Developing Mrna-Vaccine Technologies

genome. Thus, potentially detrimental genomic integration is is integration genomic detrimental potentially genome. Thus, to the virtually omnipresent ribonucleases (RNases), ribonucleases omnipresent virtually to the molecule of the sensitivity Despitethe idea. aprovocative use tic therapeu its making notoriously unstable as considered is RNA may occur in rare cases, rare in occur may molecules RNA single-stranded between recombination while Moreover, protein. encoded of the for expression required directly elements the only it harbors construct, genetic minimal the As RNA Biology9:11,1319–1330;No www.landesbioscience.com advantages. safety strong offers mRNA vaccine, as a specifically of a broadly applicable in vitro transfection technique. transfection vitro in applicable of abroadly thedevelopment in promoted first after 1989 was a therapeutic subunit vaccines like defined composition and safety. and composition defined like vaccines subunit or of killed those with induction Tcell and expression antigen endogenous as such vaccines attenuated of live features nological - immu the together it brings that sense the in ideal being perhaps platform, avaccine as advocated was later, mRNA couple of years RNA; EMCV, encephalomyocarditis virus; GMP, good manufacturing practice; HPLC, high performance liquid chromatography; chromatography; liquid performance high HPLC, practice; GMP, manufacturing good virus; EMCV, encephalomyocarditis RNA; http://dx.doi.org/10.4161/rna.22269 09/17/12 09/17/12; Accepted: Revised: 08/15/12; Submitted: Email: rna [email protected] *Correspondence to: Thomas Schlake; Abbreviations: an mRNA-based vaccine technology. vaccine mRNA-based an developing when be considered thatshould aspects different regarding knowledge current the we outline Here, platform. technology vaccine agame-changing of basis the become thatmay well vector a promising presents together, mRNA development. to Taken respect with flexibility maximum offer also vaccines mRNA process, production the to adjust need the without mRNA from expressed be can any protein Because genome. the with interact not thatdoes information of carrier transient only and is as it aminimal vector safe intrinsically an is mRNA addition, In restriction. haplotype to MHC subject not while immunity humoral and cellular both comprising response immune abalanced inducing of capable are vaccines mRNA expression, protein situ in on Based vaccines. genetic of flexibility unmet the and profile safety outstanding an with properties immunological desirable combine vaccines mRNA ORF, open reading frame; pAPC, professional antigen presenting cell; pDNA, plasmid DNA; PEG, polyethylenglycol; siRNA, siRNA, PEG, polyethylenglycol; DNA; plasmid pDNA, cell; presenting antigen professional pAPC, frame; ORF, reading open IRES, internal ribosome entry site; mRNA, messenger RNA; MHC, major histocompatibility complex; miRNA, microRNA; microRNA; miRNA, complex; major histocompatibility MHC, RNA; messenger mRNA, site; entry ribosome internal IRES, Particularly compared with DNA as a therapeutic or more a therapeutic DNA as with compared Particularly Keywords:

review Developing mRNA-vaccine technologies mRNA, adjuvant, vaccine, mRNA production, mRNA design, mRNA uptake, formulation, protein expression formulation, uptake, mRNA design, production,mRNA mRNA vaccine, adjuvant, mRNA, ARCA, anti-reverse cap analog; cDNA, complementary DNA; CTL, cytotoxic T cell; dsRNA, double-stranded double-stranded dsRNA, Tcell; cytotoxic CTL, DNA; complementary cDNA, analog; cap anti-reverse ARCA, small interfering RNA; tRNA, transfer RNA; TLR, toll-like receptor; UTR, untranslated region untranslated UTR, receptor; toll-like TLR, RNA; transfer tRNA, RNA; interfering small

Introduction Thomas Schlake,* Andreas Thess, Mariola Fotin-Mleczek and Karl-Josef Schlake,*Thomas Kallen Fotin-Mleczek Andreas Mariola Thess, 6,7 mRNA does not interact with the the with not interact does mRNA vember 2012;©2012LandesBioscience CureVac GmbH; Tübingen, Germany Tübingen, GmbH; CureVac 1 3,4 mRNA as as mRNA 2 Only a Only RNA Biology - 5

three forms of pDNA (supercoiled, relaxed circle or linear) in in or linear) circle relaxed of pDNA forms (supercoiled, three DNA and genomic of bacterial traces pDNA contains ished However, of pDNA. unpol manufacture than more effort to require appear thus might of mRNA Manufacture mRNA. the of production in step first of pDNA the is preparation the DNA (pDNA), using a bacteriophage RNA polymerase. RNA abacteriophage using DNA (pDNA), plasmid typically of template, acDNA transcription vitro by in obtained be may mRNA synthetic Functional synthesis. mRNA of mRNA-vaccines. properties immunological and expression antigen/protein as well as lation formu and format mRNA quality, and manufacturing mRNA as topicssuch important touching technology mRNA-vaccine an developing when considered to be has on what light casting the basis of a “disruptive technology.” of a“disruptive basis the to become potential the has that molecules of therapeutic class lack. vaccines self-adjuvanting, to be designed be may vaccines mRNA and receptors recognition to pattern binds mRNA addition, In restriction. haplotype MHC lack vaccines mRNA to peptides, contrast In to DNA. opposed envelope as nuclear the do not to cross need they that fact the fromprofit therapeutics mRNA-based of efficacy, terms In platforms. vaccine other with compared cost reducing and time saving adjustment, any without process production established same the using manufactured be can products diverse fected, unaf largely characteristics physico-chemical its leaving ecule, mol RNA of the sequence the alter protein just encoded of the changes Since therapies. protein replacement as well as cancer and infections as diverse as diseases fighting vaccines therapeutic and development of the prophylactic principle enabling in mRNA, by expressed and encoded be protein can Any application. and to production respect with flexibility by agreat characterized is of information. carrier decaying within a few days afew within decaying metabolically as well as non-replicative being mRNA with tion combina in of integration genomic lack this Finally, excluded.

All in all, mRNA presents a promising, even if challenging, challenging, if even apromising, presents mRNA all, in All mRNA as the technological basis of therapeutics and vaccines vaccines and of therapeutics basis technological the as mRNA 9 a property which peptide- and protein-based protein-based and peptide- which aproperty mRNA Production 8 makes mRNA a merely transient transient amerely mRNA makes 10 In the following we are we are following the In 11 Hence, Hence, REVIEW 1319 - - - - -

©2012 Landes Bioscience. Do not distribute 1320 enzyme that leaves a3'-overhang leaves that enzyme an with template DNA linearized from arise transcripts nated desig than Longer elongation. during termination premature from arise transcripts designated than Shorter simple means: by transcript correct the from separated be cannot that species steps. extraction of precipitation and combination by a sample the removed from be may contaminants These tein. terms of protein expression in vivo. in of protein expression terms in several-fold molecules of mRNA activity the increased mRNA for process production GMP a within purification matographic riophage polymerases. of bacte activity polymerase RNA RNA-dependent due to the produced also are transcripts Undesirable template DNA. ized scribed bacteriophagepolymerase, using RNA pDNA tran is linearized if hand, other onnot the aconcern, of pDNA are heterogeneity the DNA and of bacterial Remains demanding. is for avaccine, required as pDNA, invariant and of pure reproducible the preparation Hence, proportions. variable 5'-5' triphosphate, 5'-5' 5'-end to joined the residue a i.e., a7-methyl-guanosine via a“cap,” mRNA: eukaryotic of mature function for the tial essen elements two by minimum the at flanked (ORF) frame below). (see steps processing further removed during is a pure single mRNA product. mRNA single pure a yielding transcripts, longer and shorter removed both to size ing transcripts. contaminating to removesuch further purified to be have will mRNA stance, for luciferase or erythropoietin were purified by HPLC. by were purified or erythropoietin for luciferase coding transcripts when observed also was of mRNA cation expected simply based on the removal of incorrect transcript. transcript. of incorrect removal on the simply based expected be would than much higher was protein expression in increase than GTP, yielding capped mRNA. capped GTP, yielding than rather analog cap the with initiates of GTP, excess transcription in following) may be included in the reaction. the in included be may following) scripts from abortive cycling during initiation, during cycling abortive from scripts tran abortive short oligodeoxynucleotides, nucleotides, various including mixture acomplex within transcript mRNA desired by DNase. digested DNA is bacterial contaminating as well pDNA template as the transcription, Following template. it pDNA not if by is provided the transcription post added be also may tail Apoly(A) transcription. post enzymatically added be cleotide m cleotide dinu the like To analog acap end, by transcription. this mRNA capped to It obtain possible is triphosphates. nucleoside and SP6) (T7, or T3 polymerase RNA recombinant containing a mixture template). by the not is encoded cap (the transcription of termination defined to ensure plasmid of the for linearization site restriction aunique and into poly(A) transcribed sequence promoter, apoly[d(A/T)] ORF, optionally bacteriophage an a atleast contains transcription a pDNA vitro template for in However, the sample includes additional contaminating RNA RNA contaminating additional However, includes sample the Synthetic mRNA contains a protein-encoding open reading reading open aprotein-encoding contains mRNA Synthetic A single chromatographic step that separates mRNA accord mRNA separates that step chromatographic A single Increased protein expression as a result of stringent purifi of stringent aresult as protein expression Increased mRNA purification. mRNA The linearized pDNA template is transcribed into mRNA in in into mRNA pDNA transcribed template is linearized The

7 G(5')-ppp-(5')G (called “regular cap analog” in the the in analog” cap “regular (called G(5')-ppp-(5')G 12 and a poly(A) tail at the 3'-end. atthe tail apoly(A) and 18 Accordingly, to be used as a drug sub adrug as used to be Accordingly, At this point, the sample contains the the contains sample the point, this At 19 Implementation of such achro Implementation of such 17 8 or from traces of nonlinear traces or from 15 Alternatively, the cap may may cap the Alternatively, 14 If the cap analog is is analog cap the If 5 because all DNA all because 16 as well as pro as well as 13 Accordingly, Accordingly, 20 The The RNA Biology ------

tation so that the m the that so tation orien reverse the in incorporated often is analog cap regular the that found However, been it reaction. has the in analog acap ing prolonged. and increased is protein expression as rise will vaccine mRNA an of efficacy the that assumed it widely is as vaccines, mRNA ter bet yield also likely will identified thus formats Improved mRNA cell. the inside molecules mRNA of synthetic stability and tion improvein ordertransla elements to mRNA beneficial identify such destabilizing signals. and miRNA binding sites binding miRNA and by exonucleases. predominantly catalyzed is decay where cytosol, the in mRNA to stabilize required also are tail poly(A) and 5'- Cap Positioned very atthe 3'-end and of mRNA, third of mRNA molecules are not methylated at their cap. attheir not methylated are molecules of mRNA third about one As a result, nucleotide. transcribed first the instead is also has a longer half-life in cultured cells. cultured in a half-life longer has also ARCA with vitro in transcribed mRNA that shown been it has specific cis-acting destabilizing sequences like elements like AU-rich sequences destabilizing cis-acting specific mRNA lacking methylation of the cap base is not is translated. base cap of the methylation lacking mRNA may also impair translation or mRNA stability. or mRNA translation impair also may the ORF. the to flank (UTRs) regions 5' 3' untranslated and requires mRNA translation. for efficient required are they because elements essential are tail poly(A) and Cap mRNA. blueprint of eukaryotic the following designed general in is for therapy mRNA Synthetic expression. protein reducing thereby sensors, immune innate activates that RNA e.g., double-stranded, ing, contaminat of the removal due to also was purification HPLC after protein expression increased that demonstrated authors The 2'-O-methylation of the first transcribed nucleotide, resulting in resulting nucleotide, transcribed first of the 2'-O-methylation by enzymatic increased further be can mRNA capped cally complex. capping virus vaccinia the using capped subsequently and analog cap without transcribed be may mRNA pared with mRNA capped by regular cap analog. cap by regular capped mRNA with pared com lysate rabbitin reticulocyte doubled efficiency more than at translated was mRNA ARCA-capped guanosine. methylated non- of atthe anucleotide addition allows only guanosine ated base-methyl of the 3'-O-methylation ARCA, common most the In (ARCAs). analogs cap of “anti-reverse” use the with obtained scale. industrial at an production,particularly complicate may step enzymatic However, additional molecule. an RNA an 5'-triphosphate to the cap of anatural adds activity yltransferase with triphosphatase, guanylyltransferase and (guanine-7-)meth they may also be found in ORFs. in found be also may they and prolong protein expression in cultured cells. cultured in prolongand protein expression increase to both reported been has mRNA study, ARCA-capped

Cap. to made been have efforts considerations, these Following However, to further increase both translation and stability, stability, and translation both increase However, to further Protein expression from in vitro transcribed, enzymati transcribed, vitro in from Protein expression orientation, by reverse cap orderIn to avoid unmethylated Alternatively, a cap exclusively in the correct orientation is is orientation correct the in exclusively acap Alternatively, mRNA may be capped during transcription by includ transcription during capped be may mRNA 24-27 UTRs have to be carefully chosen because they they because chosen carefully to be have UTRs 7 G nucleotide does not constitute the cap but cap the not constitute does G nucleotide mRNA Design 30,31 mostly reside in UTRs, although although UTRs, in reside mostly 32 Care must be taken to avoid taken be must Care 36 In an independent independent an In 37 28 35 V 22,23 In particular, particular, In 34 olume 9Issue 11 This complex complex This In addition, addition, In 33 Such Such 2,15,21 34 29 ------

©2012 Landes Bioscience. Do not distribute www.landesbioscience.com globin 3'-UTR, known to stabilize mRNA, to stabilize known 3'-UTR, αglobin the and lation tails on expression. protein tails poly(A) longer of even effect the by investigating extended ther the poly(A) tail from 54 to 98 residues. 54 from tail poly(A) the by lengthening slightly increased still cells into cultured fected different cell types cell different in erythropoietin expressing mRNA in included been has and anomas. - mel metastatic against vaccines as for use cRNA tumor-derived amplified from of alibrary construction the in used been has ARCA. with compared cells cultured in half-life elongated and efficiency translation increased further both with mRNA yielded ARCAs Phosphorothioate-modified the context of genetic vaccination. of genetic context the in valuable to be predicted was and cells mammalian in mRNA of translation protein 70 shock enhanced heat 5'-UTR of human ARCA co-transcriptionally. ARCA with capped mRNA from to that comparable protein expression ing poly(A) tail length. tail poly(A) ing increas further with but declined residues, up to 60 only tail poly(A) of the length increasing with increased transfection post 16 of protein levels UMR-106 h cells, upon transfection trast, By con expression. peak in increase moderate additional to an led polyadenylation by enzymatic tail poly(A) of the extension to 150 64 from Further extended residues. was tail poly(A) the doubled when was into cells, of mRNA electroporation after day transcribed mRNA including RNA for immune therapy. for immune RNA including mRNA transcribed to 68 residues. to 68 up tail poly(A) of the length increasing with increased formation polysome of efficiency the noted that it was initiation, translation activity. biological different of their because synthesis after separated be must that reomers diaste two of mixture as a are obtained ARCAs ate-modified means to achieve expression of therapeutic proteins. For instance, For proteins. instance, of therapeutic expression to achieve means complementary and/or alternative an be can mRNA transcribed cells mammalian in mRNA transcribed vitro of in translation enhances virus 5'-UTR etch The of tobacco of mRNA. value tic - therapeu of the for investigations used mRNAs transcribed vitro Xenopus. of gene the βglobin those of 5'- specifically 3'-UTRs, tained and boranophosphate). and phosphoroselenoate phosphorothioate, (e.g., oxygen or anon-bridging imidodiphosphate] and nate [e.g., (methylenebis)phospho oxygen for abridging substituted either Modifications of ribosomes. recruitment the involved in 4E factor initiation of eukaryotic binding increase and mRNA corresponding of the decapping order to in inhibit linkage strongly dependent on the bacterial strain. bacterial dependent on the strongly and demanding is sequences of long poly[d(A/T)] maintenance line. cell 3T3fibroblast NIH mouse the in mRNA on heterologous efficiency translational much greater to impart demonstrated Poly(A) tail. Inclusion of an internal ribosomal entry site (IRES) in in vitro vitro in in site (IRES) entry ribosomal internal ofInclusion an in in included been also have non-globingenes from UTRs UTRs. triphosphate the within modified further been have ARCAs 2 globin 5'-UTR, improving trans 5'-UTR, improving βglobin of the Acombination 43 Globin UTRs are still in widespread use in in vitro vitro in in use widespread in still are Globin UTRs Already early on, in vitro transcribed mRNA con mRNA transcribed on, vitro in early Already 11 globin 5'- βglobin were 3'-UTRs and Xenopus the Both 15 Translation of in vitro transcribed mRNA trans mRNA transcribed vitro of Translation in When the poly(A) tail was unveiled to enhance to enhance unveiled was tail poly(A) the When 20 and mice. and 42

In practical terms, it is noteworthy that that it noteworthy is terms, practical In 38 38 The peak protein level, reached one reached protein level, peak The 47 Furthermore, a structure of the of the astructure Furthermore, 48 40 However, phosphorothio However, 41 42 This study was fur was study This 38,44,45 RNA Biology 46 27 39 ------

tion or mRNA stability. ortion mRNA transla increase either that elements for sequence transcriptomes cells. oftion melanoma injec upon intravenous metastasis from mice protected mRNA cap-less IRES-containing, such with transfected cells dendritic and a phosphorothioate modified anti-reverse cap. anti-reverse modified a phosphorothioate and of 120 residues tail poly(A) long transcribed a rather 3'-UTRs, type. cell dependent on the strongly was combination or their alone by element either afforded protein level in rise of the magnitude The types. cell tested all in seen was ment protein expression in improve substantial avery tail, poly(A) A64 ashorter with ing end and analog cap regular with capped mRNA with Compared protein expression from mRNA lacking acap. lacking mRNA from protein expression tion. transfec vivo in of successful probability improve may the which envelope but nuclear not membrane, the plasma the to cross have only vaccines RNA toHowever, DNA vaccines, opposed as cells. of target cytosol the to reach has mRNA-vaccine an response, immune antigen-specific an elicit and To translated be ( more days for many lasts and injection mRNA 24hafter to 48 protein levels maximum gives prolonged translation strongly injection, mRNA optimized. expression. gene and levels tRNA with not correlate does bias usage codon However,in species. humans many in translation of efficiency cells. to pluripotentstem fibroblasts to reprogram used factors scription tran for four coding mRNAs in included was IRES EMCV the sequence origin. lian) - mamma (or even of human already is ORF the if particularly humans, in translation improve mRNA to (generally) expected expression by several orders of magnitude. by several expression protein total increasing of expression, duration and level the both we were able to improve molecule, of the parts noncoding and injection. mRNA after up to 72 h demonstrated was at 8h and peaked expression tein pro node, lymph into the of Upon mRNA injection cells. dritic - den transfected in prolonged protein expression and increased in HeLa) as well as immature and mature human dendritic cells. dendritic human mature and immature as well as HeLa) and HepG2 (JawsII, lines cell immortalized in tested was mRNA luciferase-encoding Such of 100 residues. tail poly(A) scribed joined. been have elements mRNA beneficial different platforms, mRNA. the in present be should codon, start correct the

Completely novel UTRs may be provided by screening whole by screening provided be may novelCompletely UTRs globin βglobin consecutive two combined coworkers and Sahin ORF. Obviously, the start codon should be part of a Kozak of aKozak part be should codon Obviously, start the Using our proprietary mRNA technology modifying coding coding modifying technology mRNA proprietary our Using Capping with ARCA has been combined with a long tran with combined been has ARCA with Capping design. Combinatorial 60 49 As early as 1990, the uptake of mRNA by mouse muscle muscle by mouse of mRNA 1990, as uptake the early As The EMCV IRES has even been used successfully to direct to direct successfully used been even has IRES EMCV The Codon usage is also considered as a factor affecting the the affecting a factor as considered also is usage Codon 54 and the sequence surrounding the stop codon may be be may stop codon the surrounding sequence the and 55 52,53 Fig. 1 Fig. In addition, no upstream start codons, preceding preceding codons, start no upstream addition, In In conclusion, codon optimization cannot be be cannot optimization conclusion, codon In ). mRNA Uptake 51 In order to obtain effective vaccine vaccine effective order In to obtain 59 Upon intradermal Upon intradermal 50 Vaccination with Vaccination with 58 This resulted resulted This 1321 56 57 ------

©2012 Landes Bioscience. Do not distribute 1322 special delivery systems, was demonstrated. was systems, delivery special help from additional uponi.e., simple any injection, without cells mRNA differs from these types of molecules due to its unique due unique to its of molecules types these from differs mRNA ment in endocytic or lysosomal compartments. or lysosomal ment endocytic in entrap i.e., an localization, avesicular showed frequently and or species type cell respective dependent on the strongly often pathways, endocytic or diverse mechanisms controlled fusion by dif entered cells molecules The emerged. picture complex a and or long dsRNA siRNA DNA oligonucleotides, pDNA, of routes uptake into the looked of Most them nucleic acids. mRNA-vaccines. development of the more efficient facilitate to route important is uptake the understanding and elucidating However, initially. unclear remained enters cells mRNA naked taken up by cells in target tissues. target in up by cells taken is mRNA naked administered locally that confirmed studies ous until 9 d after mRNA injection. Background signal was set to1. set was signal Background injection. mRNA 9dafter until to 48 h after mRNA injection. ( injection. mRNA hafter to 48 24 levels protein maximal showing points, time selected at expression ( in animal by the living imaging. optical visualized was expression luciferase injection, mRNA after points time sites). At various (4 aBALB/c in injection mouse intradermally injected was stability, and translation for optimized mRNA, luciferase-encoding Firefly days. many for lasts and technology mRNA CureVac’s proprietary Figure 1. A plethora of studies investigated the cellular entry of entry cellular the investigated of studies A plethora

Protein expression in vivo is strongly prolonged using using prolonged isstrongly vivo in expression Protein B ) Time course of luciferase expression expression luciferase of course ) Time 8,62–65 ) Visualization of luciferase luciferase of A) Visualization The mechanism by which by which mechanism The 61 Later on, numer Later 66–73 However, However, RNA Biology - - - tion of DNA are suitable for complexation of mRNA. Different Different of mRNA. for complexation suitable oftion DNA are or polymers. lipids either with complexed often is mRNA end, To this cytoplasm. the in machinery translation to the delivery improve and/or by cells uptake enhance also may Complexation degradation. from RNA protect which agents complexing from significantly benefit may vaccines mRNA of efficacy the cellular ribonucleases before being taken up by cells. taken being before ribonucleases cellular by ubiquitous extra by rapiddegradation threatened is mRNA cytosol. the reaches that of mRNA fraction the increasing fromapproaches profit may strongly vaccines mRNA Hence, vesicles. endosomal in trapped to stay appears mRNA the be linear but show an optimum. but show an linear be mRNA uptake by dendritic cells upon intranodal injection. upon intranodal cells by dendritic uptake mRNA pressure and transfection efficiency/protein expression may not expression efficiency/protein transfection and pressure conditions. optimal under vitro in transfected cells with even comparable and vitro) in cells by uptake spontaneous than more efficient ization of negatively charged macromolecules. charged of negatively ization - internal facilitate and recognize to preferentially and caveolae in to concentrate known are which scavenger-receptor(s) by (a) ated appeared to enter cells via caveolae/lipid rafts, caveolae/lipid via to enter cells appeared mRNA of Most the dependent. dose and temperature it also is that anddemonstrated of uptake saturability confirmed efforts intravenous administration. intravenous target cell transfection in case of local injections of local case in transfection cell target somal pathway. somal endo an up via taken are both albeit components, two for the of uptake kinetics and routes different reveals mRNA complexed protamine- and of naked consisting vaccine two-component developed arecently For instance, vaccines. mRNA formulated at looking when more complicated even becomes picture The tion by injection. by tion - administra intradermal investigating study by amouse gained or aptamers. RNA antisense like RNAs single-stranded other from mRNA distinguishes nucleotides thousand to several dred hun of afew length its Finally, respectively. molecules, RNA and DNA double-stranded from unknown completely structures, tertiary and fold into secondary complex mRNA lets nature 2'-position single-stranded The atthe ribose. of the hydroxylated is aC3'-endo and adopts conformation preferentially midine, of deoxythy instead uridine contains mRNA to DNA, contrast In parameters. structural and of physico-chemical combination lines. cell and cells primary of different uptake mRNA involved in enon among primary cells and cell lines of diverse types. of diverse lines cell and cells primary enon among phenom awidespread is mRNA of naked uptake that revealed workvitro in movement More the elaborate of vesicles. with ated associ and improvable by calcium out saturable, to be turned (pAPCs) cells presenting antigen professional not exclusively

Importantly, not all complexing agents that promote transfec that agents complexing not all Importantly, mRNA uptake and expression in vivo is quite efficient (much quite efficient is vivo in expression and uptake mRNA First insight into the uptake mechanism of naked mRNA was was mRNA of naked mechanism uptake into the insight First To a minor degree, macropinocytosis also appeared to be to be appeared also To macropinocytosis degree, a minor 74 By contrast, macropinocytosis apparently predominates predominates apparently macropinocytosis By contrast, 8,61 In part, hydrodynamic pressure may contributeto may pressure hydrodynamic part, In 9,80 8 Local entry into cells of the dermis which were which dermis of the into cells entry Local Formulation ofmRNA 82 However, the correlation between between However, correlation the 83 Anyway, a large amount of amount a large Anyway, 74 75–78 most medi likely 81 as it does upon it does as V olume 9Issue 11 84 74 Thus, Thus, These These 79 ------

©2012 Landes Bioscience. Do not distribute www.landesbioscience.com is not released in the cytosol if bound to large polycations. to large bound if cytosol the in notis released Likely,mRNA translation. for efficient allowed polycations much smaller Only cells. inside as well as systems translation cell-free in of mRNA translation inhibit to strongly shown were reagents, proven DNA transfection all polycations, large proteins. of RNA-encoded expression to the rise gave molecules these that Xenopus from laevis tively, into oocytes respec RNA, virus encephalomyocarditis and mRNA globin of rabbit 1970s. hemo of early preparations the Microinjection in applied first was concept this For mRNA, nucleic molecules. acid exogenous by introducing of interest proteins to produce cells to force means attractive principle an in atleast is mRNA DNA, like Hence, templatefor protein expression. as used is mRNA endogenous information, of genetic carrier intermediate an As vaccines. mRNA of efficacy increased to contributeto certain is improved delivery ahead, Looking RNA. by endogenous polycations large from cytosol the in released be DNA may Interestingly, tive approaches to targeting of drugs to particular cell types. cell to particular of drugs to targeting approaches tive intravenous injection of antigen-encoding mRNA. of antigen-encoding injection intravenous and intradermal for the used for instance lipids, of cationic use by serum components. by serum degradation against mRNA stabilize efficiently to also shown was spermatogenesis, DNA during stabilizes protein which nuclear arginine-rich asmall protamine, with of mRNA complexation progress in the drug delivery field is steady,is field delivery drug the in progress icity, particularly for high molecular weight compounds. weight molecular for high icity, particularly by tox hampered often is vivo in agents of complexing use the addition, In by cells. uptake their prevented skin the in matrix lar extracellu charged negatively to the nanoparticles cationic of the charge. surface their to shield nanoparticles upon PEGylation of the successful but was ionic nanoparticles into cat formulated when failed by tattooing skin into mouse of aDNA vaccine However, delivery for vaccination. suitable gen presenting cells in the skin, the in cells presenting gen anti of professional route abundance ofto the Due delivery. gp100 spleenof mice. into the antigen melanoma encoding case) this in (replicating mRNA to inject were used liposomes Such mechanism. fusion virus-cell of a by means vivo in of cells host cytoplasm into the directly cargo their to deliver reported been have (HVJ)-liposomes Japan of virus Hemagglutinating vaccines. of mRNA for formulation used been have cytosol to the of nucleic acids improve delivery liposomes. in encapsulated mRNA used vivo in of mRNA injection upon direct response immune of an induction the demonstrating report first the enhanced, uptake and tected used to formulate antigen-encoding mRNA. antigen-encoding to formulate used content been have histidine high due to their membranes somal of endo of permeation by means nucleic delivery acid cytosolic Complexing agents may have to be tailored to the specific specific to the tailored to be have may agents Complexing to further designed rationally agents complexing addition, In pro be should mRNA that conception general the with line In 97,98 mRNA-Mediated ProteinExpression More than ten years later, in vitro transcribed RNA RNA transcribed later, vitro in years ten More than 63

89 91 Alternatively, vectors improving improving vectors Alternatively, this organ may be particularly particularly be may organ this 86 provided clear evidence evidence clear provided 94,95 92 90 Likely, adsorption Likely, adsorption including innova 87 Common is the the is Common 88 However, However, 93 Still, Still, RNA Biology 96 85 ------

proven. was mice in injection upon intradermal protein expression ful overexpression of proteins after delivery. of after overexpression proteins significant enabled biology into mRNA insights opments and important feature required for a potent vaccine (see next section). next (see for apotent vaccine required feature important self-adjuvanticity, an with of mRNA-vaccines design the with interfere they therapies, for replacement protein beneficial be may which is consistent with in vitro data. vitro in with consistent is which vivo, in types cell by different expressed up and taken be can express mRNA. express up and take non-pAPCs II-negative class MHC various) haps expression of different proteins in mouse muscle tissue. muscle mouse in proteins of different expression to lead can mRNA of naked injection local that demonstrated efforts on. First early application vivo in direct in were interested scientists demanding, very technically and laborious are vivo ex vaccines). mRNA-based section (see humans in trials clinical several in investigated was and ogy Boczkowski et al. et Boczkowski by described originally mRNA antigen-encoding with cells dritic in developmental biology. developmental in tool awell-established became embryos or early oocytes tilized into fer injection mRNA one On hand, the settings. vivo into in expanded have protein expression of mRNA-mediated approaches based cell meanwhile, vitro, in place taking exclusively expression RNAs. those from expression of protein indication unequivocal an infectious, to be shown were cDNA poliovirus and (BMV) virus bromefrom mosaic relevant increase of reticulocytes in mice ( mice in of reticulocytes increase relevant to led abiologically mRNA (Epo)-encoding of erythropoietin injection intramuscular asingle that demonstrate ogy, we could mRNA-technol proprietary our Using of protein expression. level required of the terms in protein supply more be demanding may by mRNA-mediated effect atherapeutic more, achieving even and, response immune effective an However, raising responses. immune detectable raise to sufficient is expression that onstrate dem they addition, In possible. generally is vivo in expression rise to protein expression in liver and epidermis. and liver in to protein expression rise to give demonstrated and developed was gun gene via istration admin aparticle-mediated delivery, to improveattempt mRNA modifications can enhance translation of the mRNA the of translation enhance can modifications such While expression. protein increase to nucleotides modified harboring deployed mRNA studies work, tothese our contrast in future. the in relevance of be some medical may that protocols ming reprogram of cell establishment the in culminated of mRNA use tion in a surfactant protein B-deficient mouse model. mouse protein B-deficient asurfactant in tion of protein complementa analysis by an underlined further was cationic lipofection for the delivery of RNA. delivery for the lipofection cationic and electroporation as such methods transfection of efficient adaptation the with changed This missing. were still expression tool for protein ageneral as of mRNA use allowing techniques studies. independent two by were confirmed Epo-mRNA using effects

Since these semi-in vivo applications introducing the mRNA mRNA the introducing applications vivo semi-in these Since Whereas all these examples cover mRNA-mediated protein mRNA-mediated cover examples these all Whereas These results conclusively show that mRNA-mediated protein mRNA-mediated show that conclusively results These 49,103 63 47,65 Using this administration route, it was shown that (per that shown route, it was administration this Using The potency of mRNA-mediated protein expression protein expression of mRNA-mediated potency The 8 Together, these findings suggest that mRNA mRNA that suggest Together, findings these 105 became a widely used approach in immunol in approach used awidely became 104 On the other hand, loading of den loading hand, other On the 99,100 However, at that time viable viable However, time atthat 74 102 Finally, the in vitro vitro in the Finally, Fig. 2 Fig. 2,101 106 Further devel Further Later, success ). Therapeutic Therapeutic ). 65 However, However, 107,108 61,62 In an an In and and 1323 ------

©2012 Landes Bioscience. Do not distribute 1324 RNA can also confer adjuvanticity to, e.g., protein vaccines. adjuvanticity confer also can RNA note, protamine-formulated Of signal. immunostimulatory contributeastrong complexes protamine the mRNA, naked the by driven supply mainly is antigen while other; each plementing components com of two consists vaccine mRNA resulting The cal modification. cal or chemi bycomplexation either stabilized it if was adjuvant, an of studies. aplethora forth bringing decade last the during larly particu re-stimulated then was immunostimulant scription, as tran vitro by in produced mainly RNA, of synthetic idea The approach, combining naked and protamine-formulated mRNA. protamine-formulated and naked combining approach, vaccine asimplified in culminated RNA protamine-complexed pattern may depend on particle size. on depend particle may pattern cytokine induced and selectivity of cell terms in activation cell that indicated protamine and RNA of single-stranded plexes IFN to TNF leading mRNA to protamine-condensed ascribed intravenous injection into rabbits. into injection intravenous upon interferon inducing RNA double-stranded synthetic from teract viral infections by orchestrating the innate and adaptive adaptive and innate the by orchestrating infections viral teract - coun and to detect evolved of molecules class diverse particularly of these early RNA adjuvants soon limited their further use. further their limited soon adjuvants RNA early of these exogenous RNA extracted from viruses. from extracted RNA exogenous to of cells upon exposure induction of interferon observation the by discovered were first of RNA properties immunostimulatory immune response in addition to an appropriate antigen. to an addition in response immune adaptive of the support and initiation for the signal adanger ing adjuvantsupply strong a contain should Tovaccines efficient, be recombinant protein injected intramuscularly. proteinrecombinant injected and mRNA by comparably raised are levels Reticulocyte Epo. functional of expression the causes stability, and translation for optimized mRNA, (Epo)-encoding BALB/c in erythropoietin of mice injection muscular intra- Asingle technology. mRNA CureVac’s using proprietary mice Figure 2. In 2004, in vitro transcribed mRNA was shown to serve as as to serve shown was mRNA transcribed vitro in 2004, In Among potent adjuvant targets, RNA-sensing receptors are a are receptors RNA-sensing potent targets, adjuvant Among

secretion by various cells. by various α secretion A biologically relevant increase of reticulocytes is induced in in isinduced reticulocytes of increase relevant Abiologically Adjuvanticity ofmRNA (Vaccines) 113 One year later, a strong danger signal was was signal later, danger astrong One year 114 111 A thorough analysis of com analysis Athorough However, severe side effects However, side effects severe 115 110 Recently, research on Recently, research Further support came came support Further 109 and α and The The RNA Biology 116 112 ------9

onstrated to elicit NP-specific cytotoxic T cells (CTLs). cells T cytotoxic NP-specific to elicit onstrated dem was virus of influenza nucleoprotein (NP) the encoding mRNA of liposome-encapsulated injection subcutaneous when 1993 in reported was success First immediately. almost pursued respectively, in the endosome. the in respectively, 3, and 7/8 (TLR) receptors by toll-like recognized are RNAs mouse epidermis. mouse into delivery mRNA particle-mediated using first onstrated dem was mRNA with solely induced response antibody specific antigen- An challenge. tumor against protected was animals the is critical for priming immune responses. immune for priming critical is TLR3 potentially and of TLR7 activation that demonstrated was cules harboring a5'-triphosphate moiety. harboring cules mole RNA uncapped recognizes RIG-I helicase cytosolic The well. as vaccine) (and adjuvant RNA-based of an functionality system. immune of the arms tration of naked mRNA via intradermal injection. intradermal via mRNA of naked tration adminis the allowing protocol for vaccination anew ducing bors distinct nucleotide modifications. nucleotide distinct bors har RNA the if impaired is RNA and receptors cytoplasmic as well as endosomal between interaction the investigated, as far as Notably, of debate. a matter still is vaccines) (and adjuvants based by RNA- immunostimulation to the non-TLR RNA-sensors family whose members can all bind double-stranded RNA, bind double-stranded all can members whose family areceptor forms RIG-I MDA5homologous proteins LGP2, and mentioned here as well. mentioned here as oftion eIF-2 by phosphoryla translation inhibiting synthetase, oligoadenylate 2'-5'- and PKR sensors RNA cytoplasmic the of completeness, by in vitro transcription. vitro by in or produced cells from released either by mRNA but also viruses, for many intermediary an RNA, by double-stranded activated plete adaptive immune response consisting of antigen-specific of antigen-specific consisting response immune plete adaptive acom elicit could yet equipment boost, or heterologous special reagents, transfection any not require did design vaccination sible, proven fea to be was of mRNA administration vivo in After mRNA-vaccines. self-adjuvanting of thedesign impairs RNA modified such stimulation of several blood cell types. cell blood of several stimulation by the indicated as TLRs with interaction its to preserve appears stranded RNA. for single- identified be could binding for receptor motifs sequence vaccination, challenge with tumor cells). tumor with challenge vaccination, RNA intramuscular (repeated schedule prime-boost erologous to a het in response antibodies of antigen-specific induction the triggered mRNA of naked use the afterwards, Shortly setting. this in CTLs specific to raise failed mRNA naked contrast, 2'-O-methylation status of the cap structure. cap of the status 2'-O-methylation ribose on the based of RNAs discrimination the involved in is MDA5 For example, patterns. recognition but additional do have

Other pattern recognition receptors may be important for the for the important be may receptors recognition pattern Other In 2000, the field of mRNA vaccines was advanced by intro by advanced was vaccines mRNA field of the 2000, In 61 the concept of using mRNA as a basis for vaccines was was for vaccines abasis as mRNA of using concept the and activating RNase L, respectively, should be be should respectively, L, RNase activating α and 123-126 106 mRNA-Based Vaccines 134,135 122 However, the contribution of these However, of contribution these the Protamine-complexation of RNA of RNA Protamine-complexation 117,118 119-121 Single- and double-stranded double-stranded and Single- However, TLR3 is not is only However, TLR3 108,136,137 114 127–129 For mRNA vaccines it vaccines For mRNA 62 9,45 However, none of none However, Notably, optimal Notably, optimal As a consequence, a consequence, As Together the with 133 V For the sake For sake the olume 9Issue 11 63 This basic basic This 130–132 87 By By ------

©2012 Landes Bioscience. Do not distribute www.landesbioscience.com immune responses. cellular antigen-specific to stimulate applications of such ity capabil the demonstrated DCs mRNA-transfected telomerase utilizing trial aclinical For instance, clinic. the in vaccination for mRNA approach used frequently most the is oftion pAPCs ficking signal for increased antigen presentation. antigen increased for signal ficking traf Imolecule class MHC an by adding antigen the engineered mary CTL response in vitro. in response CTL mary 16 a pri induced type papillomavirus of human antigen E6 the or CEA encoding mRNA with transfection DCs, human Using alone. treatment each with compared as improved effects achieving thereby chemotherapy, as such therapies standard, other, with combined be can vaccine of mRNA type new this treatment Notably, for tumor effects. immunostimulatory strong contributes mRNA protamine-complexed the expression, antigen optimal confers mRNA naked the while functions: complementary fulfill components two the vaccine, this In mice. in settings therapeutic as well as prophylactic in protection tumor and responses immune strong of inducing capable vaccine atwo-component in results mRNA protamine-formulated with mRNA naked Combining (OVA). ovalbumin antigen model the encoding mRNA complexed lipid- and transcribed vitro of in injection intravenous as well as by intradermal achieved also was Tumor inhibition growth leading to successful immunization by intradermal injection. by intradermal immunization to successful leading EG.7-OVA B16 and models. melanoma in immunity anti-tumor to elicit were shown (DCs) cells dritic den murine mRNA-transfected vivo. ex mRNA with fected trans pAPCs with by vaccination induced be also may response node. lymph into the injections frequent and repeated after onstrated dem be could hemagglutinin virus A influenza and ovalbumin against immunogenicity good vaccines mRNA of naked delivery had been transfected with total RNA derived from tumor cells. tumor from derived RNA total with transfected been had that of group pAPCs, to the belong which cells, for Langerhans enriched cells epidermal of injection upon reduced significantly from progression but also from metastasis. from but also progression from not prevent B16 only could mRNA melanoma MART1 that revealed injection for systemic lipopolyplexes histidylated using approach Asimilar mice. TRAMP in self-antigen to this ance toler to break not sufficient was tumor/self-antigen for a model lesions, whereas in a later study a cocktail of protamine-complexed of protamine-complexed acocktail study alater in whereas lesions, melanoma from derived libraries deployed autologous mRNA trial first The published. been have vaccines mRNA-based preparations. cell gous) (autolo patient-specific needs and laborious consuming, time induce immunity to a subsequent challenge with the tumor. the with challenge to asubsequent immunity induce to shown S1509 the was from line isolated cell tumor RNA total gen anti model the against activity lytic with Tcells and antibodies tions. injec or intradermal subcutaneous upon perinodal, responses immune not such elicit could authors the injection, intranodal As an alternative to direct injection of mRNA, an immune immune an of mRNA, injection to direct alternative an As for routes the administration of different acomparison In Very few clinical studies of the direct administration of administration direct of the studies Very clinical few β 45 45 -galactosidase. Directly thereafter, intradermal injection of injection intradermal thereafter, Directly -galactosidase. Recently, an alternative, simplified approach was described described was approach simplified alternative, Recently, an In order to optimize the vaccine’s potency, the authors the order In to optimize 88 However, analogous vaccination with mRNA coding coding mRNA with vaccination However, analogous 140 However, the underlying procedure is very very is procedure However, underlying the

139 Today, transfec mRNA vivo ex 105 Tumor growth was also also Tumor was growth 90 44 Unlike with with Unlike 59 RNA Biology 138 138 ------9

shifted toward a Th1-biased response by IL-2. aTh1-biased response toward shifted for Helicobactercine pylori aDNA vac While indications. among differs ofmum which opti the parameter vaccine important an response, immune of the polarization the to affect demonstrated was IL-2 kine mRNA vaccines. mRNA of model potency on the by mRNA, encoded all CD80, and IL-2 of GM-CSF, effect the investigating astudy from came idea this for support First approach. much more elegant and afeasible be to protein appears as than rather mRNA via molecules adjuvant auxiliary providing protein expression), mRNA-mediated tion sec (see vivo in and vitro in administration sion upon mRNA improved T and B cell responses. Bcell improved Tand (GM-CSF) factor colony-stimulating macrophage granulocyte cytokine the and antigens or viral tumor of DNA encoding Co-injection DNA vaccines. enhance DNA could as encoded of molecules adjuvant inclusion Indeed, cytokines. and ecules mol of co-stimulatory or, more co-expression delivery elegantly, by co- response immune to improve the were made efforts great immunogenicity, show native also pDNA vaccines Although performance. excellent to their contributing self-adjuvanticity to possess designed be can mRNA-vaccines discussed, As section. next the in detail Gag DNA vaccine. Gag HIV-1 to an response immune the enhanced ligand superfamily TNF soluble multi-trimeric a recombinant DNA expressing fragment. IgG4-Fc to human protein fused FLT3 ligand for human strated demon was mice in vaccine mRNA of anaked effect tumor using an intensified treatment regimen. treatment intensified an using intradermally given was antigens different six encoding mRNAs response. to aTh1 aTh2 by shifting of immunity polarization affected β antigen model to the response immune the enhanced GM-CSF recombinant mice, In investigations. of object various the been have molecules adjuvant additional DNA vaccine. HBV to an response improved immune to led an turn in which CD40 of activating were capable of which part antibodies, anti-CD40 induced plasmid of aCD40-expressing delivery co- the For instance, feasible. also are approaches more indirect molecule, adjuvant the aDNA encoding administering directly vaccine was already tested in a clinical trial. aclinical in tested already was vaccine co-stimulatory molecule activating pAPCs activating molecule co-stimulatory a as ligand To CD40 we included end, this by mRNA. encoded molecule adjuvant accessory of an effect anti-tumor the tigated We inves recently have cells. of memory generation enhanced an indicated Moreover, response manner. CTL a more durable adose-dependent in activity of CTL induction improved the with six antigens, six with vaccination 24 hafter adjuvant as of GM-CSF administration the included IV stage carcinoma cell renal with patients with trial

In an analogous fashion, RNA vaccines supplemented with supplemented with vaccines RNA fashion, analogous an In 64 151 In addition, GM-CSF as a supplement of an mRNA asupplement mRNA as of an GM-CSF addition, In Adjuvanted mRNA-BasedVaccines However, in light of getting good protein expres good of However, getting light in 150 88 143 Among these adjuvants, GM-CSF mRNA mRNA GM-CSF adjuvants, these Among an approach that will be discussed in more in discussed be will that approach an 148 Moreover, DNA encoding the cyto the Moreover, DNA encoding elicited a strong Th2 response, it was it was response, Th2 astrong elicited 144-147 43,141,142 As a further example, example, further a As 152 143 which may establish establish may which An improved anti- improved An -galactosidase and -galactosidase A further clinical clinical Afurther 149 In addition to addition In 1325 ------

©2012 Landes Bioscience. Do not distribute 1326 and practical, treatment regimens. treatment practical, and i.e., effective atsuitable, of arriving terms in challenging remains however, which, approach promising avery is molecules adjuvant auxiliary encoding mRNAs additional and vaccines of mRNA ( vaccine mRNA non-adjuvanted two-component the with pared com- as reduction growth improvedtumor significantly mRNA as supplied ligand CD40 presentation. antigen enhancing by response immune the improving circuit autoregulatory an Fig. 3 Fig. vaccination. mRNA anti-tumor therapeutic the of efficacy the increases vaccination OVA with mRNA together mRNA ligand-encoding CD40 of combination The control. the as served buffer with treated Mice schedule. indicated tothe according ligand CD40 for coding mRNA with combination in or alone vaccine OVA-mRNA either with intradermally vaccinated were 7, day on Commencing 0. day mice on cells E.G7-OVA tumor syngenic with subcutaneously challenged were group) (n Mice =8per vaccine. mRNA two-component of a effect anti-tumor the increases mRNA by Figure 3.

). In summary, these data suggest that the combination combination the that suggest data these summary, In ). CD40 ligand as an accessory adjuvant molecule encoded encoded molecule adjuvant accessory an as ligand CD40 RNA Biology infectious diseases. infectious against or therapeutic, prophylactic either for vaccination, also immunotherapy, but not for cancer just technology” a “disruptive become could it Hence, safe. and effective flexible, of being light the in vector vaccine a promising offers Taken together, mRNA development more the challenging. making regimen treatment the and/or vaccine of the complexity the increase would this potency. However, greatest the yield likely most will therapies anti-tumor other Moreover, with combination treatments. the challenging of particularly case in effects optimal for achieving option interesting an be may vaccine mRNA into an molecules mRNA of accessory inclusion The addressed. to be have likely would issue this and vaccine by the immunostimulation weaken severely may receptors RNA endosomal with of interaction lack the expression, antigen enhance certainly would cytosol into the delivery direct adjuvanticity. While vaccine affect severely may modes noveldelivery mentioned modifications, nucleotide ously previ to the addition in on. For instance, early considered fully care be should and RNA-sensors with interactions its and/or production on mRNA major have implications may parameters to these However, changes any administration. and formulation mRNA as as well designs RNA by novel influenced be can which expression antigen for efficient parameters critical are mRNA the of uptake and format the discussed, As vaccines. mRNA-based development/improvements of further for field awide still is there But data. efficacy clinical for first waiting eagerly is nity Today, applications. commu prophylactic - as scientific well the as for therapeutic platform technology vaccine game-changing a to become promise vaccines mRNA-based vivo, in mRNA of administration successful first the after decades two About critical reading of the manuscript. of the reading critical and comments for helpful Scheel Birgit colleague our We thank disclosed. were interest of conflicts No potential

Disclosure of Potential Conflicts of Interest of Conflicts ofPotential Disclosure Concluding Remarks Acknowledgments V olume 9Issue 11 - -

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