a self-assembled non-viral vector as potential platform for mrna-based ... · potential platform...

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2017Vol. 8 No. 3: 119

DOI: 10.21767/2172-0479.100119

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Research Article

Translational BiomedicineISSN 2172-0479

iMedPub Journals

Stefano Persano1,2*

1 InstitutoItalianodiTecnologia(IIT),Genova,16163,Italy

2 DepartimentodiMatematicaeFisica"EnniodeGiorgi",Universitàdelsalento,Lecce,73100,Italy

*Corresponding author: StefanoPersano

stefano.persano@iit.it

InstitutoItalianodiTecnologia(IIT),Genova,16163,Italy.

Citation:PersanoS(2017)ASelf-AssembledNon-ViralvectorasPotentialPlatformformRNA-BasedVaccines.TranslBiomed.Vol.8No.3:119

IntroductionSeveral studies have demonstrated the immunogenicity oftumors, therefore, cancer cells may potentially be recognizedandattackedby immune system, andmelanoma is oneof thebest-definedimmunogenictumor[1-4].

Essential components of the mammalian immune system arethe antigen presenting cells (APCs), a heterogenous group ofimmune cells specialized to process and presenting antigensfor recognition by certain lymphocytes such as T-cells[5,6].Therefore, they represent a vital component of the innateimmunityandplayapivotalroleinthegenerationandregulationof adaptive immunity by cell-cell interaction (e.g. APC-T-cellinteraction)andreleaseofregulatorycytokines(e.g.IL-12,IL-18andIFN-αor-β)[7,8].ClassicalAPCsincludedendriticcells(DCs),macrophages andB-cells, althoughDCs are themostpowerfulAPC of the mammalian immune system [9]. Represent a keylinkbetween innateandadaptive immunity, given their innateimmunity features. Hence, DCs are an optimal target in thedevelopmentofanti-microbialoranti-cancervaccines.

Essentially, it is possible to resume the innate properties ofDCs in the following sentinel and sensor roles in the immunesystem:(i)Capture,processandpresentGantigens(ii)Migrateintolymphoidorgans,wherenaïveT-cellsareretained,and(iii)

Received: July03,2017; Accepted: July24,2017; Published: July30,2017

AbstractHere,weshowauniversalanti-cancervaccine,basedonantigen-mRNA-loadedself-assembledpolyplexnanocarrier.Theestablishmentofantigen-specificT-cells,asconsequenceofthevaccination,performedfollowingasubcutaneousrouteofadministration,wasconfirmedbydetectionofIFN-γ/IL-2producingT-cellsinthespleenofthetreatedmice.Moreover,ahighreleaseofTh1-releatedIgGisotypes(IgG2bandIgG2c)wasobserved,indicatingapredominantlyTh1response.Finally,OVA-mRNA-based vaccine formulation has been employed for the treatmentofmelanoma lungmetastasis of B16-OVA challengedmice, inducing amarkedreductionofmetastaticnodulesupto93%.

Ascertainedthatanypolypeptide-basedantigencanbeencodedasRNA,potentiallyourplatformcanrepresentauniversalstrategysuitableforthedevelopmentofanymRNA-basedvaccine.

Keywords:mRNA;Vaccine;Immunotherapy;Cancervaccines

A Self-Assembled Non-Viral vector as Potential Platform for mRNA-Based Vaccines

Rapiddifferentiationormaturationandproductionof immuneenhancingcytokines,inresponsetoseveralstimulirangingfrominnateimmunityreceptors(e.g.Toll-likereceptors,TLRs)[10,11].Pattern recognition receptors (PRRs) represent the sentinelsof the innate immune system, expressed essentially onto thesurface of dendritic cells and macrophages and they are ableto recognize specific pathogen associated molecular patterns(PAMPs)[12].

ThefamilyofTLRs istheclassofPRRsthat ithasbeenstudiedmostextensivelyandrepresentanimportantgroupofreceptors,as previous studies demonstrated that innate immune systemactivation can be used as vaccine adjuvants to potentiate theadaptive immuneresponseduetheirability toactivatevariousimmune cells simultaneously, through the release of specificcytokines[13,14].

Theuseofnucleicacid-basedvaccines(DNAorRNAencodingforanantigen)isaveryattractivestrategy,ascaninduceefficientlyboth humoral and cellular immune responses, which makestumor escape less likely [15,16]. Additionally, nucleic acid-based vaccine oppositely tomore classic and diffused vaccineformulationcontainingproteinorpeptideantigens,offerseveraladvantages:(i)Unlikepeptide-basedvaccines,theydonotrequirepriorknowledge;(ii)Theyarenotrestrictedbythepatient’sHLAtype; (iii)Finally,nucleicacidscanactasadjuvantbyproviding

2 This article is available in: www.transbiomedicine.com

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costimulatorysignals, forexample,viatoll-likereceptors(TLRs)[16-19].ThenucleicacidvaccinesconsistofthedeliveryofDNAorRNA,encodingforaspecificantigen,intotheAPCs;usingoneofthefollowingstrategies:(i)ex-vivotransfectionofAPCs;or(ii)in vivoAPC-targeting.

Despite the promising features of DNA vaccines, in general,theyhavebeenfoundtoelicitarelativelackimmuneresponsecomparetoRNA-basedvaccines[19-22].Thereasonsforthisarenotcompletelyclear,butpossibleexplanationsincludeinefficientdeliveryofDNAintohumancells,duetothefactthatDNAneedsto cross both cell and nuclear membranes to be transcribed,thelowexpressionofDNA-sensingmachinery,andthedifferingexpression of nucleic acid sensing PRRs [17,18]. The relativelypoorimmunogenicityofDNAvaccines,combinedwithconcernsabout their potential for oncogenesis via integration into thehostgenome,hasledtoashiftfromDNAvaccinestowardsRNAvaccines.

In the past, several viral vectors were proposed as DC-transfectionsystemsforthedeliveryofantigen-mRNA,butthesekinds of vectors shown some limitations [23]. First, a poorlyunderstood butwell-documented immunological phenomenoncalled immunodominancemay occur,whereby other antigenicproteins,suchasviralantigens,maskorsuppresstheresponsetothelesspotenttumourantigens[24].Second,activatedCytotoxicT lymphocyte (CTLs)may recognize and kill antigen-presentingDCs, because DCs expressing both viral and tumour antigens,thereforeDCscanbeeliminatedbyvirus-specificTcellsbeforetheycanactivateatumour-specificresponse[25].Ifaviralvectoris used to deliver the tumour antigen, this elimination effectwouldbeamplifiedwithsubsequentimmunizations,asitislikelythatthevirus-specificT-cellpoolwouldbeexpandedwitheachvaccination.Hence,itseemsdesirabletodevelopmethodsofDCtransfection that lead to theexpressionofonly thoseproteinstowardwhichanimmuneresponseisdesired.

Non-viralgenedeliverysystemswere introducedasalternativeto viral-based systems. These vectors have many advantages,suchaseasyoffabricationandlowimmuneresponse[26].Thebiggestdisadvantageofnon-viralvectors inclinicaluse is theirlow in vivo transfectionefficiency.But, contrarily to theclassicgene therapy approaches, for gene-based immunotherapy arelatively low transfection efficiency can be sufficient to elicita strong humoral and cell-mediated antigen-specific immuneresponseandpromotetumorregressionandthegenerationofmemorythatcanpreventrelapse.

cell/tissue targeting is not requested, because it wasdemonstrated that DC targeting and activation is possible bysimple systemic delivery of the mRNA-loaded carrier, withoutthe need for ligand functionalization [27]. Recently, non-viralvectorshavebeenwidelyexploredasvehiclesformRNAdeliveryin the area of mRNA-based immunotherapy, but their clinicalapplicationislimited,becauseusually,anelevateddoseofmRNAisneededforantigen-specificimmunization[27-30].

Hereweproposeaself-assembledPEG-PAsp(DET)(polyethyleneglycol-b-poly{N’-[N-(2-aminoethyl)-2-aminoethyl] aspartamide})

block copolymer/ PAsp (DET) (poly{N’-[N-(2-aminoethyl)-2-aminoethyl] aspartamide}) homopolymer polyplex as efficientplatform for mRNA-based vaccine (Figure 1A). Although thispolymer has been widely explored as an alternative for genedelivery vector, it hasneverbeenusedasplatform fornucleicacid-based vaccination. The ability of the proposed system toinduce a specific immunization was confirmed employing anmRNAencodingforOvalbumin(OVA)chickenprotein,anantigencommonlyusedasamodelproteinforstudyingantigen-specificimmuneresponses inmice. Interestingly,weshowourvaccineformulation has an intrinsic self-adjuvant propriety given itsabilityto lead, inthetransfectedDCs,arobustreleaseof IL-12and IFN-β,whichare recognizedasapotent vaccineadjuvant,by TLR 7/8 pathway activation. Moreover, OVA-mRNA-loadednanovectorwasabletoinduceahighcell-mediatedandhumoralantigen-specificimmuneresponse.

Finally,amurineOVA-B16lungmetastaticmelanomamodelwasemployed to investigate the anti-tumor effect of the antigen-mRNA loaded polyplex vaccine. Immunization of B16-OVA-bearing mice with OVA-mRNA loaded Polyplex significantlyreducedupto93%thenumberoflungmetastases.

Material and MethodsPolyplex (PAsp (DET)/mRNA) and PEG-Polyplex (PEG-PAsp (DET)/PAsp (DET)/ mRNA) preparation and characterizationPEG-PAsp(DET) block polymer, MW of PEG~12,000 Da, DP ofPAsp(DET)~70andPAsp(DET)homopolymerwithDPofca.50,were purchased from NOFAmerica corporation. One volumeof PAsp(DET) and/or PEG-PAsp(DET) solution (PAsp(DET)/PEG-PAsp(DET)moleratioof100:1and50:1,forPEG-polyplexAandBrespectively)wasmixedwithtwovolumeofmRNAsolutionin10mMTris-HClbuffer (pH7.4) toafinalvolumeof90µl.ThePolyplexeswerepreparedusingdifferentN/Pratio(aminegrouponPAsp(DET)andphosphategroupsonRNA).Afterincubationat roomtemperature for30minutes, thecomplexesarereadytouse.ThesizedistributionandzetapotentialweremeasuredusingaMalvernZetasizerNanoZSdynamiclightscattering(DLS)instrument (Malvern Instruments, Worcestershire, UK). ThemRNA (1 mg/mL) was purchased from Trilink Biotechnologies(SanDiego,CA).

Gel retardation assayPolyplexes and PEG-Polyplexes (250 ng of eGFP-mRNA) wereloadedwith1xRNA loadingbuffer (BioRad,Hercules,CA)andrunona0.7%agarosegel in standard1 x TBEbuffer (BioRad,Hercules,CA)at75mVfor1hour.TheRNAbandswerestainedwithGelRed nucleic acid gel stain (Biotium,Hayward, CA) andthendetectedbyaGelDocsystem(BioRad,Hercules,CA).

Atomic Force Microscopy (AFM)Siliconsubstratewastreatedwithpiranhasolution(H2SO4/H2O2,1:2(vol/vol))for1hourat80°Candwashedwithdistilledwaterthoroughlyandblow-dry.Then,thesubstratewasfunctionalized

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with 2-(carbomethoxy) ethyltrimethoxysilane (CETES). Briefly,the silane was diluted to a final concentration of 2% in 95%IsopropylAlcohol(IPA)(madefreshlybyadding5%waterto100%IPA). Then the substratewas submerged in the silane solutionandincubatedundershakinginaThermomixerat1400rpmfor2hoursat35°C.Thesubstratewaswashed3timesRinsewith100%IPAandblow-dry.Then,20µlofdilutedpolyplexessolution(1:100 or 1:200) was dropped onto the silanes-silicon surfaceand incubated for20minutes.After thesurfacewaswashed3times with distilled water and the substrate was immediatelytransferredontotheAFMstagetobeanalyzed.

Stability of polyplexes against heparinPolyplexeswerepreparedandincubatedfor30minwithheparinsolutions of different concentrations, expressed as heparin/mRNA(w/w)ratio(1,2,3,4and5).Thesampleswererunona0.7%agarosegel1xTBEbuffer(BioRad,Hercules,CA)at75mVfor1hour.RNAbandswerestainedwithGelRednucleicacidgelstainandthendetectedbyaGelDocsystem.

Generation of bone marrow derived cells (BMDCs)Dendritic cells from C57BL/6 mice were obtained from bonemarrowaspreviouslydescribed[31].Briefly,BMDCsfromfemurandtibiawereflushedoutusing1%FBS-containingPBSusinga5-mlsyringe.ThecellsweretreatedwithACKlysisbuffer(LonzaInc.) to remove red blood cells and centrifuged at 500 g for 4minutes. The BMDCs were resuspended in RPMI-1640 culturemedium supplemented with 10% FBS (Atlas Biological, FortCollins,CO),0.1%β-mercaptoethanol,1%penicillin/streptomycin(Pen/Strep,10,000unitsofpenicillinand10mgofstreptomycin,Sigma-Aldrich, Saint Louis, MO). The cytokines Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) (20 ng ⁄ ml)andIL-4(20ng⁄ml)wereaddedandcellsweresetatadensityof1x106cells ⁄ml.Themediumwaschangedeverytwodaysandnon-adherentcellswereharvestedasimmatureBMDCs.Thecellswereusedforallthetransfectionexperimentsatday5.ThedendriticcellshavebeenroutinelytestedforCD11cexpressionand≥85%ofcellsarepositiveforthismarker.

In vitro transfection using an eGFP-mRNABMDCswere seeded in a 24-well plate at density of 3 x 105inRPMI-1640media supplementedwith 10% fetal bovine serum(FBS, Atlas Biological, Fort Collins, CO), 1% streptomycin/Penicillin (Sigma-Aldrich) and 0.1% β-mercaptoethanol (Sigma-Aldrich). After 24 hours of incubation, the dendritic cellswereincubated for with PEG-Polyplexes prepared using differentN/P ratios (80, 120 and 160) and loaded with 0.5 µg eGFP-mRNA.Fluorescence imageswerecapturedusingafluorescentmicroscopy (Eclipse TE2000-S microscope Nikon Corporation,Tokyo, Japan). Flow cytometry analysis was used to quantifythetransfectionandtranslationefficiencyinBMDCs.CellswereharvestedandresuspendedinPBSwith2%FBSandcytometricanalysis was performed using a BD Accuri C6 flow cytometer(BectonDickinson,BD,FranklinLakes,NJ,USA).Gatesweresettoexcludenecroticcellsandcellulardebrisandthefluorescence

intensityofeventswithinthegatedregionswasquantified.Datawerecollectedon20,000eventsforeachsample.

Cytotoxicity test by MTS assayCytotoxicityassayswereperformedusingtetrazoliumcompoundbased CellTiter 96® AQueous One Solution Cell Proliferation(MTS)assay(Promega).Totestforcellularcytotoxicity inducedby polyplexes, the cells were cultured in 96-well plate, withappropriated complete media (Final volume 200 µl) at aconcentration of 104 cells/well. Then, 0.1 µg mRNA loadedpolyplexes were added and MTS assay was then performedaccording to the manufacturer’s instruction at 24 hours aftertreatment.

Cellular uptake mechanismDC2.4cells(ATCC)wereseededin24-wellplatesatadensityof1.5x105cells/mlandincubatedfor24h.Then,theyweretreatedwithPEG-PolyplexespreloadedwitheGFPmRNAafter30minutesof incubation with different inhibitors such as amiloride (0.2mM),chloroquine(100mM),genistein(50µm),chlorpromazine(15 µm) pimozide (10 µm). The cells were washed with ice-coldPBSandthemeanfluorescenceintensitywasobservedbyfluorescent microscopy (Eclipse TE2000-S microscope NikonCorporation,Tokyo,Japan).

Evaluation of dendritic cells maturation markers by flow cytometry analysisDendritic cell activation was achieved by in vitro transfectionusingOVA-mRNA.DC2.4cellswereseededin24-wellplatesatadensityof1.5×105cell/wellin1mlofRPMIcompletemedium(10%FBS,1%Pen/Strep,0.1%β-mercaptoethanol)24hpriortotransfection. Then, PEG-polyplexes containing 0.5 μg ofmRNAwere incubatedwithDC2.4cellsat37°C for24hours.After24hours, the cellswerewashedwithPBSand stained forCD11c,CD40,CD86andMHCII.ThecellswereanalyzedbyBDAccuriC6flowcytometer(BectonDickinson,BD,FranklinLakes,NJ,USA).AlltheantibodiesusedforthisexperimentwerepurchasedfromBDBioscience.

OVA-antigen presentation by dendritic cellsBMDCswere transfectedwith PEG-polyplexes loadedwith 0.5µgofOVA-mRNA,andafter48hoursthecellswerestainedfor10 minutes at room temperature with 25-D1.16 monoclonalantibody(MHCclassIH-2Kb,BDBioscience).Subsequently,thecellswerestained30minuteswithananti-CD11c(BDBioscience)andthenevaluatedusingaBDAccuriC6flowcytometer(BectonDickinson,BD,FranklinLakes,NJ,USA).

MHC I and II–restricted antigen presentation assaysBMDCswereseededataconcentrationof3x105cells/mlina24-wellplateandincubatedfor24hrat37°CinRPMI-1640medium(10%FBS,1%Pen/Strepand0.1%β-mercaptoethanol).Then,atday5ofculture,thedendriticcellsweretransfectedwithPEG-Polyplexesloadedwith0.5µgofOVA-mRNA.After48hours,thetransfected dendritic cells were washed and co-cultured with

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B3Z, T–T hybridomas against OVA (257–264)/H-2Kb complex,andDOBW,T–ThybridomaagainstOVA(323–339)/I-Adcomplex,T-cellsusingaT-cell/dendriticcellsratioof1:1.T-cellactivationwasmonitored at 24hours bymeasuring IL-2 accumulation inthe supernatant by ELISA (eBioscience). Data shown are fromtriplicateexperiments.

Innate immunity activationDC2.4cellswereculturedin24-wellplatesatadensityof1.5x105 cells in 1ml of completemedium in thepresenceof PEG-Polyplexes loaded with 0.5 µg OVA-mRNA. LPS was used at100ng/mlasapositivecontrol.After24hours incubation, thesupernatantswere collected, then IL-6, TNF-α, IFN-β and IL-12concentrations weremeasured using sandwich ELISA kit (R&DSystems,Minneapolis,MN).

TLR inhibitorsTLR7 (ODN 2087) and TLR7/8 (ODN 20959) inhibitors werepurchased from Militenly Biotec (San Diego, CA). DC2.4 cellswereseededina24-wellplateatadensityof1.5x105in1mlofRPMI-1640completemediumand incubated24hoursat37°C.Then,thedendriticcellswerepre-incubatedfor1hourat37with2.5 µm of TLR inhibitor. Subsequently, PEG-Polyplexes loadedwith0.5µgofOVA-mRNAwereadded intothewellsandafter24hours theculturemediumwascollected tobeanalysed forIL-12 and IFN-β levels by ELISA kit. The dendritic cellswithoutpre-incubationwithTLRinhibitorwereusedaspositivecontrol,whereasthedendriticcellsincubatedinabsenceofOVA-mRNAloaded PEG-Polyplexes with/without inhibitor represent thenegativecontrols.

Mice immunization by two dose injections of OVA-mRNA loaded polyplexesPEG-Polyplex and Polyplex vectors were prepared using anN/P ratio of 120 following the procedure described above.Then, two groups of five BALB/c mice were injected s.c. withpolyplexesandPEG-Polyplexesloadedwith2µgofOVA-mRNA.Aseconds.c.injectionwasperformedafter3daysfromthefirstadministration.ControlmicewereinjectedinthesamemannerwithPBS.Themiceweresacrificed7daysafterthefirstinjectiontoallowspleen, lymphnodes,andbloodcollection.Protaminesulfate/mRNA polyplexes were prepared, mixing protaminesulfate(SigmaAldrich)andOVA-mRNA(TrilinkBiotechnologies)atanw/wratioof2in10mMTris-HClbuffer,pH7.4,followedby20minutesofincubationatroomtemperature.

Ex-vivo detection of antigen-specific T-cells by ELISPOTSpleensandlymphnodeswereharvestedfromimmunizedandcontrol groups of mice. Single-cell suspensions were obtainedand blood cells were lysed using ACK buffer and the cellsuspensions passed through a 70µm cell strainer (BD Falcon).ToquantifytheIFN-γproductionbyex-vivostimulatedantigen-specific T-cells, cells were plated in RPMI-1640 medium at adensityof3x104cells/wellinUNIFILTER®96-wellELISpotplates.Consecutively,cellswerestimulatedwith2ng/mlOVApeptides

(OVA257-264,OT-I,andOVA323-339,OT-II)andOVAprotein.Followingovernightincubation(15–18hr)at37°C,thecellswerewashed3 times with PBS containing 0.05% Tween-20. Plates wereincubated for3 to4hrat roomtemperaturewithbiotinylatedmousesecondaryantibodyforIFN-γ(BioLegend).After3washes,asbefore,theplateswerefurtherincubatedfor2hrwithanti-rabbitIgGHRPO(SIGMA)andthenprocessedforstainingusingan AEC kit according to manufacturer's instructions (VectorLaboratories).Thenumberof spots formingcellswasanalyzedusingImmunoSpotsoftware(CellularTechnology).

Measure ex-vivo IFN-γ release by ELISA assayT-cellswereisolatedfromspleenandlymphnodesaspreviouslydescribed.Thecollectedcellswereculturedin24-wellplatesatadensityof1x106cells/mlandincubatedwith2ng/mlofOVApeptides(OVA257-264specificforMHCclassIandOVA323-339,specificforMHCclassII)andOVAproteinat37°C.After24hincubation,theculturesupernatantwasharvestedandthepresenceofIFN-γwastestedusingacommercialmouseIFN-γimmunoassayELISAkit(BDBioscience)accordingtothemanufacturer'sinstructions.The concentrations of IFN-γ in the samples were determinedfromthestandardcurves.

Measure of OVA-antibodies tittersOVA-specific antibodiestittersweremeasured in the serumofimmunizedmice.Bloodwascollectedby retro-orbitalbleedingon day 14 after immunization, and allowed to clot overnightat4°C.Theserumwasseparatedbycentrifugationat1000xgfor20min. Serumwas collectedandanti-OVAantibodytitterswere determined by enzyme-linked immunosorbent assay, aspreviously described. Briefly, 96-well plates (Nunc-Immuno)werecoatedbyovernightincubationat4°Cwith100μl/wellofPBScontainingOVAat40μg/ml.Plateswereblockedwith1%bovineserumalbumininPBSfor2h,andserialtwofolddilutionsof serum samples in PBSwere added to thewells. After a 2-hincubation,plateswerethenwashedwithPBScontaining0.05%Tween20andincubatedfor1hatroomtemperaturewithHRP-conjugated goat anti-mouse immunoglobulin G (IgG), IgG1,IgG2c,IgG2bandImmunoglobulinM(IgM)antibodies(SouthernBiotechnology Associates). After three additional washes, theplateswerethenincubatedwithtetramethylbenzidinesubstrate.The reaction was stopped by the addition of 1N HCl (Sigma).Absorbancewasreadat450nmwithamicroplatereader(Bio-Rad).AbTiterswerecalculatedastheinversedilutionatwhichthe absorbance equaled that of control mice plus 2 standarddeviations.

In vivo toxicity of PEG-PolyplexCytokineanalysiswasperformedtoassessthecytokineresponseinducing by PEG-Polyplex exposure. A MAP Mouse Cytokine/ChemokineMagneticBeadPanel-Premixed32Plex-ImmunologyMultiplexAssay(EMDMillipore,Darmstadt,Germany)wasusedtomeasurethecytokinelevelssuchasIL-1b,IL-2,IL-6,IL-12(p70),TNF-α,IFN-γ,andRANTES.BALB/cmiceweres.c.injectedwith2µgOVA-mRNAloadedmicellesandafter24hoursserumsampleswereanalyzedinduplicatewellsaccordingtothemanufacture'sinstruction.

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In vitro CD8+ T-cell mediated anti-cancer activityDC2.4 cellswere seeded at a density of 1.5 × 105 cells/well in24-wellplatewith1mlofRPMI-1640medium.Afterovernightincubation, dendritic cellswerepre-incubatedwith TLR7 (ODN20958, Miltenyi Biotec, Germany) and TLR7/8 (ODN 2087,Miltenyi Biotec, Germany) inhibitors for 1 hour at 37°C. Then,0.5µgofOVA-mRNAloadedpegylated-polyplexwereaddedandincubatedfor24hoursat37°C.Subsequently,OVA-specificCD8+ T-cells(B3Zcells)wereco-culturedwiththetransfectedDC2.4atT-cell/DC2.4ratioof2:1andincubatedtogetherfor24hoursat37°C.B16-OVAtumorcellswereplatedina96-welltissuecultureplateat0.5×104cellsperwell(200µl)andincubatedovernightat 37°C. The tumor cells were co-cultured with activated B3Zcellsateffector/targetratioof5:1for4,6and24hoursat37°C.Theviabilityof tumorcellswasthendeterminedusinganMTSformazan viability assay (Promega, Madison, WI). The opticaldensityofthewellswasreadat490nmonaplatereader.Themean viability of the treated tumor cells was calculated as apercentagerelativetothecontrolwellswithnon-treatedcancercells(100%survival).Allthesamplesweremeasuredintriplicate.

Metastatic lung cancer modelMicewereinjectedwith2.5×105B16-OVAmelanomatumorcellsintravenouslytoestablishlungmetastases[32].Threedayslater,tumour-bearingmiceweres.c.vaccinatedwith2μgOVA-mRNAloadedmicelles.Micewereboostedatdays7and10.Lungswereharvested18daysfollowingtumorinoculationandfixedwith4%ofparaformaldehyde.Thelungmetastaseswerecountedunderadissectingmicroscope.

TRP2-specific CD8+ T-cells detection in vivoC57BL/6 mice were immunized s.c. with TRP2-mRNA loadedPEG-Polyplexatday3,7and10using thesamedoseofTRP2-mRNA (2μg).Oneweekafter the last immunization thebloodwascollectedandanalyzedbyflowcytometryforthepresenceof tetramer positive CD8+ T-cells (H-2b/TRP2 tetramer, MBL).TetramerpositivecellsareexpressedasthepercentageofCD3+/CD8+Tcells.

Statistical analysisComparisonsbetweenexperimentalgroupsweremadebyusingthetwo-tailedStudentt-test.P<0.05wasconsideredsignificant.

Results and DiscussionCharacterization of mRNA-loaded polyplexesSize and zeta potential of the polyplexes were evaluated bydynamic light scattering (DLS) measurements (Figures 1B and 1C), which showed smaller cumulant diameter and a lowerpolydispersity index in thepegylated-polyplexes (PEG-Polyplex)compared with non-pegylated polyplexes (Polyplex). ThepolyplexessizedecreasewithincreasingofN/Pratiointherangefrom10to80,andbeyondthisrangethesizedistributionremainconstant.Butafurthersizereductionwasobservedinresponseto the pegylation and this variation is proportionated to thePEG-PAsp(DET)/PAsp(DET) ratioused forpolyplexpreparation.HigherzetapotentialwasmeasuredwithN/Pratiobetween20-80 and over this range, the superficial charge of the polyplexremainpositivealthough,decreasesignificantly.Moreover,itis

SchematicillustrationofPEG-Polyplexvaccine(A).ThePEG-PAsp(DET)/PAsp(DET)/mRNAandPAsp(DET)/mRNApolyplexcharacterizationwasperformedthroughsizedistribution(B),zetapotential(C)measuresbyDLSandAFManalysis(D-F). Inaddition,mRNAencapsulationwas confirmedbyagarosegel retardationassay (G). PEG-PolyplexAandBwerepreparedusingaN/Pratioof120.

Figure 1

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evidentthattheinclusionofPEG-PAsp(DET)blockcopolymer,intheformulation,inducearelevantreductionofthezetapotential(Table 1).

Size and morphology of the non-pegylated and pegylated-polyplexeswere confirmedbyatomic forcemicroscopy (AFM),whichconfirmeda sizedecrease in response to thepegylationand that the size variation is proportioned to the amount ofblock copolymer included in the polyplex formulation (Figures 1D-1F).Whereas, the incorporationofmRNA into thepolyplexwas confirmed by gel retardation assay (Figure 1G). The gelretardation assay revealed completemRNA incorporation intothepolyplexesalreadyemployinganN/Pratioof20(Figure 1G).

Serum and extracellular matrix proteins can lead to vectordisassembly [33]. To evaluate the possible effects of anionicproteins on PEG-Polyplex stability,we tested the susceptibilityof our Nano vector to heparin displacement. Heparin isone of the glycosaminoglycans (GAG), which are negativelychargedpolysaccharidesthatarethemajorcomponentsoftheextracellular matrix in many tissues and it is also present onthecellsurface[34].Polyplexeswereincubatedwithincreasingheparinconcentrations.PolyplexdissociationintermsofmRNArelease was shown by electrophoresis of the samples on angelred-containingagarosegel(Figure 2).Anydissociationdidnotoccurevenathighheparin/mRNA(w/w)ratios.

In vitro optimization of transfection conditionsPrimarycellsareknowntoberesistanttotransfection,therefore,afteroptimizedthepolyplexformulation,wedecidedtoevaluatethetransfectionefficiencyofeGFP-mRNA-loadedpolyplexeswithprimarydendritic cells,which representour target topromotean antigen-specific immune response. BMDCs (Bone marrow-derived cells)were obtained from femurs of BALB/cmice andculturedwithgranulocytemacrophage-colonystimulatingfactor(GM-CSF)andIL-4toinducedendriticcellsdifferentiation.

Thedendriticcellsatdayfiveweretransfectedwithpolyplexesloadedwith0.5µgofeGFP-mRNAand48hourspost-transfectionGFP-positive cells were detected by fluorescence microscopy(Figures 3A-3E).Threedifferentpolyplex formulation,obtainedusinganN/Pratioof80,120and160,weretestedfortransfectionability with dendritic cells. The transfection efficiency wasmeasuredbyFACSanalysisandahighernumberofGFP-positive

cells were observed using an N/P ratio of 120 (Figure 3F).Importantly,MTSassaydidnotdetectanysignificantcytotoxicity,thereforeaspreviouslydemonstratedPAsp(DET)polymerhasanoptimalbiocompatibility(Figure 3G)[35,36].

After having confirmed that polyplexes efficiently deliver themRNA into the DCs, we wanted to elucidate the mechanismbywhichthiscellsuptakemRNA-loadedpolyplexes.Therefore,we examined the different cellular uptake pathways forthe polyplexes using various endocytic inhibitors; each onespecific for a particular endocytic pathway (Figures 3H-3P).The amiloride (blocks macropinocytosis by Na+/H+ exchangerinhibition) significantly decreased the cellular transfectionby 70% (Figure 3P), proving that our polyplexes are able toreleaseefficiently themRNA into the cytosolmainly exploitingmicropinocytosispathway.Aprominentroleofmicropinocytosisin the PEG-Polyplex entry into dendritic cells was confirmedby the substantial reduction (~45%) in transfection efficiencyfollowingpre-treatmentwithcytochalasinDaninhibitorofactinpolymerization(Figure 3P).

Moreover,thisstudyhasrevealedthataddinggenistein(inhibitscaveolin-mediated endocytosis), chlorpromazine (inhibitsclathrin-mediatedendocytosis),Pimozide(inhibitsphagocytosis)did not significantly altered the cellular transfectionefficiency.On the contrary, chloroquine, known to prevent endosomeacidification and maturation, induced a 2-fold improvedtransfection efficiency. Still, it has not been clarified themechanism by which chloroquine increase the transfectionefficiency,butitseemslikelythatendolysosomalaccumulationof

Polyplex Z-Average(nm) PDI (Polydispersity index)N/P=10 1298.3 0.643N/P=20 737.3 0.568N/P=40 329.6 0.335N/P=80 231.4 0.267N/P=120 158.4 0.259N/P=160 157.7 0.228

PEG-Polyplex Z-Average (nm) PDI (Polydispersity index)FormulationA 147.9 0.224FormulationB 96.45 0.186

Table 1Averagesizeandpolydispersityindex(PDI)forthesizeintensitydistribution plots shown in Figure 1B. The polyplexeswere preparedusinganeGFP-mRNA(996nt).

HeparindisplacementassayPEG-Polyplexeswere prepared at N/P ratio of 120 andincubatedfor30minat37°Cinthepresenceofincreasingamountsofheparin,expressedasheparin/siRNA(w/w)ratio.

Figure 1

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chloroquineinhibitacidificationbyproton-bufferingwhichinducetoincreasetheosmoticpressurewithintheendosomethatleadtoendosomalmembranedisruptionwiththeconsequentantigenescapeintothecytosol,enablingitstranslationandprocessing.

In vitro validation of PEG-Polyplex for mRNA-based vaccine applicationsWith the ability to deliver antigen-mRNA into the cytosol ofdendriticcells,wethendeterminedthatthispropertycouldbecombinedtoelicitdendriticcellsmaturation.Dendriticcellsweretransfectedwith0.5μgofmRNAencodingforOvalbumin(OVA-mRNA),achickenproteinusedasamodelantigenforstudyingantigen-specific immune responses in mice. 48 hours post-treatment dendritic cells displayed a shift from the immatureto the mature phenotype through upregulation of the co-stimulatorymoleculesCD40,CD86,andMHCII(Figure 4A).

MHCclass IImolecules are crucial for the activationof CD4+ Tcells,whichiswellknownfortheircriticalroleintherisingandregulationofadaptiveanti-tumourresponse.Tcellscandirectlymediatecytotoxicityagainsttumorcells.

To test the ability of activated DCs to process antigen thepresence of dendritic cells presenting OVA257– 264 peptide(SIINFEKL)on themajor histocompatibility complex I (MHC-I)was analyzed 48 hours after treatment. The mRNA-basedvaccine was capable of generating a significant number ofSIINFEKL-MHC-I+DCs(Figure 4A).

Primed-T-cells secrete several cytokines, such as interlukin-2(IL-2), which influence various lymphocyte subsets during theimmune responses. For example, IL-2 has a crucial role in themaintenanceofregulatoryT(Treg)cellsandforthedifferentiationofCD4+T cells into specificeffectorT-cell subsets in responseto antigen-mediated activation [37]. Furthermore, IL-2 signalfor CD8+ T-cells regulate both effector T-cell generation anddifferentiationintomemorycells[38].TheelevatedlevelsofIL-2measured in themedium, using an ELISA assay after dendriticcells-T cells co-culture confirm the ability of thedendritic cellstoactivateT-cellsbyMHC-antigenpresentationafterstimulationwith OVA-mRNA-loaded polyplex (Figure 4B). An importantadvantage of mRNA-based immunotherapies over traditionalrecombinant protein vaccines is the ability to activate innate

TransfectionofBMDCswasachievedusingPEG-Polyplexesloadedwith0.5µgofeGFP-mRNAandpreparedemployingdifferentN/P ratio (N/P=80,120and160).Fluorescencemicroscopy imagesshowBMDCs transfectedwith0.5µgofGFP-mRNAloadedPEG-Polyplex(A-E).TheFigureBisthenegativecontrolandFigureC,DandEshowthetransfectionwithPEG-PolyplexobtainedusingN/Pratioof80,120and160respectively..TransfectionefficacyofthePEG-Polyplex,preparedusingaN/Pratioof80,120and160,wasconfirmedandquantifiedusingflowcytometryanalysis(F).ALossinmetabolicactivitywasquantifiedusinganMTSassaywithcolorimetricreadout,measuredbyamultiplatereader(G).Thecytotoxicityassaywasperformedintriplicateforeachsample.EffectoftreatmentwithdifferentendocyticinhibitorsontransfectionefficiencyofeGFP-mRNAloadedPEG-PolyplexbyDC2.4cells.FluorescenceimageofDC2.4cellstransfectedwith PEG-Polyplex loadedwith 0.5μgof eGFP-mRNAand treatedwith different inhibitors (mock, control, amiloride,chlorpromazine,chloroquine,genistein,pimozideandcytocalasinD,figureH, I, J, K, L, M and O respectively).InthefigureP isreportedthe GFPexpressionmediatedbyPEG-Polyplexeswithoutandinthepresenceofendocytosisinhibitors.Thedataisthemeanofthreeindependentexperiments±standarddeviations.

Figure 3

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Upregulationofthematurationmarkers,includingCD40,CD86,andMHCIIinresponseto24hourstreatmentofDC2.4cellswithPEG-Polyplex(0.5µgofOVA-mRNA,N/Pratioof120)(A).PositivecellsweregatedfromtheCD11c+population.Percentageofantigen-presentingcellswereanalyzedbystainingBMDCswithanti-CD11cand25D1.16(OVA257-264/H-2Kbcomplex)antibodies.Thepercentageof25D1.16positivecellsweregatedfromtheCD11c+population.(B)CD8+orCD4+T-cell/DendriticcellcocultureafterBMDCstransfectionwith0.5µgofOVA-mRNA.ThelevelofIL-2intheculturemediawasmeasuredbyELISAkit.ThelevelofIL-12,IL-6,TNF-αandIFN-βintheculturemediapost-transfectionofDC2.4cells(C).EffectofTLR7 (ODN20958)andTLR7/8 (ODN2087) inhibitionon thepromotionof IFN-βand IL-12 release (D andE,respectively).Non-transfectedDC2.4cellswithout/withTLR7andTLR7/8inhibitorspre-treatmentwereusedasnegativecontrols.

Figure 4

immunity by TLR signalling. TLRs were found act as adjuvantreceptors,thatpromoteandpotentiateadaptiveimmunesystembystimulatingthereleaseofseveralcytokines, includingtypeIinterferonandIL-12.TypeIinterferonandIL-12arerecognizedasapowerfuladjuvantthatpotentiatetheantigen-specificimmuneresponse,becausecapableofactivatingT-andNK-cellsforIFN-γproductionandtopromoteCD+Th1immunity[38,39].Immunedeviation towards Th1 responses results in tumor rejection asTh1 pathways typically produce activation of cytotoxic T-celllymphocytes (CTL), natural killer (NK) cells, macrophages andmonocytes, all of which can attack cancer cells and generallydefendagainsttumors.Asignificantup-regulationofIFN-β(1.7fold)andarobustsecretionofIL-12(8fold)wereobservedaftertreatmentofDCswithOVA-mRNA-loadedpolyplexes.Whereas,a non-significant change in the levels of TNF-α and IL-6 wasdetectedinresponsetothetreatmentwithOVA-mRNA-loadedpolyplexes. Interestingly,naked-mRNAandPAsp(DET)polymerwere not able to induce IFN-β and IL-12 release (Figure 4C). TheroleofTLRsinthepromotionofIL-12andTypeIinterferonsecretion, by OVA-mRNA-loaded polyplexes treated DCs, wasprovedusingTLR7(ODN20958)andTLR7/8inhibitors(ODN2087).The release of IL-12 and IFN-β is significantly compromisedbytheinhibitionofTLR7/8pathway(Figures 4D and 4E).Therefore,we can conclude that mRNA-loaded polyplexes induce innateimmunesystemactivationbyTLR7/8signalling.

In vivo antigen-specific immunization by administration of OVA-mRNA loaded PEG-polyplexesToassessthesuccessofin vivoimmunizationinresponsetos.c.injection ofOVA-mRNA-loaded polyplexes,we determined thepresenceofantigen-specificIFN-γsecretingT-cellsinthespleenandlymphnodesoftreatedmice.Themicewereimmunizedbysubcutaneously (s.c.) injection of 2 μgOVA-mRNA, boosted atday3withthesamedoseand,spleensand lymphnodeswereharvested at day 7. The presence of OVA-, OTI-, OTII- specificIFN-γsecretingT-cellswasconfirmedbyELISAandELIspotassays(Figures 5A-5D).High levelsof IFN-γandsignificantnumberofantigen-specificIFN-γsecretingT-cellsweredetectedbothinthespleen(Figures 5A and 5B)andlymphnodesofimmunizedmice,confirming the ability of the proposed vaccine formulation topromoteanantigen-specificadaptiveimmuneresponse.

Induction of OVA-specific immunoglobulin G (IgG, IgG1, IgG2bandIgG2c)andIgMweredetectedintheserumatday7afterfirstOVA-mRNAvaccineadministration(Figures 5E and 5F).Giventhehigh levelsofOVA-specific IgGand IgMdetected in the serumofthevaccinatedmice,wecanaffirmthattheproposedvaccineefficiently induces apotent antigen-specifichumoral response.Moreover, s.c. injection induced high OVA-specific IgG1 andIgG2a titers using only a small amount of OVA-mRNA (2 μg),

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Measureex-vivoofIFN-γinspleen(AandB)andlymphnode(CandD)byELISA(AandC)andELIspot(BandD)assays.Thecellswerestimulatedwith0.2ng/mLofOVA,OTI,andOTII.AmeasureofIgGs(E)andIgM(F)levels7dayspost-treatmentwith2μgofOVA-mRNAloadedintoPS/mRNAandPEG-Polyplexvectors.Chemokynes(G)andcytokines(J)serumlevels48hoursafteradministrationofOVA-mRNA(2μg).

Figure 5

demonstratingthatthemRNA-basedimmunotherapypromotesbothTh1andTh2typeofantibody-mediatedimmunity.

With the purpose to demonstrate that the proposed systemsrepresent an important advancement of the current state-of-the-art in the area ofmRNA-base vaccine implementation,wedecidedtocompareourvaccineformulationagainstprotaminesulfate/mRNA polyplex (PS/mRNA), a non-viral vector widelyemployedforantigen-mRNAdeliveryandcurrentlyunderstudyinseveralclinical trials foranti-cancer immunotherapy [40-42].ThedetectionofOVA-specificIFN-γsecretingcellsinthespleenand lymph nodes of vaccinated mice, revealed a significantlyhighernumberofprimedT-cell in themice treatedwithOVA-mRNAloadedPEG-PolyplexthanmicevaccinatedwithPS/OVA-

mRNApolyplex. Contrarily, equivalenttiters of IgG and IgM, itwas found in both vaccination with OVA-mRNA loaded PEG-PolyplexandwithPS/OVA-mRNAcomplex.

A major barrier to the clinical application of nanomaterials istheir potential toxicity. Therefore, we assessed the toxicityassociated with OVA-mRNA-loaded polyplex using wild-typemice. A 32-plex Luminex assay was employed to measurecytokineandchemokineserumlevels,includingIL-1b,IL-2,IL-6,IL-12(p40),IL-12(p70),TNF-α,IFN-γ,RANTES,EotaxinG-CSF,GM-CSF,M-CSF,VEGF,andIP10.AsreportedintheFigures 5G and 5J,24hourspost-treatmentwecanexcludeanypolyplex-relatedpro-inflammatoryortoxicity.

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Anticancer effect of OVA-mRNA loaded PEG-Polyplex in vitro and in vivoTomeasuretheantigen-specificcell-mediatedimmuneresponsepromoted by immunization with OVA-mRNA-loaded PEG-Polyplexvaccine,weemployedanin vitroCytotoxicTLymphocyte(CTL) cytotoxicity assay. OVA-mRNA transfected DCs were co-incubated with OVA-specific CD8+ T-cells using a ratio of 1:2,then primed-T cellswere co-culturewith B16-OVA tumor cellsat effector/target cells ratio of 5:1. CD8+ T-cell-mediated anti-tumoractivitywasestimatedmeasuringtumorcellviabilityat4,6and24hoursbyMTSassay(Figure 6A).UsingTLR7/8inhibitors,weprovedtheconceptthatinhibitionofthispathway,withtheconsequent block of type-I IFN and IL-12 secretion, affect theanti-tumourcytotoxicimmuneresponse.Indeed,itiswellknown

that IL-12 enhances the function and anti-tumour activity inmurineandhumanCD8+T-cells[43].

Anti-cancermRNA-based immunotherapy is able to induce anantigen-specific cell-mediated and humoral immune responsessufficienttoobtaintumorregression.Toaddressthisquestion,we generated an OVA-B16 melanoma lung metastasis tumormodelandtreateditatday3throughs.c.injectionofOVA-mRNA-loadedpolyplexes,followedbytwoboosterdosesatdays7and10.Themiceweresacrificedatday18post-tumorinoculation.AsshowninFigure 6B,micewithoutvaccination(injectedwithPBS),developedextensivepulmonarymetastases.However,amarkedreductioninpulmonarymetastases(upto93%)wasobservedinmicetreatedwithOVA-mRNA-loadedpolyplexes(Figure 6B).

Tumourcellskillingactivitybyantigen-specificCD8+T-cellswasmonitoredatdifferenttimes(4,6and24hours)byMTSassay(A).ThetumorcellviabilityisreportedasarelativepercentageofB16-OVAcellstreatedwithnon-activatedT-cells.Anti-tumouractivityofOVA-antigenspecificT-cellswasinhibitedby1hourpre-incubationwithTLR7(ODN20958)andTLR7/8 (ODN2087) inhibitors.Thephotographic imagesshowthe lungsharvested fromtumour-bearingmice injectedwith PBS (negative control) andOVA-mRNA loadedPEG-Polyplexes 18 days post-tumor challenge (B). Thehistogramshows thequantificationof B16 lungmetastatic-like tumor foci. Data are presented asmean± standarddeviationofn=5micepergroup.AnalysisoftheTRP2antigen-specificCD8+T-cellfromperipheralbloodofvaccinatedmicebyflowcytometry(C).Mice(n=4pergroup)werevaccinatedwithPBS,TRP2-peptideorTRP2-mRNAloadedPEG-Polyplex.

Figure 6

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ConclusionInconclusion,wedemonstrated,thatusingalowdoseofantigen-mRNA, the administration of antigen-mRNA-loaded polyplexescan induce a potent anti-tumor immunity, effective for cancertreatment.Our results contribute to a relatively small numberof studies published on mRNA-loaded nanoparticle mediatingtumor vaccination. In addition, our findings also support theconcept that mRNA-based vaccine offers the advantages topromotesimultaneouslyantigen-specificadaptivecell-mediated,humoral immune response, and it can act as self-adjuvant,promotinginnateimmunitybyTLRs.TLRsignallingpromotesthereleaseofseveralcytokines,suchastypeI interferonandIL-12

that have the capacity to potentiate the anti-tumor adaptiveimmuneresponse.

Self-assembledarefastandinexpensivetoprepareandpotentiallycan be used as a universal platform for anti-tumour mRNA-based immunotherapyapproaches, given that anypolypeptidecan be encoded by mRNA. Thus, the mRNA-loaded polyplexintroducedherecanrepresentanovelandvalidplatformforthedevelopmentofmRNA-basedvaccines.

AcknowledgementThisworkwas fundedby the ItalianFlagshipProjectNanoMax(S.P.)

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