also produced an RNA molecule. However, an additional enzymatic step may due to the RNA-dependent RNA polymerase activity of bacte- complicate production, particularly at an industrial scale. riophage polymerases.18 Accordingly, to be used as a drug sub- Alternatively, a cap exclusively in the correct orientation is stance, mRNA will have to be purified further to remove such obtained with the use of “anti-reverse” cap analogs (ARCAs). In contaminating transcripts. the most common ARCA, 3'-O-methylation of the base-methyl- A single chromatographic step that separates mRNA accord- ated guanosine only allows addition of a nucleotide at the non- ing to size removed both shorter and longer transcripts, yielding methylated guanosine. ARCA-capped mRNA was translated at a pure single mRNA product.19 Implementation of such a chro- more than doubled efficiency in rabbit reticulocyte lysate com- matographic purification within a GMP production process for pared with mRNA capped by regular cap analog.34 In addition, mRNA increased the activity of mRNA molecules several-fold in it has been shown that mRNA transcribed in vitro with ARCA terms of protein expression in vivo.8 also has a longer half-life in cultured cells.36 In an independent Increased protein expression as a result of stringent purifi- study, ARCA-capped mRNA has been reported to both increase cation of mRNA was also observed when transcripts coding and prolong protein expression in cultured cells.37 for luciferase or erythropoietin were purified by HPLC.20 The Protein expression from in vitro transcribed, enzymati- increase in protein expression was much higher than would be cally capped mRNA can be further increased by enzymatic expected simply based on the removal of incorrect transcript. 2'-O-methylation of the first transcribed nucleotide, resulting in 1320 RNA Biology Volume 9 Issue 11 protein expression comparable to that from mRNA capped with the EMCV IRES was included in mRNAs coding for four tran- ARCA co-transcriptionally.38 scription factors used to reprogram fibroblasts to pluripotent stem ARCAs have been further modified within the triphosphate cells.49 The EMCV IRES has even been used successfully to direct linkage in order to inhibit decapping of the corresponding protein expression from mRNA lacking a cap.50 Vaccination with mRNA and increase binding of eukaryotic initiation factor 4E dendritic cells transfected with such IRES-containing, cap-less involved in the recruitment of ribosomes. Modifications either mRNA protected mice from metastasis upon intravenous injec- substituted for a bridging oxygen [e.g., (methylenebis)phospho- tion of melanoma cells. nate and imidodiphosphate] or a non-bridging oxygen (e.g., Completely novel UTRs may be provided by screening whole phosphorothioate, phosphoroselenoate and boranophosphate).39 transcriptomes for sequence elements that either increase transla- Phosphorothioate-modified ARCAs yielded mRNA with both tion or mRNA stability.51 further increased translation efficiency and elongated half-life in ORF. Codon usage is also considered as a factor affecting the cultured cells compared with ARCA.40 However, phosphorothio- efficiency of translation in many species. However, in humans ©2012 Landes Bioscience. Do not distribute ate-modified ARCAs are obtained as a mixture of two diaste- codon usage bias does not correlate with tRNA levels and gene reomers that must be separated after synthesis because of their expression.52,53 In conclusion, codon optimization cannot be different biological activity. expected to (generally) improve mRNA translation in humans, Poly(A) tail. When the poly(A) tail was unveiled to enhance particularly if the ORF is already of human (or even mamma- translation initiation, it was noted that the efficiency of polysome lian) origin. formation increased with increasing length of the poly(A) tail up Obviously, the start codon should be part of a Kozak to 68 residues.15 Translation of in vitro transcribed mRNA trans- sequence54 and the sequence surrounding the stop codon may be fected into cultured cells still increased slightly by lengthening optimized.55 In addition, no upstream start codons, preceding the poly(A) tail from 54 to 98 residues.41 This study was fur- the correct start codon, should be present in the mRNA. ther extended by investigating the effect of even longer poly(A) Combinatorial design. In order to obtain effective vaccine tails on protein expression.38 The peak protein level, reached one platforms, different beneficial mRNA elements have been joined. day after electroporation of mRNA into cells, was doubled when Capping with ARCA has been combined with a long tran- the poly(A) tail was extended from 64 to 150 residues. Further scribed poly(A) tail of 100 residues. Such luciferase-encoding extension of the poly(A) tail by enzymatic polyadenylation led mRNA was tested in immortalized cell lines (JawsII, HepG2 and to an additional moderate increase in peak expression. By con- HeLa) as well as immature and mature human dendritic cells.56 trast, upon transfection of UMR-106 cells, protein levels 16 h Compared with mRNA capped with regular cap analog and end- post transfection increased with increasing length of the poly(A) ing with a shorter A64 poly(A) tail, a very substantial improve- tail only up to 60 residues, but declined with further increas- ment in protein expression was seen in all tested cell types. The ing poly(A) tail length.42 In practical terms, it is noteworthy that magnitude of the rise in protein level afforded by either element maintenance of long poly[d(A/T)] sequences is demanding and alone or their combination was strongly dependent on the cell strongly dependent on the bacterial strain.42 type. UTRs. Already early on, in vitro transcribed mRNA con- Sahin and coworkers combined two consecutive β globin tained 5'- and 3'-UTRs, specifically those of the β globin gene 3'-UTRs, a rather long transcribed poly(A) tail of 120 residues57 of Xenopus.11 Both the Xenopus β globin 5'- and 3'-UTRs were and a phosphorothioate modified anti-reverse cap.58 This resulted demonstrated to impart much greater translational efficiency in increased and prolonged protein expression in transfected den- on heterologous mRNA in the mouse NIH 3T3 fibroblast cell dritic cells. Upon injection of mRNA into the lymph node, pro- line.2 A combination of the β globin 5'-UTR, improving trans- tein expression peaked at 8 h and was demonstrated up to 72 h lation and the α globin 3'-UTR, known to stabilize mRNA,27 after mRNA injection. has been used in the construction of a library from amplified Using our proprietary mRNA technology modifying coding tumor-derived cRNA for use as vaccines against metastatic mel- and noncoding parts of the molecule, we were able to improve anomas.43 Globin UTRs are still in widespread use in in vitro both the level and duration of expression, increasing total protein transcribed mRNA including RNA for immune therapy.38,44,45 expression by several orders of magnitude.59 Upon intradermal UTRs from non-globin genes have also been included in in mRNA injection, strongly prolonged translation gives maximum vitro transcribed mRNAs used for investigations of the therapeu- protein levels 24 to 48 h after mRNA injection and lasts for many tic value of mRNA. The 5'-UTR of tobacco etch virus enhances more days (Fig. 1). translation of in vitro transcribed mRNA in mammalian cells46 and has been included in mRNA expressing erythropoietin in mRNA Uptake different cell types20 and mice.47 Furthermore, a structure of the 5'-UTR of human heat shock protein 70 enhanced translation of To be translated and elicit an antigen-specific immune response, mRNA in mammalian cells and was predicted to be valuable in an mRNA-vaccine has to reach the cytosol of target cells. the context of genetic vaccination.48 However, as opposed to DNA vaccines, RNA vaccines only have Inclusion of an internal ribosomal entry site (IRES) in in vitro to cross the plasma membrane, but not the nuclear envelope transcribed mRNA can be an alternative and/or complementary which may improve the probability of successful in vivo transfec- means to achieve expression of therapeutic proteins. For instance, tion.60 As early as 1990, the uptake of mRNA by mouse muscle www.landesbioscience.com RNA Biology 1321  combination of physico-chemical and structural parameters. In contrast to DNA, mRNA contains uridine instead of deoxythy- midine, preferentially adopts a C3'-endo conformation and is hydroxylated at the 2'-position of the ribose. The single-stranded nature lets mRNA fold into complex secondary and tertiary structures, completely unknown from double-stranded DNA and RNA molecules, respectively. Finally, its length of a few hun- dred to several thousand nucleotides distinguishes mRNA from other single-stranded RNAs like antisense RNA or aptamers. First insight into the uptake mechanism of naked mRNA was gained by a mouse study investigating intradermal administra- tion by injection.8 Local entry into cells of the dermis which were ©2012 Landes Bioscience. Do not distribute not exclusively professional antigen presenting cells (pAPCs) turned out to be saturable, improvable by calcium and associ- ated with the movement of vesicles. More elaborate work in vitro revealed that uptake of naked mRNA is a widespread phenom- enon among primary cells and cell lines of diverse types.74 These efforts confirmed saturability of uptake and demonstrated that it is also temperature and dose dependent. Most of the mRNA appeared to enter cells via caveolae/lipid rafts,74 most likely medi- ated by (a) scavenger-receptor(s) which are known to concentrate in caveolae and to preferentially recognize and facilitate internal- ization of negatively charged macromolecules.75–78 To a minor degree, macropinocytosis also appeared to be involved in mRNA uptake of different primary cells and cell lines.74 By contrast, macropinocytosis apparently predominates mRNA uptake by dendritic cells upon intranodal injection.79 The picture becomes even more complicated when looking at formulated mRNA vaccines. For instance, a recently developed two-component vaccine consisting of naked and protamine- complexed mRNA reveals different routes and kinetics of uptake Figure 1. Protein expression in vivo is strongly prolonged using CureVac’s proprietary mRNA technology and lasts for many days. Firefly for the two components, albeit both are taken up via an endo- luciferase-encoding mRNA, optimized for translation and stability, was somal pathway.9,80 injected intradermally in a BALB/c mouse (4 injection sites). At various mRNA uptake and expression in vivo is quite efficient (much time points after mRNA injection, luciferase expression was visualized more efficient than spontaneous uptake by cells in vitro) and in the living animal by optical imaging. (A) Visualization of luciferase comparable even with cells transfected in vitro under optimal expression at selected time points, showing maximal protein levels 24 to 48 h after mRNA injection. (B) Time course of luciferase expression conditions.8,61 In part, hydrodynamic pressure may contribute to until 9 d after mRNA injection. Background signal was set to 1. target cell transfection in case of local injections81 as it does upon intravenous administration.82 However, the correlation between pressure and transfection efficiency/protein expression may not cells upon simple injection, i.e., without any additional help from be linear but show an optimum.83 Anyway, a large amount of special delivery systems, was demonstrated.61 Later on, numer- the mRNA appears to stay trapped in endosomal vesicles. Hence, ous studies confirmed that locally administered naked mRNA is mRNA vaccines may profit strongly from approaches increasing taken up by cells in target tissues.8,62–65 The mechanism by which the fraction of mRNA that reaches the cytosol. naked mRNA enters cells remained unclear initially. However, elucidating and understanding the uptake route is important to Formulation of mRNA facilitate the development of more efficient mRNA-vaccines. A plethora of studies investigated the cellular entry of mRNA is threatened by rapid degradation by ubiquitous extra- nucleic acids. Most of them looked into the uptake routes of cellular ribonucleases before being taken up by cells.84 Thus, pDNA, DNA oligonucleotides, siRNA or long dsRNA and a the efficacy of mRNA vaccines may benefit significantly from complex picture emerged. The molecules entered cells by dif- complexing agents which protect RNA from degradation. fusion controlled mechanisms or diverse endocytic pathways, Complexation may also enhance uptake by cells and/or improve often strongly dependent on the respective cell type or species delivery to the translation machinery in the cytoplasm. To this and frequently showed a vesicular localization, i.e., an entrap- end, mRNA is often complexed with either lipids or polymers. ment in endocytic or lysosomal compartments.66–73 However, Importantly, not all complexing agents that promote transfec- mRNA differs from these types of molecules due to its unique tion of DNA are suitable for complexation of mRNA. Different 1322 RNA Biology Volume 9 Issue 11 large polycations, all proven DNA transfection reagents, were from brome mosaic virus (BMV) and poliovirus cDNA were shown to strongly inhibit translation of mRNA in cell-free shown to be infectious, an unequivocal indication of protein translation systems as well as inside cells. Only much smaller expression from those RNAs.99,100 However, at that time viable polycations allowed for efficient translation. Likely, mRNA techniques allowing use of mRNA as a general tool for protein is not released in the cytosol if bound to large polycations.85 expression were still missing. This changed with the adaptation Interestingly, DNA may be released in the cytosol from large of efficient transfection methods such as electroporation and polycations by endogenous RNA.86 cationic lipofection for the delivery of RNA.2,101 Further devel- In line with the general conception that mRNA should be pro- opments and insights into mRNA biology enabled significant tected and uptake enhanced, the first report demonstrating the overexpression of proteins after delivery.102 Finally, the in vitro induction of an immune response upon direct injection of mRNA use of mRNA culminated in the establishment of cell reprogram- in vivo used mRNA encapsulated in liposomes.87 Common is the ming protocols that may be of some medical relevance in the use of cationic lipids, for instance used for the intradermal and future.49,103 ©2012 Landes Bioscience. Do not distribute intravenous injection of antigen-encoding mRNA.88 However, Whereas all these examples cover mRNA-mediated protein complexation of mRNA with protamine, a small arginine-rich expression exclusively taking place in vitro, meanwhile, cell based nuclear protein which stabilizes DNA during spermatogenesis, approaches of mRNA-mediated protein expression have expanded was shown to also efficiently stabilize mRNA against degradation into in vivo settings. On the one hand, mRNA injection into fer- by serum components.63 tilized oocytes or early embryos became a well-established tool In addition, complexing agents rationally designed to further in developmental biology.104 On the other hand, loading of den- improve delivery of nucleic acids to the cytosol have been used dritic cells with antigen-encoding mRNA originally described by for formulation of mRNA vaccines. Hemagglutinating virus of Boczkowski et al.105 became a widely used approach in immunol- Japan (HVJ)-liposomes have been reported to deliver their cargo ogy and was investigated in several clinical trials in humans (see directly into the cytoplasm of host cells in vivo by means of a section mRNA-based vaccines). virus-cell fusion mechanism. Such liposomes were used to inject Since these semi-in vivo applications introducing the mRNA mRNA (replicating in this case) encoding melanoma antigen ex vivo are laborious and technically very demanding, scientists gp100 into the spleen of mice.89 Alternatively, vectors improving were interested in direct in vivo application early on. First efforts cytosolic nucleic acid delivery by means of permeation of endo- demonstrated that local injection of naked mRNA can lead to somal membranes due to their high histidine content have been expression of different proteins in mouse muscle tissue.61,62 In an used to formulate antigen-encoding mRNA.90 attempt to improve mRNA delivery, a particle-mediated admin- Complexing agents may have to be tailored to the specific istration via gene gun was developed and demonstrated to give route of delivery. Due to the abundance of professional anti- rise to protein expression in liver and epidermis.106 Later, success- gen presenting cells in the skin,91 this organ may be particularly ful protein expression upon intradermal injection in mice was suitable for vaccination. However, delivery of a DNA vaccine proven.63 Using this administration route, it was shown that (per- into mouse skin by tattooing failed when formulated into cat- haps various) MHC class II-negative non-pAPCs take up and ionic nanoparticles but was successful upon PEGylation of the express mRNA.8 Together, these findings suggest that mRNA nanoparticles to shield their surface charge.92 Likely, adsorption can be taken up and expressed by different cell types in vivo, of the cationic nanoparticles to the negatively charged extracellu- which is consistent with in vitro data.74 lar matrix in the skin prevented their uptake by cells. In addition, These results conclusively show that mRNA-mediated protein the use of complexing agents in vivo is often hampered by tox- expression in vivo is generally possible. In addition, they dem- icity, particularly for high molecular weight compounds.93 Still, onstrate that expression is sufficient to raise detectable immune progress in the drug delivery field is steady,94,95 including innova- responses. However, raising an effective immune response and, tive approaches to targeting of drugs to particular cell types.96 even more, achieving a therapeutic effect by mRNA-mediated Looking ahead, improved delivery is certain to contribute to protein supply may be more demanding in terms of the required increased efficacy of mRNA vaccines. level of protein expression. Using our proprietary mRNA-technol- ogy, we could demonstrate that a single intramuscular injection mRNA-Mediated Protein Expression of erythropoietin (Epo)-encoding mRNA led to a biologically relevant increase of reticulocytes in mice (Fig. 2). Therapeutic As an intermediate carrier of genetic information, endogenous effects using Epo-mRNA were confirmed by two independent mRNA is used as template for protein expression. Hence, like studies.47,65 The potency of mRNA-mediated protein expression DNA, mRNA is at least in principle an attractive means to force was further underlined by an analysis of protein complementa- cells to produce proteins of interest by introducing exogenous tion in a surfactant protein B-deficient mouse model.65 However, nucleic acid molecules. For mRNA, this concept was first applied in contrast to our work, these studies deployed mRNA harboring in the early 1970s. Microinjection of preparations of rabbit hemo- modified nucleotides to increase protein expression. While such globin mRNA and encephalomyocarditis virus RNA, respec- modifications can enhance translation of the mRNA107,108 and tively, into oocytes from Xenopus laevis provided clear evidence may be beneficial for protein replacement therapies, they interfere that these molecules gave rise to the expression of RNA-encoded with the design of mRNA-vaccines with self-adjuvanticity, an proteins.97,98 More than ten years later, in vitro transcribed RNA important feature required for a potent vaccine (see next section). www.landesbioscience.com RNA Biology 1323  arms of the immune system.117,118 Single- and double-stranded RNAs are recognized by toll-like receptors (TLR) 7/8 and 3, respectively, in the endosome.119-121 However, TLR3 is not only activated by double-stranded RNA, an intermediary for many viruses, but also by mRNA either released from cells or produced by in vitro transcription.122 Protamine-complexation of RNA appears to preserve its interaction with TLRs as indicated by the stimulation of several blood cell types.114 For mRNA vaccines it was demonstrated that activation of TLR7 and potentially TLR3 is critical for priming immune responses.9,45 Notably, optimal sequence motifs for receptor binding could be identified for single- stranded RNA.123-126 ©2012 Landes Bioscience. Do not distribute Other pattern recognition receptors may be important for the functionality of an RNA-based adjuvant (and vaccine) as well. The cytosolic helicase RIG-I recognizes uncapped RNA mole- cules harboring a 5'-triphosphate moiety.127–129 Together with the homologous proteins MDA5 and LGP2, RIG-I forms a receptor family whose members can all bind double-stranded RNA,130–132 Figure 2. A biologically relevant increase of reticulocytes is induced in but do have additional recognition patterns. For example, MDA5 mice using CureVac’s proprietary mRNA technology. A single intra- is involved in the discrimination of RNAs based on the ribose muscular injection in BALB/c mice of erythropoietin (Epo)-encoding mRNA, optimized for translation and stability, causes the expression of 2'-O-methylation status of the cap structure.133 For the sake functional Epo. Reticulocyte levels are raised comparably by mRNA and of completeness, the cytoplasmic RNA sensors PKR and 2'-5'- recombinant protein injected intramuscularly. oligoadenylate synthetase, inhibiting translation by phosphoryla- tion of eIF-2α and activating RNase L, respectively, should be Adjuvanticity of mRNA (Vaccines) mentioned here as well.134,135 However, the contribution of these non-TLR RNA-sensors to the immunostimulation by RNA- To be efficient, vaccines should contain a strong adjuvant supply- based adjuvants (and vaccines) is still a matter of debate. Notably, ing a danger signal for the initiation and support of the adaptive as far as investigated, the interaction between endosomal as well immune response in addition to an appropriate antigen.109 The as cytoplasmic receptors and RNA is impaired if the RNA har- immunostimulatory properties of RNA were first discovered by bors distinct nucleotide modifications.108,136,137 As a consequence, the observation of interferon induction upon exposure of cells to such modified RNA impairs the design of self-adjuvanting exogenous RNA extracted from viruses.110 Further support came mRNA-vaccines. from synthetic double-stranded RNA inducing interferon upon intravenous injection into rabbits.111 However, severe side effects mRNA-Based Vaccines of these early RNA adjuvants soon limited their further use.112 The idea of synthetic RNA, mainly produced by in vitro tran- After in vivo administration of mRNA was proven to be fea- scription, as immunostimulant was then re-stimulated particu- sible,61 the concept of using mRNA as a basis for vaccines was larly during the last decade bringing forth a plethora of studies. pursued almost immediately. First success was reported in 1993 In 2004, in vitro transcribed mRNA was shown to serve as when subcutaneous injection of liposome-encapsulated mRNA an adjuvant, if it was stabilized by either complexation or chemi- encoding the nucleoprotein (NP) of influenza virus was dem- cal modification.113 One year later, a strong danger signal was onstrated to elicit NP-specific cytotoxic T cells (CTLs).87 By ascribed to protamine-condensed mRNA leading to TNFα and contrast, naked mRNA failed to raise specific CTLs in this IFNα secretion by various cells.114 A thorough analysis of com- setting. Shortly afterwards, the use of naked mRNA triggered plexes of single-stranded RNA and protamine indicated that the induction of antigen-specific antibodies in response to a het- cell activation in terms of cell selectivity and induced cytokine erologous prime-boost schedule (repeated intramuscular RNA pattern may depend on particle size.115 Recently, research on vaccination, challenge with tumor cells).62 However, none of protamine-complexed RNA culminated in a simplified vaccine the animals was protected against tumor challenge. An antigen- approach, combining naked and protamine-formulated mRNA.9 specific antibody response induced solely with mRNA was dem- The resulting mRNA vaccine consists of two components com- onstrated first using particle-mediated mRNA delivery into plementing each other; while antigen supply is mainly driven by mouse epidermis.106 the naked mRNA, the protamine complexes contribute a strong In 2000, the field of mRNA vaccines was advanced by intro- immunostimulatory signal. Of note, protamine-formulated ducing a new protocol for vaccination allowing the adminis- RNA can also confer adjuvanticity to, e.g., protein vaccines.116 tration of naked mRNA via intradermal injection.63 This basic Among potent adjuvant targets, RNA-sensing receptors are a vaccination design did not require any transfection reagents, particularly diverse class of molecules evolved to detect and coun- special equipment or heterologous boost, yet could elicit a com- teract viral infections by orchestrating the innate and adaptive plete adaptive immune response consisting of antigen-specific 1324 RNA Biology Volume 9 Issue 11 antibodies and T cells with lytic activity against the model anti- mRNAs encoding six different antigens was given intradermally gen β-galactosidase. Directly thereafter, intradermal injection of using an intensified treatment regimen.43,141,142 A further clinical total RNA isolated from the S1509 tumor cell line was shown to trial with patients with renal cell carcinoma stage IV included the induce immunity to a subsequent challenge with the tumor.138 administration of GM-CSF as adjuvant 24 h after vaccination Tumor growth inhibition was also achieved by intradermal as with six antigens,143 an approach that will be discussed in more well as intravenous injection of in vitro transcribed and lipid- detail in the next section. complexed mRNA encoding the model antigen ovalbumin (OVA).88 However, analogous vaccination with mRNA coding Adjuvanted mRNA-Based Vaccines for a model tumor/self-antigen was not sufficient to break toler- ance to this self-antigen in TRAMP mice. A similar approach As discussed, mRNA-vaccines can be designed to possess using histidylated lipopolyplexes for systemic injection revealed self-adjuvanticity contributing to their excellent performance. that MART1 mRNA could not only prevent B16 melanoma Although pDNA vaccines also show native immunogenicity, ©2012 Landes Bioscience. Do not distribute from progression but also from metastasis.90 great efforts were made to improve the immune response by co- In a comparison of different administration routes for the delivery or, more elegantly, co-expression of co-stimulatory mol- delivery of naked mRNA vaccines good immunogenicity against ecules and cytokines. Indeed, inclusion of adjuvant molecules ovalbumin and influenza A virus hemagglutinin could be dem- encoded as DNA could enhance DNA vaccines. Co-injection onstrated after repeated and frequent injections into the lymph of DNA encoding tumor or viral antigens and the cytokine node.45 In order to optimize the vaccine’s potency, the authors granulocyte macrophage colony-stimulating factor (GM-CSF) engineered the antigen by adding an MHC class I molecule traf- improved T and B cell responses.144-147 As a further example, ficking signal for increased antigen presentation.44 Unlike with DNA expressing a recombinant soluble multi-trimeric TNF intranodal injection, the authors could not elicit such immune superfamily ligand enhanced the immune response to an HIV-1 responses upon perinodal, subcutaneous or intradermal injec- Gag DNA vaccine.148 Moreover, DNA encoding the cyto- tions.45 Recently, an alternative, simplified approach was described kine IL-2 was demonstrated to affect the polarization of the leading to successful immunization by intradermal injection.9 immune response, an important vaccine parameter the opti- Combining naked mRNA with protamine-formulated mRNA mum of which differs among indications. While a DNA vac- results in a two-component vaccine capable of inducing strong cine for Helicobacter pylori elicited a strong Th2 response, it was immune responses and tumor protection in prophylactic as well as shifted toward a Th1-biased response by IL-2.149 In addition to therapeutic settings in mice. In this vaccine, the two components directly administering a DNA encoding the adjuvant molecule, fulfill complementary functions: while the naked mRNA confers more indirect approaches are also feasible. For instance, the co- optimal antigen expression, the protamine-complexed mRNA delivery of a CD40-expressing plasmid induced anti-CD40 contributes strong immunostimulatory effects. Notably, for tumor antibodies, part of which were capable of activating CD40 treatment this new type of mRNA vaccine can be combined with which in turn led to an improved immune response to an HBV other, standard, therapies such as chemotherapy, thereby achieving DNA vaccine.150 improved effects as compared with each treatment alone.59 In an analogous fashion, RNA vaccines supplemented with As an alternative to direct injection of mRNA, an immune additional adjuvant molecules have been the object of various response may also be induced by vaccination with pAPCs trans- investigations. In mice, recombinant GM-CSF enhanced the fected with mRNA ex vivo. mRNA-transfected murine den- immune response to the model antigen β-galactosidase and dritic cells (DCs) were shown to elicit anti-tumor immunity in affected polarization of immunity by shifting a Th2 to a Th1 EG.7-OVA and B16 melanoma models.105 Tumor growth was also response.64 In addition, GM-CSF as a supplement of an mRNA significantly reduced upon injection of epidermal cells enriched vaccine was already tested in a clinical trial.143 An improved anti- for Langerhans cells, which belong to the group of pAPCs, that tumor effect of a naked mRNA vaccine in mice was demon- had been transfected with total RNA derived from tumor cells.138 strated for human FLT3 ligand protein fused to human IgG4-Fc Using human DCs, transfection with mRNA encoding CEA or fragment.151 However, in light of getting good protein expres- the E6 antigen of human papillomavirus type 16 induced a pri- sion upon mRNA administration in vitro and in vivo (see sec- mary CTL response in vitro.139 Today, ex vivo mRNA transfec- tion mRNA-mediated protein expression), providing auxiliary tion of pAPCs is the most frequently used approach for mRNA adjuvant molecules via mRNA rather than as protein appears to vaccination in the clinic. For instance, a clinical trial utilizing be a feasible and much more elegant approach. First support for telomerase mRNA-transfected DCs demonstrated the capabil- this idea came from a study investigating the effect of GM-CSF, ity of such applications to stimulate antigen-specific cellular IL-2 and CD80, all encoded by mRNA, on the potency of model immune responses.140 However, the underlying procedure is very mRNA vaccines.88 Among these adjuvants, GM-CSF mRNA time consuming, laborious and needs patient-specific (autolo- improved the induction of CTL activity in a dose-dependent gous) cell preparations. manner. Moreover, a more durable CTL response indicated an Very few clinical studies of the direct administration of enhanced generation of memory cells. We have recently inves- mRNA-based vaccines have been published. The first trial tigated the anti-tumor effect of an accessory adjuvant molecule deployed autologous mRNA libraries derived from melanoma encoded by mRNA. To this end, we included CD40 ligand as a lesions, whereas in a later study a cocktail of protamine-complexed co-stimulatory molecule activating pAPCs152 which may establish www.landesbioscience.com RNA Biology 1325  Concluding Remarks About two decades after the first successful administration of mRNA in vivo, mRNA-based vaccines promise to become a game-changing vaccine technology platform for therapeutic as well as prophylactic applications. Today, the scientific commu- nity is eagerly waiting for first clinical efficacy data. But there is still a wide field for further development/improvements of mRNA-based vaccines. As discussed, the format and uptake of the mRNA are critical parameters for efficient antigen expression which can be influenced by novel RNA designs as well as mRNA formulation and administration. However, any changes to these ©2012 Landes Bioscience. Do not distribute parameters may have major implications on mRNA production and/or its interactions with RNA-sensors and should be care- fully considered early on. For instance, in addition to the previ- ously mentioned nucleotide modifications, novel delivery modes may severely affect vaccine adjuvanticity. While direct delivery into the cytosol would certainly enhance antigen expression, the lack of interaction with endosomal RNA receptors may severely weaken immunostimulation by the vaccine and this issue would likely have to be addressed. The inclusion of accessory mRNA Figure 3. CD40 ligand as an accessory adjuvant molecule encoded molecules into an mRNA vaccine may be an interesting option by mRNA increases the anti-tumor effect of a two-component mRNA for achieving optimal effects in case of particularly challenging vaccine. Mice (n = 8 per group) were challenged subcutaneously with treatments. Moreover, the combination with other anti-tumor syngenic E.G7-OVA tumor cells on day 0. Commencing on day 7, mice therapies will most likely yield the greatest potency. However, were vaccinated intradermally with either OVA-mRNA vaccine alone or in combination with mRNA coding for CD40 ligand according to the this would increase the complexity of the vaccine and/or the indicated schedule. Mice treated with buffer served as the control. The treatment regimen making the development more challenging. combination of CD40 ligand-encoding mRNA together with OVA mRNA Taken together, mRNA offers a promising vaccine vector in the vaccination increases the efficacy of the therapeutic anti-tumor mRNA light of being flexible, effective and safe. Hence, it could become vaccination. a “disruptive technology” not just for cancer immunotherapy, but also for vaccination, either prophylactic or therapeutic, against an autoregulatory circuit improving the immune response by infectious diseases. enhancing antigen presentation. CD40 ligand supplied as mRNA significantly improved tumor growth reduction as com- Disclosure of Potential Conflicts of Interest pared with the non-adjuvanted two-component mRNA vaccine No potential conflicts of interest were disclosed. (Fig. 3). In summary, these data suggest that the combination of mRNA vaccines and additional mRNAs encoding auxiliary Acknowledgments adjuvant molecules is a very promising approach which, however, We thank our colleague Birgit Scheel for helpful comments and remains challenging in terms of arriving at suitable, i.e., effective critical reading of the manuscript. and practical, treatment regimens. 1326 RNA Biology Volume 9 Issue 11  20. 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