s41392-024-02002-z by Biogenomics

Signal Transduction and Targeted Therapy

REVIEWARTICLE

ProgressandprospectsofmRNA-baseddrugsinpre-clinical andclinicalapplications

YingyingShi1,MeixingShi1,YiWang 1 ✉ andJianYou

Inthelastdecade,messengerribonucleicacid(mRNA)-baseddrugshavegainedgreatinterestinbothimmunotherapyandnonimmunogenicapplications.Thissurgeininterestcanbelargelyattributedtothedemonstrationofdistinctadvantagesofferedby variousmRNAmolecules,alongsidetherapidadvancementsinnucleicaciddeliverysystems.Itisnoteworthythatthe immunogenicityofmRNAdrugspresentsadouble-edgedsword.Inthecontextofimmunotherapy,extrasupplementationof adjuvantisgenerallyrequiredforinductionofrobustimmuneresponses.Conversely,innon-immunotherapeuticscenarios, immuneactivationisunwantedconsideringthehosttolerabilityandhighexpressiondemandformRNA-encodedfunctional proteins.Herein,mainlyfocusedonthelinearnon-replicatingmRNA,weoverviewthepreclinicalandclinicalprogressand prospectsofmRNAmedicinesencompassingvaccinesandothertherapeutics.Wealsohighlighttheimportanceoffocusingonthe host-speciﬁcvariations,includingage,gender,pathologicalcondition,andconcurrentmedicationofindividualpatient,for maximizedefﬁcacyandsafetyuponmRNAadministration.Furthermore,wedeliberateonthepotentialchallengesthatmRNA drugsmayencounterintherealmofdiseasetreatment,thecurrentendeavorsofimprovement,aswellastheapplicationprospects forfutureadvancements.Overall,thisreviewaimstopresentacomprehensiveunderstandingofmRNA-basedtherapieswhile illuminatingtheprospectivedevelopmentandclinicalapplicationofmRNAdrugs.

SignalTransductionandTargetedTherapy (2024)9:322 ;https://doi.org/10.1038/s41392-024-02002-z

INTRODUCTION

Recently,messengerribonucleicacid(mRNA)therapyrepresentsa novelapproachfortreatingawiderangeofdiseases,encompassingbothimmune-relatedandnon-immuneconditions. AmidsttheCOVID-19pandemic,mRNAvaccineshaveachieved remarkableadvancements,owingtotheunwaveringdedication ofnumerousscientistswhohavebeenattheforefrontofmRNA researchfordecades.

However,despiteaplethoraofpreclinicalstudiesconducted, successfultranslationofmRNAmedicinesintoclinicalapplications remainslimited,probablyduetothesuboptimaldesignand administrationofmRNAdrugstopatientswithspecificphysiologicalandpathologicalconditions.DistinctcharacteristicsofmRNA drugsaredemandedindifferenttherapeuticindications.1 Specifically,inimmunogenicapplicationsthatincludecancer immunotherapiesandinfectiousdiseasevaccines,appropriate incorporationofadjuvantsisrequiredforelicitingaugmentedhost immuneresponses.Incontrast,innon-immunotherapiesencompassingproteinreplacement/supplementationtherapy,regenerativemedicinetherapy,andgeneticediting,theimmunogenicityof mRNAdrugsisunfavorable,whichmayleadtodiminishedprotein expressionandevencauseadversereactions.Ontheotherhand, thehostindividualvariations,suchas,age,gender,diseaseand medicalhistorymayinfluenceboththeefficacyandsafetyof mRNAmedication.Forexample,comparedtohealthyadults, immunocompromisedpatientsexhibitinadequateimmune

responsesfollowinginitialmRNAvaccineinoculation,where repeatedvaccinationsarerecommendedfortheestablishment ofsufﬁcientimmuneprotection.2 Nevertheless,repeateddoses maycausepotentialrisksthatexacerbatethepathological burden.3 Todate,thereisstillalackofsystemicunderstanding overthefateandoutcomesofmRNAdrugsinpersonalized recipient.

Inthisreview,we ﬁrstsummarizethepreclinicalandclinical applicationsofmRNAdrugspertainingtoimmunotherapyand non-immunotherapy,thendiscusstheimpactfrompatient physiologicalandpathologicalcharacteristics.Finally,weprovide insightsintothefuturedirectionsandresearchprioritiesofmRNA drugs.

EVOLUTIONANDMILESTONESINMRNA-BASEDDRUGS

BriefhistoryofmRNAdrugdevelopment

Chronologically(Fig. 1),mRNAwas ﬁrstdiscoveredbypioneering researchesin1961.4 However,itwasnotuntil1990whenWolff etal.successfullyexpressedproteinsbyinjectingmRNAintothe body5 thatmRNAgraduallygainedrecognitionasatherapeutic modality.1 In1999,mRNA-engineereddendriticcells(DCs)entered clinicaltrialasantitumorvaccinesforthe ﬁrsttime (NCT00004211).6–8 In2004,Weideetal.conductedtheinitial clinicaltrialinvolvingthedirectinjectionofprotamine-stabilized mRNAintothehumanbodytotargetmetastaticmelanoma

Correspondence:YiWang(zjuwangyi@zju.edu.cn)orJianYou(youjiandoc@zju.edu.cn)

Theseauthorscontributedequally:YingyingShi,MeixingShi

Received:2June2024Revised:3September2024Accepted:26September2024 1CollegeofPharmaceuticalSciences,ZhejiangUniversity,866YuhangtangRoad,Hangzhou,Zhejiang,P.R.China; 2StateKeyLaboratoryforDiagnosisandTreatmentofInfectious Diseases,79QingchunRoad,ShangchengDistrict,Hangzhou,Zhejiang,P.R.China; 3TheFirstAfﬁliatedHospital,CollegeofMedicine,ZhejiangUniversity,79QingChunRoad, Hangzhou,Zhejiang,P.R.Chinaand 4JinhuaInstituteofZhejiangUniversity,498YiwuStreet,Jinhua,Zhejiang,P.R.China

Fig.1 ChronologicaldevelopmentofmRNAdrugs.Yellowbox,commoneventsofmRNAdrugs;greenbox,mRNA-basednonimmunotherapy;redbox,mRNA-basedimmunotherapy.From1961to1990:mRNAdiscoveryandthematurationofIVTmRNAtechnology, includingthediscoveryofmRNA,4 purifiedmRNAcouldbetranslatedintoproteinsinthemammaliancell-freesystem,518 discoveryofmRNA cap,519 discoveryofsingle-strandedcircularRNA,520 capanalogcommercialized,18 syntheticmRNAwas firstproducedinthelaboratoryby IVT,374 T7RNApolymerasescommercialized.335 From1990to2019:theexplorationofmRNAvaccines,particularlyforcancertherapy, includingIVTmRNAinjectedintothemouseskeletalmuscleachievedproteintranslation, 5 vasopressinmRNAinjectedintothehypothalamus ofBrattlebororatswasfoundtosuccessfullyexpressvasopressin,357 mRNAvaccineencodingtumorantigeninmice,171 firstclinicaltrialof mRNA-engineeredDCsvaccinestrategy,6–8 firstDCvaccinewithautologoustumormRNAwasusedtotreatclinicalPhaseI/IIofadvanced malignantmelanomatrial(NCT01278940),332,333 firstattemptwasmadetoinjectanmRNAvaccinedirectlyintohumansto fighttumors (NCT00204607),9,10 nucleotide-modifiedRNAreducedthepotentialforimmunestimulation,11 firstpersonalizedcancermRNAvaccinein clinicaltrial(NCT02035956),521,522 firstclinicaltrialofprophylacticmRNAvaccine(CV7201)againstrabies(NCT02241135),13 NIHcalledfor gendertobeincludedinbiologicalvariablesinpreclinicalandclinicalstudies,435 firstmRNA(AZD8601)therapyencodingVEGF-Atoenterthe clinic(NCT02935712),523 firstclinicaltrialofmRNA-encodingimmunostimulant(mRNA-2416,NCT03323398). 524 From2019tothepresent: rapiddevelopmentofmRNA-basedtherapeutics,includingtwomRNAvaccines(mRNA-1273486 andBNT162b2)havebeenapprovedfor emergencyusebytheFDA,123 124 firsttouseunmodifiedmRNAinregenerativemedicine,370 forty-threeCOVID-19mRNAvaccineswerein clinicaltrials,125 firstcombiningmRNAtherapywithphotodynamictherapyto fighttumors,290 PhaseIIItrialofmRNA-4157plus Pembrolizumabinthetreatmentofmelanoma.501 IVTinvitrotranscription,NIHtheNationalInstitutesofHealth,DCsdendriticcells,FDAthe FoodandDrugAdministration.ThegraphiciscreatedwithAdobeIllustrator

(NCT00204607),9 whereinthemRNAencodedtumor-associated antigens(TAAs) Melan-A,Mage-A1,Mage-A3,Tyrosinase,gp100, andSurvivin.10 In2005,Karikóetal.demonstratedthatRNAcould evadeimmunedetectionwhenitwasnaturallymodifiedwith nucleotidessuchas5-methylcytosine(m5C),5-methyluridine (m5U), N6-methyladenosine(m6A),pseudouridine(Ψ),and2′ -OMethyluridine.11 12 Theidentificationofthisapproachtomitigate theimmunogenicityofmRNAiscrucialforbroadeningthescope ofmRNAtherapeuticsinnon-immunologicalmedical fields.In 2013,CureVacconductedaninitialevaluationofthesafetyand immunogenicityofaprophylacticmRNAvaccinecodingthe rabiesvirusglycoprotein(CV7201)inaclinicaltrial(NCT02241135) involvinghealthyadults.13 Thistrialrepresentsthe firstclinical studyutilizingmRNAvaccinestocombatinfectiousdiseases.12 Then,in2016,thetherapeuticpotentialofmRNAwas first unleashedinthe fieldofproteinsupplementationtherapy (NCT02935712).Notably,in2020,twoantivirusmRNAvaccines mRNA-1273byModerna(NCT04283461)andBNT162b2by Pfizer/BioNTech(NCT04380701),werequicklyapprovedbythe UnitedStates(U.S.)FoodandDrugAdministration(FDA)andput intouseduringtheCOVID-19outbreak,whichachievedgreat successandmarkedasignificantmilestoneinemergency responseagainstinfectiousdiseases.14 Sincethen,mRNAdrugs haveexperiencedanexplosionofdevelopment.

Classiﬁcation,advantages,andlimitationsofmRNAdrugs mRNA,asingle-strandedribonucleicacid,actsasatransient carrierforthegeneticinformationtranscribedfromDNAtoguide proteinsynthesis.15 TheexecutionofmRNAfunctionineukaryotes involvesmultiplestages,includingtranscription,posttranscriptionalprocessing,intracellulartransport,andtranslation.16 PriortothematurationofmRNA,precursormessenger RNA(pre-mRNA)mustundergothreeessentialprocessingsteps: additionofa5’ capstructure,cleavagemediatedbynumerous proteinfactors,and3’-endprocessingwhichincludesaddinga polyadenosine[poly(A)]tail.Thesepre-mRNAprocessingstepsare crucialforthegenerationofmaturemRNA.17 WiththecommercializationofCapanalogsandT7RNApolymerasein1983and 1985,respectively,thetechnologyforinvitrotranscription(IVT)of mRNAhassteadilymatured.18 ThestructuralcharacteristicsofIVT mRNAcloselyresemblethoseofendogenousmRNAfoundin naturaleukaryoticcells.19

Thelinearnon-replicatingmRNA,representativeofIVTmRNA,is composedof fivesegmentsinthe5’ to3’ direction:the5’ cap,the 5’ untranslatedregion(UTR),anopenreadingframe(ORF) encodingthetargetprotein,the3’ UTR,andapoly(A)tail.20–22 TheORFfunctionsasthecodingsequenceforproteintranslation andisanindispensableelementofIVTmRNA.TheUTR,although non-coding,playsapivotalroleinribosomerecruitmentand successfulmRNAtranslation.Furthermore,the5’ capand3’ Poly(A)tailstructuressignificantlycontributetoenhancingmRNA stabilityandtranslationefficiency.23,24 Oneofthekeyattributesof mRNAisitshighlycustomizablenature.TheoptimizationofmRNA involvesnucleotidesequencerefinement,chemicalmodifications,25 andmRNApurification.Variousfactorsmustbetaken intoconsiderationduringmRNAdesign,includingtheinfluenceof nucleotidesequenceonRNAfolding,immunogenicity,enhancementofmRNAstability,andmaximizationoftheexpressionofthe targetprotein.26,27 TheimmunogenicityofexogenousmRNA elicitsaninnateimmuneresponse,leadingtothesuppressionof exogenousmRNAtranslationinthebody.28 Substitutinguridine withmodifiednucleotidessuchasm5Uand Ψ,whicharenot recognizedbypattern-recognitionreceptors,canmitigatethe immunogenicityofmRNA.26 Inaddition,severalstudiessuggest that Ψ modificationmayenhancethestabilityoftranscripts.29 Nevertheless,therehavebeeninvestigationssuggestingthat specificcircumstancesmaydisruptthestabilityofmRNA.30 Itis noteworthythattherearecurrentlymorethan170identified typesofRNAmodifications.31 ExploringtheoptimizationofmRNA holdssubstantialresearchpotential.32

Comparedtorecombinantproteindrugs,mRNAdrugshavea shorterproductioncycle33 andarenotlimitedtoshortpeptide sequences.34 mRNAvaccinescaneasilydelivernumeroustumor antigenfragmentssimultaneously,therebyincreasingthediversity ofantigenicepitopes.33 IncontrasttoDNAvaccines,mRNA vaccinescircumventtheneedtoenterthenucleus,thereby obviatingthepotentialforgeneinsertionandsubsequent mutations.33,35 Inaddition,mRNAexhibitsmodifiableimmunogenicity36 andcanbeeffectivelypotentiatedbyadjuvantsto efficientlyelicithumoralandcellularimmunity.35 Moreover,mRNA vaccineshavethepotentialtobecomepersonalizedtherapeutic drugs,inwhichtheyhaveapplicationprospectsfortargeting specifictumors37 andtreatingrarediseases.Nevertheless,mRNAis susceptibletodegradationbynucleases33 andsuffersfrom thermalinstability,necessitatingacold-chaininfrastructurefor storageandtransportation,therebyincreasingtheoverallcost.38 Furthermore,mRNA,beingalargepolyanionicstructure,presents challengesintraversingcellandtissuebarriers. 33 Overcoming theselimitationsnecessitatesthedevelopmentofappropriate deliverystrategies.

TwoinnovativemRNAstructureshavebeendevelopedfor speciﬁcpurposes,namelyself-amplifyingmRNA(saRNA)39 and circularmRNA(circRNA).40 saRNAcanconferequivalentvaccine

protectioneffectstothoseofnon-replicatingmRNAatareduced dosage.41–43 TheprimarydistinctionbetweensaRNAandlinear non-replicatingmRNAisthatsaRNAcontainsadditionalselfamplifyingreplicongenesthatoriginatefrommultiplepositivestrandRNAviruses,44–46 resultinginalargermolecularsize.47 OwingtotheextensiveanionicstructureofsaRNA,itsdelivery vehicleoftencomprisespolycations.Nevertheless,theuseofsuch deliveryvectorsmayleadtohighchargedensitythatinduces cytotoxicity.Hence,itisimperativetooptimizeasuitabledelivery methodforenhancingboththeloadingefficiencyandtheoverall safetyofsaRNAdrugs.Dastgerdietal.48 demonstratedthat optimizingtheratioofpolyanions,suchas γ-polyglutamicacid,to polycationsinRNAformulationsisaneffectiveapproachfor improvingthedeliveryefficiencyandsafetyofsaRNA.Furthermore,giventhecontinuousgenerationofnewmRNAwithinthe bodybysaRNAvaccines,itisimportanttoremainvigilantabout safetyconcernssuchasthepotentialforseveresystemicorlocal inflammatoryresponses. 49 CircRNAisaclassofcovalentlyclosedloopsingle-strandedRNAmoleculethatisnotinitiatedbythe classicaltranslationpathwayduetotheabsenceofa5’ capanda 3’ tail.50 Theinternalloadingoftheribosomemaybethesole meansforitstranslationtocommence.51 Thecapabilitytoencode proteinswithouttheclassicalpathwayisgrantedbycircRNA’s abilitytotranslatewithouta5’ cap.50 Meanwhile,circRNAexhibits betterstructuralandbiochemicalstabilityduetoitsinherent resistanceagainstexonucleases-mediateddegradation.52 Therefore,theaveragehalf-lifeofcircRNAincellssignificantlyexceeds thatoflinearmRNA.53 Hence,cirRNAhasthecapacitytoextend antigenproductionandsustainenduringimmuneresponses.54 Nevertheless,despitethemorepersistentandrobustexpression ofencodedproteins,circRNAseemstoperformpoorlyintargeted delivery.55 Atpresent,non-replicatingIVTmRNA,whichhasbeen well-establishedinproductionandqualitycontrol,isprimarily usedinpreclinicalandclinicalpractice.

DeliverysystemandpreparationstrategyofmRNAdrugs DuetothesusceptibilityofIVTmRNAtodegradationby nucleases,itiscrucialtoselectanappropriatedeliveryvehicle tosafeguardtheintegrityofthemRNApriortoitsintracellular delivery.56 AninnovationinitiallynotintendedformRNAbut crucialformRNAdrugdevelopmentistheadvancementof deliveryvectors,particularlythedevelopmentoflipidnanoparticles(LNPs).Atpresent,mRNAdeliverysystemscanbeclassiﬁed intoviralandnonviralsystems.57

Virus-relateddeliverysystemsincludebothvirusesandviruslikeparticles(VLPs).58 Viralvectorsencompassadenoviruses, adeno-associatedviruses(AAV),lentiviruses,herpessimplexvirus (HSV),andSendaivirus.59,60 TheformationofVLPsisfacilitatedby thestructuralproteinsofavirus,whichareviralcomponents devoidofgeneticmaterial.61 Incomparisontononviralvectors, viralvectorspossesstheinherentadvantageofefficientlyentering cellsanddeliveringnucleicaciddrugs.58 Thisapproachholds significantpromiseforvaccinedevelopment.62 Severalclinical trialsarecurrentlyutilizingviralvectorstodelivergeneticmaterial forthetreatmentofdiseases.Forinstance,AAV9-mediated CLN6 genetherapy(AT-GTX-501)hasprogressedtoclinicalstagesI/II (NCT02725580),andinterim findingsindicatedthatAT-GTX-501 demonstratedfavorabletolerabilityandcouldamelioratethe deteriorationinmotorandlanguagefunctionamongpediatric patientswithvariantlateinfantileceroidlipofuscinosis6 (vLINCL6).63 Nevertheless,virus-mediateddeliverysystemspose potentialbiosecurityconcerns(e.g.,tumorgenicityandimmunogenicity),exhibitlowpackagingefficiency,andentailhigh manufacturingcosts.59 Theselimitationshavedrivenresearchers toinvestigatealternativedeliverymethodsformRNAvaccines, suchasnonviraldeliverysystems.62 Variousnonviraldeliveryvectors,suchasLNPs,64–68 polymeric nanoparticles,67,69,70 lipidenvelopedhybridnanoparticles,66

protein/peptide-basednanoparticles,67,68 lyotropicliquidcrystallinelipidnanoparticles(LCNPs),71,72 inorganicnanoparticles,65 nanoemulsions,73,74 exosomes,64,75 hydrogels,76,77 polymeric nanoparticlegel,78 andbiologicalmembrane-basedvesicles,79 havebeendevelopedforefficientandversatilemRNAdelivery. OptimizingtherouteofmRNAdrugadministrationisessentialfor enhancingtherapeuticefficacy,asitcansignificantlyimpactthe biodistributionofthedrugwithintissues.Generally,intravenously injectedmRNAdrugstendtoaccumulateintheliver,whereas locallyadministratedonesoftenprolongthedurationofprotein expressionattheinjectionsiteanddeliversustainedtherapeutic effects.Subcutaneousandintramuscularinjectionsrepresentthe predominantroutesforadministeringmRNAvaccines.80 AlternativemRNAdrugdeliverymethodsincludeaerosolinhalationto directlytargetthelungs,81–83 eyedrops,84 intravitrealadministration,subretinaladministration,80 oraladministration,85,86 transcutaneousroute,87 andinuterodelivery.88

Despitethedevelopmentofvarioustypesofdeliveryvectorsin preclinicalstudies,LNPsremaintheestablishedpreferreddelivery systemformRNAduetoitsclinicalvalidation.89 Therefore,a comprehensiveunderstandingofthedesignstrategiesemployed inLNPsisessentialfortheeffectiveimplementationofmRNA therapies.ThecurrentformulationofLNPsfornucleicaciddelivery typicallycomprisesfourkeycomponents:ionizablelipids,helper lipids,cholesterol,andpolyethyleneglycol(PEG)-lipids,90,91 with theratioof~50:10:38.5:1.5mol%.92 Ionizablelipidsplayacrucial roleinencapsulatingmRNAandfacilitatingthesuccessful transfectionofmRNAintocells.93 TheincorporationofPEGlipidsservesprimarilytoevademacrophagephagocytosisand ensureprolongedsystemiccirculationofLNPs.Theinclusionof helperlipidsandcholesterolallowsformodulationofLNPs rigidity.94 Kulkarnietal.95 demonstratedthathelperlipidsand cholesterolplaycrucialrolesintheformulationofLNPs,aidingin theencapsulationofgeneticmaterial.96 However,Suetal.97 broke awayfromthetraditionalLNPsmodelanddiscoveredthat cholesterolandphospholipidsmaynotbeessentialforLNPs.

ItisessentialtoelucidatetheconsiderationsfordesigningLNPs deliverysystems,encompassingpayloadefficiency,stability, circulationtime,facilitationofendosomalescape,biodegradability,immunogenicity,safety,andtargetingcapability.98 Theprecise structureofcationiclipidsispivotal,typicallycomprisinga positivelychargedheadandmultiplehydrophobictails.Even minoralterations,suchastheadditionorremovalofanatomora functionalgroup,cansignificantlyimpacttissuetargeting,cellular uptake,andendosomalescape.99 Thedesignofionizablelipids necessitatescarefulconsiderationofthepKavaluesofthe headgroupinordertoachieveanoptimalpH-dependent electrostaticpattern.98 Ionizablelipidsaretypicallypositively chargedinacidicpHenvironmentsandpossessthecapabilityto aggregatenegativelychargedmRNAintoLNPs.Atphysiological pH,theybecomeneutralizedtominimizetoxicityandenhance thebiocompatibilityofmRNALNPs.Uponuptakebycellsinto acidicendosomes,theyundergoprotonation,therebypromoting thetranslocationofmRNAfromendosomesintothecytosol.93 The pKavaluesofionizablelipidscanbemodifiedbyalteringthe chemicalcompositionoftheheadgroup,suchasimidazole,ester, andpiperazine.98 Thehydrophobictailsoftheionizablelipidsplay acrucialroleinfacilitatingtheassemblyofLNPsandensuringthe stableencapsulationofmRNA.Thedesignofthelinkerregion betweentheheadgroupandthelipidtailshasasignificantimpact ontheoverallpHsensitivityoftheionizablelipids,consequently influencingboththereleasekineticsandmagnitudeofmRNA.61 Theshapeofionizablelipidscanalsoimpacttheirfunctionality. Moreconicalionizablelipids,withamolecularstructurethatisnot compatiblewiththelipidbilayer,canfacilitatemRNAescapefrom lysosomes.100

ThesafetyandstabilityconcernsassociatedwithmRNAtherapy encompassvariousfacets.Withregardtosafety,itisimperativeto

assessthebiocompatibilityofmRNAanditsdeliverysystem, minimizedirectcellulardamage,andmitigatetheelicitationof unnecessaryimmuneresponses.101 However,thepresenceofpHdependentorpermanentcationicpropertiesmayresultinsafety concerns,suchasdisruptionofcellularmembranesandorganelles,releaseofdegradativeenzymesfromlysosomes,and damagetoDNA.Asaresult,biocompatiblemolecularcomponents capableofformingcovalentesteroramidebondshavebeen developedfortheproductionofbiodegradableionizablecationic lipids.102 Intermsofstability,thecarrierdesignshouldensure protectionofthemRNAfromenzymaticdegradationwithout impactingitsrelease.101 Inaddition,enhancingthethermal stabilityofmRNALNPsiscrucialforreducingstoragecosts.For example,inordertoenhancethethermostabilityofmRNALNPs, researchersdevelopedanovelionizablelipidDOG-IM4modiﬁed withimidazole.103

ModifyingthephysicalandchemicalcharacteristicsofLNPs, suchastheirsize,surfacecharge,andsurfacehydrophobicity,can impacttheirbiologicalactivity.104 Generally,hydrophilicLNPswith aneutralchargeandasizesmallerthan100nmexhibitprolonged circulationinthebloodstream.LNPswithapositivechargeare moreconducivetocellularuptake.105 Themorphologyand nanostructureofLNPsarecorrelatedwiththetransfection efficiencyofmRNA.Nevertheless,themorphologyofmRNALNPs remainsambiguous,necessitatingfurtherresearchforelucidation.106 Furthermore,thepreciselocalizationofthefourcomponentswithintheLNPsremainsunclear,posingcertainlimitations inmoleculardesign.107 Inordertotacklemoredemandingtasks forLNPs,suchastargetedfunctionality,cellularpenetration,and endosomalescape,thesurfaceofLNPscanbesubjectto modification.PolymerslikePEG-modifiedlipidsarethepredominantsurfacemodifiersutilizedforLNPs.108 Nevertheless,surfacemodifiedLNPsmaypresentpotentialadverseeffects,suchas allergicresponsestriggeredbylipidsmodifiedwithPEG.61 Furthermore,thechallengesinscalinguptheproductionof surface-modifiedLNPscouldimpedetheircommercialization. 108 Presently,thecommonlyutilizedmethodologiesforformulating LNPsencompassthethin-filmhydrationapproach,solvent injectiontechnique,reverseevaporationmethod,andmicrofluidic technology.109 Dependingonthespecificapplication,microfluidic chipscanbefabricatedfromavarietyofmaterialssuchassilicon, glass,polydimethylsiloxane(PDMS),cyclo-olefinpolymersand copolymers(COPs/COCs).Inaddition,variousfabricationmethods includingphotolithography,electronbeamlithography,wetand dryetching,andembossingcanbeemployed.Microfluidic technologyoffersnumerousadvantagesintheproductionof LNPs,facilitatingcontinuousliquid flowwithinthemicrofluidic platformtoensureconsistentnanoparticlequalityovertimeand mitigatebatch-to-batchvariations.Furthermore,thedesign flexibilityofmicrofluidicchannelsenablesadaptationtodifferent rapidmixingmodes.110 Microfluidictechnologyhasnotonly yieldedremarkableresultsinlaboratoryresearch,butalsobeen adoptedandintegratedintoindustrialproductionprocesses.The microfluidicdeviceutilizedinthepreparationofBNT162b2isan impingementjetmixer.111 Furthermore,ongoingadvancementsin automatedhigh-throughputpreparationtechniquesarecontinuouslyenhancingtheproductionofLNPs112 andothernanodeliveryvectors.113

PRECLINICALANDCLINICALAPPLICATIONSOFMRNABASEDDRUGS

Immunotherapy

mRNAvaccinesoutplayedtheirtraditionalcounterpartsinrapid developmentandcost-effectivenesstrait,114 renderingthema promisingtoolforimmunizationagainstviruses,tumors,bacteria, andparasite.DespitethefactthatmRNAwasdiscoveredin1961, therevolutionarybreakthroughofusingitasavaccinehasonly

Fig.2 mRNAcodesforimmunotherapy-associatedantigen,antibody,cytokine,ligand,tumorsuppressorprotein,andadoptivecelltherapy. RVrotavirus,VZVvaricella-zostervirus,RSVrespiratorysyncytialvirus,CMVcytomegalovirus,HPVhumanpapillomavirus,EBVEpstein–Barr virus,RABVrabiesvirus,HIVhumanimmunodeﬁciencyvirus,MPXVmonkeypoxvirus,anti-VEGFanti-vascularendothelialgrowthfactor.The graphiciscreatedwithBioRender.com

occurredinrecentyears,duetoitssusceptibilitytoenzymatic degradation,inefﬁcientinvivodelivery,andintrinsic immunogenicity.115

Inordertoachieveantiviral,antitumor,antibacterial,and antiparasiticeffects,mRNA-basedimmunotherapiesmustelicita robustandspeciﬁchostimmuneresponsetoeliminatepathogens andconferdurableprotection.28 mRNAvaccinesencoding disease-speciﬁcantigensfunctionbytransfectingantigenpresentingcells(APCs).Theproteinantigenistranslatedwithin thecellandreleasedintotheextracellularspaceordegradedby theproteasometoexposetheantigenicsites.Intrinsicantigens canbepresentedbythecellsurfacemajorhistocompatibility complexI(MHCI)toinducematurationofclusterofdifferentiation (CD)8+ Tcells,whileextracellularantigenproteinscanbe recognizedbyBcells,internalized,degradedbyAPCcells,and presentedthroughtheMHCIIclasspathwaytoactivateCD4+ T helper(Th)cells.28,116

Inadditiontodisease-speciﬁcantigens,proteinsrelatedto immunotherapyencompassantibodies,cytokines,ligands,tumor suppressorproteins,andotherfunctionalproteins.Itisworth notingthatmRNAtherapiesencodingcytokinescouldbeutilized toeitherenhanceorsuppresstheimmuneresponse.Cytokines areaclassofproteinsthatmodulateimmunecellsbyactivating downstreamcytokinereceptors.117 Stimulatorycytokinesare suitableforcombatinginfectiousdiseasesandcancer,while inhibitorycytokinesareappropriatefortreatingautoimmune diseases.Itisnoteworthythatcertaincytokines,suchas Interleukin(IL)-2,donothavea ﬁxedfunctioninregulatingthe immuneresponse;rather,theirfunctionchangeswithvariations inconcentration.118, 119 Moreover,mRNA-basedadoptivecell

therapiesmaynotbeexclusivelyutilizedforimmunemodulation orsuppression,contingentupontheproteinencodedbythe mRNA.Forinstance,BNT211isemployedinthetreatmentof CLDN6-positiveadvancedsolidtumors,120 whileDescartes-08is designatedforautoimmunedisorders.121

Ingeneral,mRNApossessesthecapacitytoencodediverse proteinsincludingantigens,antibodies,ligands,andtumor suppressorproteins.Inaddition,mRNAcanalsobeharnessedin combinationwithadoptivecelltherapy(Fig. 2),thusshowcasing immensepotentialwithintherealmofimmunotherapy(Table 1).

Disease-speciﬁcantigentherapy

VirusantigenmRNAvaccine: SARS-CoV-2: In2019,thenovel coronavirusemergedglobally,leadingtowidespreadtransmission andthetragiclossofnumerouslives,therebyposingahuge threattoglobalhealthandsanitation.Thescarcityofaspeciﬁc therapeuticagentforpost-infectiontreatmentwithSARS-CoV-2 causedglobalpanic.Inresponsetothischallenge,avarietyof SARS-CoV-2vaccineshavebeenpropelledintoanintenseand acceleratedresearchphase.mRNAvaccines,representingan innovativetechnologyforthepreventionofinfectiousdiseases, wereinitiallyanticipatedtorequire5–6yearstoreachthe market.122 Encouragingly,followingthepublicdisclosureofthe coronavirusRNAsequenceinJanuary2020,122 ittooklessthan3 monthsforthemRNAvaccines,mRNA-1273(NCT04283461,Phase IinitiatedinMarch2020)andBNT162b2(NCT04380701,PhaseI andPhaseIIinitiatedinApril2020),totransitionfromdevelopmenttoclinicaltrials.123 Remarkably,withinayear,bothmRNA vaccines(BNT162b2andmRNA-1273)rosetoprominence, securingemergencyapprovalfromtheU.S.FDAandwere

Table1. Representativecompletedandongoingclinicalstudies(immunotherapy)

PhaseStatusResultNCTnumberSponsorRef.

Study started

Administration route

Delivery system

TherapySubclassmRNAdrugApplicationmRNA-encoded protein

486

NCT04470427ModernaTX, Inc.

2020-07-27PhaseIIICompletedShow94.1% ef ﬁ cacyin preventing COVID-19disease, withnosafety concernsfound exceptfor transientlocalor systemicreactions

Intramuscular injection

LNPs(SM-102, PEG2000-DMG, DSPC, cholesterol)

mRNA-1273COVID-19Prefusionstabilized full-lengthspike protein

Virus antigen

Disease- speci ﬁ c antigen therapy

NCT04368728BioNTechSE 487

2020-04-29PhaseII/IIICompletedGoodimmune ef fi cacyagainst COVID-19witha sustainedsafety pro fi leand acceptable adverseevents, buttheimmune ef fi cacydeclines after6months

Intramuscular injection

LNPs(ALC- 0315,ALC- 0159,DSPC, cholesterol)

BNT162b2COVID-19SARS-CoV-2full- lengthspikeprotein

2023-10-06PhaseIIIRecruiting/NCT06067230ModernaTX, Inc. 479

Intramuscular injection

LNPs(ionizable lipid, phospholipid, PEGlipid, sterol)

mRNA-1345RSVRSVprefusion stabilizedF(preF) glycoprotein

NCT05566639ModernaTX, Inc. 134 , 488

2022-09-14PhaseIIICompletedAcceptablesafety andtolerability support continuedstudy

LNPsIntramuscular injection

Membrane-bound hemagglutinin(HA) surfaceglycoproteins offourin ﬂ uenza strains(A/H1N1,A/ H3N2,B/Victoria,and B/Yamagata)

mRNA-1010Seasonal in ﬂ uenza

2021-10-26PhaseIIIActive,not recruiting /NCT05085366ModernaTX, Inc. 489

LNPsIntramuscular injection

mRNA-1647CMVTwoCMVantigens (glycoproteinBand thepentameric glycoprotein complex)

NCT03014089ModernaTX, Inc. 490

2016-12-21PhaseICompletedWelltolerated,but poorZikavirus- speci ﬁ cnAb responses

LNPsIntramuscular injection

mRNA-1325ZikavirusPremembraneand envelopeEstructural proteins(prME)from aMicronesia2007 Zikavirusisolate

NCT04064905ModernaTX, Inc. 490

2019-07-30PhaseICompletedWelltolerated, inducestrong Zikavirus-speci ﬁ c serumnAb responsesafter twodosesthat supportedthe continuedstudy ofmRNA-1893

LNPsIntramuscular injection

mRNA-1893ZikavirusprMEfromtheRIO- U1Zikavirusisolate

NCT03325075ModernaTX, Inc. 491

2017-08-15PhaseICompletedGoodsafetyand immunogenicity

mRNA-1388CHIKVFullCHIKVstructural polyprotein(capsid andenvelope proteinsE3,E2,6k/TF, andE1)fromCHIKV WestAfricanstrain 37,997 LNPs (cholesterol, DPSC, ionizablelipid MC3,PEG2000- DMG) Intramuscular injection

Table1. continued

PhaseStatusResultNCTnumberSponsorRef.

Study started

Administration route

Delivery system

TherapySubclassmRNAdrugApplicationmRNA-encoded protein

492

NCT03076385ModernaTX, Inc.

2015-12PhaseICompletedWelltolerated, triggerstrong humoralimmune responses

LNPsIntramuscular injection

Full-length, membrane-bound formoftheHA glycoproteinfromthe H10N8in ﬂ uenza strain(A/Jiangxi- Donghu/346/2013)

In ﬂ uenza (H10N8)

H10N8mRNA vaccine(VAL- 506440)

492

NCT03345043ModernaTX, Inc.

2016-05-11PhaseICompletedWelltolerated, triggerstrong humoralimmune responses

LNPsIntramuscular injection

Full-length, membrane-bound formoftheHA glycoproteinfromthe H7N9in ﬂ uenzastrain (A/Anhui/1/2013)

In ﬂ uenza (H7N9)

H7N9mRNA vaccine(VAL- 339851)

NCT02241135CureVac 13

2013-10PhaseICompletedGenerallysafeand reasonably tolerated

Intradermal, intramuscular injection

Cationic protein protamineas stabilizerand adjuvant

CV7201RabiesRabiesvirus glycoprotein

493

NCT03713086CureVac

2018-10-12PhaseICompletedLowdoses(1 μ gor 2 μ g)werewell- tolerated,whereas the5 μ gdose exhibited unacceptable reactogenicity

Intramuscular injection

CV7202RabiesRabiesvirus glycoprotein LNPs (cholesterol, DSPC, PEGylated lipid,cationic lipid)

2022-05-25PhaseIActive,not recruiting /NCT05414786International AIDSVaccine Initiative 156

Intramuscular injection

mRNA-1644HIVeOD-GT860merSelf- assembling nanoparticles

494 , 495

2021-11-12PhaseIActive,not recruiting /NCT05001373International AIDSVaccine Initiative

HIVCore-g28v260merLNPsIntramuscular injection

mRNA- 1644v2-Core

2023-09-21PhaseIRecruiting/NCT05988203BioNTechSE 167

LNPsIntramuscular injection

BNT166aMPXVMPXVantigensA35, B6,H3,andM1

2022-11-18PhaseIRecruiting/NCT05714748WestChina Hospital 496

UndisclosedUndisclosedIntramuscular injection

EBV-positive advanced malignant tumors

EBVmRNA vaccine

NCT04534205BioNTechSE 497

2021-01-07PhaseIIRecruitingAcceptablesafety pro ﬁ le

LiposomalIntravenous injection

HPV16oncoproteins E6andE7

BNT113HPV16positive headandneck squamouscell carcinoma (HNSCC)that expressesPD- L1

498

2023-02-15 (estimated) PhaseIRecruiting/NCT05738447WestChina Hospital

HBsAgLNPsIntramuscular injection

HBV-associated refractory hepatocellular carcinoma

HBVmRNA vaccine

NCT02410733BioNTechSE 174

2015-03PhaseICompletedStrong immunogenicity andpromising clinicalactivity

LipoplexesIntravenous administration

BNT111MelanomaMelanomaTAAs:New Yorkesophageal squamouscell carcinoma1(NY-ESO- 1),tyrosinase, melanoma-associated antigen3(MAGE-A3), andtransmembrane phosphatasewith tensinhomology (TPTE)

Tumor antigen

Table1. continued

PhaseStatusResultNCTnumberSponsorRef.

Study started

Administration route

Delivery system

TherapySubclassmRNAdrugApplicationmRNA-encoded protein

NCT04382898BioNTechSE

2019-12-19PhaseI/IITerminatedAcceptablesafety pro ﬁ le,induce robustprostate antigen-speci ﬁ c immune responsesin patientswith advancedprostate cancer

LipoplexesIntravenous bolusinjection

BNT112ProstatecancerProstatecancerTAAs: kallikrein-2,kallikrein- 3,acidphosphatase prostate,homeobox B13(HOXB13),and NK3homeobox1

2022-06-17PhaseIRecruiting/NCT05142189BioNTechSE

UndisclosedIntravenous injection

Sixsharedantigens frequentlyexpressed innon-smallcelllung cancer

BNT116Non-smallcell lungcancer

481

NCT03897881ModernaTX, Inc.

2019-07-18PhaseIIbRecruitingSigni ﬁ cantly extendeddistant metastasis-free survivalin patientswith resectedhigh-risk melanomaas comparedwith Pembrolizumab monotherapy

LNPsIntramuscular injection

mRNA-4157MelanomaUpto34patient- speci ﬁ ctumor neoantigens

501

2023-07-19PhaseIIIRecruiting/NCT05933577MerckSharp &DohmeLLC

LNPsIntramuscular injection

mRNA-4157MelanomaUpto34patient- speci ﬁ ctumor neoantigens

502

NCT04161755Memorial Sloan Kettering Cancer Center

Preliminarily showntobesafe incombination with Atezolizumaband mFOLFIRINOXand todelay recurrencein patientswith surgically removed pancreaticductal adenocarcinoma (PDAC)

2019-12-13PhaseIActive,not recruiting

Intravenous delivery

NeoantigenLipoplex

nanoparticles

Pancreatic cancer

Autogene cevumeran (RO7198457)

503

2023-04-20NotapplicableNotyet recruiting /NCT05761717Shanghai Zhongshan Hospital

LNPsSubcutaneous injection

LivercancerPersonalizedtumor neoantigen

Personalized tumor neoantigen

mRNA vaccine

504

2019-10-18NotapplicableRecruiting/NCT03908671Stemirna Therapeutics

TumorneoantigenLNPsSubcutaneous injection

Advanced esophageal cancerand non-smallcell lungcancer

Tumor neoantigen

mRNA vaccine

505

NCT03639714Gritstonebio, Inc.

2019-02-13PhaseI/IICompletedWelltolerated, induce neoantigen- speci ﬁ cCD8 + T-cellresponsein allpatients, subsequentPhase II/IIIinitiated (NCT05141721)

Intramuscular injection

Chimpanzee adenovirus vector

SolidtumorsNeoantigen (#samRNA)

GRT-C901 andGRT- R902

Table1. continued

PhaseStatusResultNCTnumberSponsorRef.

Administration route Study started

TherapySubclassmRNAdrugApplicationmRNA-encoded protein Delivery system

2023-07-26PhaseI/IIActive,not recruiting /NCT05975099ModernaTX, Inc 188

LymediseaseUndisclosedUndisclosedIntramuscular injection

mRNA-1975 andmRNA- 1982

Bacterial antigen

2023-07-31PhaseI/IIRecruiting/NCT05547464BioNTechSE 453

TuberculosisUndisclosedUndisclosedIntramuscular injection

BNT164a1 and BNT164b1

2022-12-15PhaseIActive,not recruiting /NCT05581641BioNTechSE 201

LNPsIntramuscular injection

BNT165b1MalariaPartofthe Plasmodium falciparum circumsporozoite protein(PfCSP)

Parasite antigen

506

NCT03829384ModernaTX, Inc.

2019-01-22PhaseICompletedAcceptablesafety pro ﬁ le,produce highlevelsof functionally neutralizing antibodies

Intravenous infusion

AntibodymRNA-1944CHIKVLightandheavy chainsofahuman monoclonalantibody (CHKV-24IgG) targetingtheCHIKV E2glycoprotein LNPs(a proprietary IAL;a proprietary high-purity PEG-2k- stearate monoester; cholesterol, DOPE)

Therapeutic antibody therapy

2022-01-18PhaseI/IITerminated/NCT04683939BioNTechSE 507

LNPsIntravenous injection

Anti-CLDN18.2 antibodies

BNT141Unresectable ormetastatic CLDN18.2- positivegastric, pancreatic, ovarianand biliarytract tumors

2022-03-28PhaseI/IIRecruiting/NCT05262530BioNTechSE 508

LNPsIntravenous bolus/infusion

BNT142CLDN6-positive solidtumors T-cell-engaging bispeci ﬁ cantibody againstCLDN6and theT-cell receptor –associated moleculeCD3

2010-01PhaseITerminated/NCT01216436Duke University 236

DCsIntranodal injection

MelanomaAnti-CTLA-4andanti- GITRmAband melanomaTAAs MART,tyrosinase,and gp100,andMAGE-3

RNA- transfected mature autologous DC

2019-05-08PhaseICompleted/NCT03946800MedImmune LLC 259

MEDI1191SolidtumorsIL-12LNPsIntratumoral injection

CytokineImmune- potentiating

NCT03739931ModernaTX, Inc. 509

2018-11-27PhaseIActive,not recruiting Tolerated,maybe associatedwith tumorshrinkage

OX40L,IL-23,andIL- 36 γ LNPsIntratumoral injection

mRNA-2752Advancedsolid tumorsand lymphoma

NCT03871348Sano ﬁ 263

2019-01-03PhaseITerminatedWelltolerated (clinicaltrialin17 patients;July 2020),supporting furtherclinical studies

Intratumoral injection

Saline- formulated mixture

SAR441000SolidtumorsIL-12singlechain,IFN ɑ -2b,GM-CSF,andIL- 15sushi

SolidtumorsIL-2,IL-7LNPsIntravenous2021-06-08PhaseIRecruiting/NCT04710043BioNTechSE 510

BNT153and BNT152

511

2021-07-28PhaseICompleted/NCT04916431ModernaTX, Inc.

LNPsSubcutaneous injection

Humanserum albuminIL-2mutein fusionprotein(HSA- IL2m)

mRNA-6231Healthy volunteers

Immune- suppressing

Table1. continued

PhaseStatusResultNCTnumberSponsorRef.

Study started

Administration route

Delivery system

TherapySubclassmRNAdrugApplicationmRNA-encoded protein

Ziekenhuis Brussel 512

2018-11-12PhaseIRecruiting/NCT03788083Universitair

UndisclosedIntratumoral administration

LigandLigandTriMixBreastcancerDC-activating[CD40 ligand(CD40L),CD70, andconstitutively activeToll-like receptor4(TLR4)] proteins

120

NCT04503278BioNTechCell &Gene Therapies GmbH

2020-09-16PhaseI/IIRecruitingGoodsafety pro ﬁ leand encouraging ef ﬁ cacy

CLDN6LipoplexesIntravenous injection

CLDN6-positive advancedsolid tumors

CAR-TCLDN6CAR-T cells + / CARVac

Adoptive celltherapy

NCT04146051Cartesian Therapeutics

Infusion2019-12-04PhaseIb/IIaRecruitingSafeandwell- tolerated

AutogolousT cells

Anti-BCMAtargeting CARprotein

NCT02719782LionTCRPte. Ltd. 513

Welltolerated, havenoadverse effectsonthe transplantedliver

Infusion2015-07-02PhaseIUnknown status

121 TCR-TLioCyx-MRecurrent hepatocellular carcinoma HBV-speci ﬁ cTCRAutologousT cells

329

NCT03608618MaxCyte,Inc.

2018-08-27PhaseITerminatedMediateeffective andlong-term antitumor response

PBMCsIntraperitoneal infusion

Anti-mesothelinCAR (Meso-CAR)

CAR-TDescartes-08Myasthenia gravis

CAR-PBMCMCY-M11Advanced ovariancancer andperitoneal mesothelioma

DCsUndisclosed2008-02PhaseIICompleted/NCT00672191Argos Therapeutics 514

DCsAGS-004HIVCD40Landthreeor fourautologousHIV-1 antigens

DCsUndisclosed2012-10PhaseIIActive,not recruiting /NCT01686334Zwi Berneman 515

WT1antigenmRNA loadedautologous DCs(by electroporation)

Acutemyeloid leukemia(AML)

WT1antigen- targetedDC vaccine

2009-09PhaseICompleted/NCT00978913IngeMarie Svane 516

DCsIntradermal injection

Survivin,hTERTand p53

Metastatic breastcancer ormalignant melanoma

DCvaccine (DCs transfected withSurvivin, hTERTand p53mRNA)

NCT02366728Mustafa Khasraw, MBChB,MD, FRCP,FRACP 517

2015-10-12PhaseIICompletedEffectiveGBM inhibition

GBMCMVproteinpp65DCsIntradermaland bilateral administration atthegroinsite (dividedequally tobothinguinal regions)

HumanCMV pp65-LAMP mRNA-pulsed autologous DCs

LNPs lipidnanoparticles, PEG polyethyleneglycol, DSPC distearoylphosphatidylcholine, RSV respiratorysyncytialvirus, CMV cytomegalovirus, CHIKV chikungunyavirus, HIV humanimmunode ﬁ ciencyvirus, MPXV monkeypoxvirus, EBV Epstein –Barrvirus, HPV humanpapillomavirus, HBV hepatitisBvirus, TAAs tumor-associatedantigens, DOPE 1,2-dioleyl-sn-glycero-3-phosphoethanolamine, CLDN6 claudin6, DCs dendritic cells, IL interleukin, IFN interferon, GM-CSF granulocyte-macrophagecolony-stimulatingfactor, TriMix mRNA-encodingCD40L,CD70,andTLR4, CAR chimericantigenreceptor, BCMA B-cellmaturationantigen, TCR T-cellreceptor, PBMC peripheralbloodmononuclearcells, WTI Wilms ’ tumor, GBM glioblastoma

Fig.3 ApplicationofmRNAvaccineinimmunotherapy. a Typesof COVID-19vaccinesinclinicaltrials(fromWHO;March30,2023). b mRNA-baseddrugsinPhaseIII/IVtrials.Onclinicaltrial.gov,search for “mRNA” astheonlykeywordandthesearchcriteriaarelimitedto “Phase3” and “Phase4”.Notethatthissearchmethodcannot ﬁndall mRNAvaccines,andsomemRNAvaccinesdonotcontaintheword “mRNA”,sothe ﬁgureshouldbevieweddialectically.PSprotein subunit,VVnrviralvector(non-replicating),IVinactivatedvirus,VVr viralvector(replicating),VLPvirus-likeparticle,VVr + APCVVr + antigen-presentingcell,LAVliveattenuatedvirus,VVnr + APC VVnr + antigen-presentingcell,BacAg-SpVbacterialantigen-spore expressionvector

authorizedforglobalmassvaccinationagainstSARS-CoV-2.122,124 AccordingtotheconsolidatedinformationprovidedbytheWorld HealthOrganization(WHO),asofMarch30,2023(Fig. 3a),atotal of183vaccineshadenteredtheclinicalstage,withRNA-based vaccinesaccountingforabout24%(43intotal),rankingsecond onlytoproteinsubunitvaccineswhichaccountforaround32% (59intotal).125

Rotavirus: ThesuccessfulapplicationofmRNAvaccinesagainst COVID-19diseasehaspromptedresearcherstoswiftlyexpand themtootherantiviral fields.Rotaviruses(RVs)areresponsiblefor causingdiarrheainchildrenworldwideandcanresultin subsequentgastroenteritis,yetthereisnospecifictreatmentfor thisvirus.Luetal.14 designedanmRNAvaccineencodingtheVP7 protein,whichisoneofthecapsidproteinsofRVsandplaysan importantroleintheinfectionoftargetcells,forpreventingRVs infection.TheVP7mRNAvaccinewasinoculatedtomicevia intramuscularorsubcutaneousinjection(threedoses).The findingsdemonstratedthattheVP7mRNAvaccineeffectively stimulatedT-cellimmuneresponsesandelicitedRV-specific antibodies.

Varicella-zostervirus: Varicella-zostervirus(VZV),amemberof thealphaherpesvirussubfamily,isaneurotropichumanherpesvirus.126 Theinitialinfectionmanifestsaschickenpoxsymptoms, afterwhichthevirusremainslatentinthebody.Reactivationof thevirusoccurswhentheimmunesystemweakensduetoaging orcompromisedimmunity.127 In2024,Huangetal.128 developed anmRNA-basedVZVvaccinecalledZOSAL,whichemployed ionizableLNPsforencapsulatingsequence-optimizedmRNA encodingthefull-lengthglycoproteinE.Inthisstudy,the immunogenicity,safety,andimmunemechanismsofZOSALwere comparedwiththoseofthelicensedprotein-basedvaccine Shingrixinmiceandrhesusmacaques.The findingsdemonstrated thatZOSALexhibitedsuperiorimmunogenicity,safetyprofile,and capacitytoinducevirus-specificT-cellimmunitywhencompared toShingrix.

Respiratorysyncytialvirus: Respiratorysyncytialvirus(RSV)is anenveloped,single-strandedRNAvirus,whichcancause respiratoryillnesses.129 RSVcanresultinfataloutcomesfor

pediatric,geriatric,andimmunocompromisedindividuals.130 One ofthehighlyprogressivevaccinesaimedatcombatingRSVis mRNA-1345,whichispresentlybeingdevelopedbyModernaTX, Inc.Thisvaccineencodesthemembrane-anchoredRSVprefusion stabilizedF(preF)glycoprotein.131 Currently,themRNA-1345 vaccineforRSVisundergoingPhaseIIItrial(NCT05330975, NCT06067230),andithasalreadyreceivedFDAapprovalviafasttrackdesignationforadministrationinindividualsaged60years andolder.132

Influenzavirus: Influenzaviruseshavetheabilitytoinfecta widerangeofvertebratesandareresponsibleforcausing seasonalinfluenzaaswellasinfluenzapandemicsinhumans.133 ModernaTX,Inc.developedaquadrivalentmRNAvaccine(mRNA1010)targetingseasonalinfluenza,encodingthefourhemagglutinin(HA)surfaceglycoproteinsofinfluenzastrains(A/H1N1,A/ H3N2,B/Victoria,andB/Yamagata).Thevaccinehasundergone testinginhealthyadultsduringPhaseI/IItrial(NCT04956575),with interimresultsdemonstratingfavorablesafety.134 Further,ithas currentlyenrolled22,510adultsaged50yearsandolder (NCT05566639)135 and8400adultsaged18yearsandolder (NCT05827978)136 toinitiatePhaseIIIsafetyandefficacystudies.

Cytomegalovirus: Cytomegalovirus(CMV)isanincredibly prevalentvirus,withestimatedincidenceratesof60%in developedcountriesand90%indevelopingcountries.Although asymptomaticinhealthyindividuals,infectionwithCMVcanpose asignificantrisktoimmunocompromisedpatients(suchasthose undergoingorgantransplantation),potentiallyleadingtograft rejectionandevenlife-threateningcomplications.Inaddition,itis imperativetoacknowledgetheissueofverticaltransmissionof CMVfrompregnantwomentofetuses,whichcanresultin prematurebirthandprofoundpermanentdisabilitiesinneonates, encompassingcognitiveimpairmentandvisualdeficits.Inorderto tacklethisconcern,ModernaTX,Inc.developedanmRNAvaccine (mRNA-1647)thatencodestwoCMVproteins,namelyglycoproteinB(gB)andpentamericgH/gL/UL128/UL130/UL131Aglycoproteincomplex(pentamer).Thevaccinehassuccessfully completedPhaseIclinicaltrial(NCT03382405),demonstrating thatadministrationofthreedosesofmRNA-1647(180µg)could effectivelyelicithightitersofneutralizingantibodies(nAbs),broad neutralizationactivity,robustT-cellresponse,andlong-lasting memoryBcellsinhealthyadults,137 whilemaintainingan acceptablesafetyprofile.138 Huetal.139 comparedmRNA-1647 (NCT03382405)withMF59-adjuvantedgBsubunit(gB/MF59) vaccine(NCT00133497),andobservedthatalthoughthegBspecificIgGresponseswerelowerafterinoculationwithmRNA1647vaccine,itelicitedpersistentHCMV-specificantibody responsesandhigherantibody-dependentcellularcytotoxicity responses.Inaddition,aPhaseIIItrialwasconductedinhealthy femaleparticipantsaged16–40yearswithmRNA-1647 (NCT05085366).140

Humanpapillomavirus: Thedistinctivepropertiesofcertain viruses,suchascarcinogenicity,rendermRNAvaccinesencoding viralproteinsapromisingstrategyforcombatingtumors.HPV infectioniswidelyrecognizedastheprimaryfactorcontributingto cervicalcancer.141,142 TheE6andE7proteinsofhuman papillomavirus(HPV)havebeenestablishedtomodulatecell cycleandexhibitahighassociationwithcervicalcancer.Inlightof this,Leeetal.143 designedanmRNAvaccineencodingtheE6and E7proteins,whichwassubsequentlyadministeredtoC57BL/6J miceviaintramuscularorsubcutaneousinjection.Their findings revealedthatthemRNAvaccinemarkedlyelicitedrobustT-cellmediatedimmuneresponsesintumor-bearingmice,leadingtoa significantinhibitionoftumorgrowth.Importantly,theexperimentalresultsdemonstratedthatthevaccinenotonlyexhibited therapeuticpotentialagainstexistingtumorsbutalsodisplayed preventiveefficacy.

Epstein–Barrvirus: Epstein–Barrvirus(EBV),anoncogenicvirus inhumans,isfrequentlyassociatedwithnasopharyngeal

carcinoma(NPC).LMP2isoneofthemainviralproteins.Xiang etal.144 deliveredLMP2-mRNALNPstotumor-draininglymph nodes(TDLN)forthepurposeofinducingactivationand cytotoxicityofCD8+ TcellsagainsttumorcellsexpressingLMP2. Zhaoetal.145 developedthreetherapeuticEBVmRNAvaccines thatencodedtruncatedlatentEBVproteinregionscontaining abundantT-cellepitopes,includingtruncatedformsoflatent membraneprotein2A(LMP2A),EBVnuclearantigen1(EBNA1), andEBVnuclearantigen3A(EBNA3A).Their ﬁndingsdemonstratedthattheseEBVmRNAvaccinescouldeffectivelyelicit antigen-speciﬁcimmuneresponses,therebysuppressingtumor progressionandprolongingthesurvivaltimeoftumorbearingmice.

Rabiesvirus: Theneurotropicrabiesvirus(RABV),amemberof theLyssavirusfamilyandasingle-strandedRNAvirus,146 hasthe abilitytoinfectallwarm-bloodedanimals,147 includinghumans, leadingtothedevelopmentofrabies.Rabiesisafatalneurological disease,andoncetheinitialsymptomsmanifest,thereisminimal timetopursuetreatmentoptions,asthemortalityrate approaches100%.146 Rabiescausesanestimated59,000fatalities annuallyonaglobalscale.148 Thecurrentlymostwidelyused rabiesvaccinesareallinactivated,requiringindividualstoreceive threeto fivedosesforoptimalimmuneprotection. 149,150 In summary,despiteadvancementsinrabiesvaccinedevelopment, thecurrentavailabilityofvaccinesisconstrainedbyfactorssuch ashighcosts,limitedproductioncapacityandstoragerequirements,aswellasthenecessityformultipledoses.147 ThemRNA vaccinesrepresentapromisingapproachthathasthepotentialto reducecostsandlowertherequireddosageforachieving effectiveimmuneprotection. 150 RABVglycoprotein(RABV-G), beingtheonlyvirion-surfaceproteinofRABV,playsacrucialrole asanantigeninvaccinedevelopment.151 In2013,CureVac conductedtheinitialPhaseIclinicaltrialforanmRNA-based rabiesvaccineinhealthyadults(NCT02241135).Thetrial findings indicatedthatthemRNAvaccine(CV7201)encodingtheRABV-G demonstratedafavorablesafetyprofile.13 Subsequently,CureVac alsoprogressedamRNAvaccinenamedCV7202,encodingthe RABV-Gprotein,intoPhaseIclinicaltrial(NCT03713086).Currently, researchonthemRNAvaccineforRABV-Gisongoing.Caoetal.152 developedanovelmRNALNPsthatencodesRABV-Gwiththe H270Pmutation,whichcanstabilizetheprefusionconformation ofRABV-G.Their findingsdemonstratedthatthisvaccineelicited superiorhumoralandcellularimmuneresponsescomparedto mRNALNPsencodingRABV-G,indicatingthatstructured-guided vaccinedesignmaybethefuturedirectionforvaccine development.

Humanimmunodeficiencyvirus: Acquiredimmunodeficiency syndrome(AIDS),resultingfrominfectionwiththehuman immunodeficiencyvirus(HIV),remainsafataldiseasefor humans.153 Althoughcurrentantiretroviraltherapies154 and therapeuticvaccinescanimproveHIV-relatedmorbidityand mortality,theyarestillunabletocompletelyeradicateHIV.155 At present,theHIVmRNAvaccinesthathavebeendevelopedand enteredclinicaltrialsincludetheeOD-GT860mermRNAvaccine (mRNA-1644,NCT05414786)156 andtheCore-g28v260mermRNA vaccine(mRNA-1644v2-Core,NCT05001373).157 AsofJune2024, thesevaccinesarestillundergoingPhaseIclinicaltrials.Despite theevaluationofvariousHIVvaccinesinpreclinicalandclinical studies,theoveralloutcomeshavebeenunsatisfactory. 158 Further endeavorsareimperativeforthedevelopmentofaneffectiveHIV vaccine.Mandaletal.159 designedLNPscontainingmRNAthat encodedmultipleepitopesofHIVviralproteasecleavagesites. ThemRNALNPsexhibitedlong-termstabilityatcold-chain temperaturesandcouldelicitpotentcellularimmunity,making itasapromisingcandidateforaprophylacticHIVmRNAvaccine.A majorobstacleintheeradicationofHIVliesinthevirus’sabilityto concealitselfwithinhostcells,includingmyeloidcellsandCD4+ Tcells.160 Itappearsthatvaccinesalonemaynotbesufficientfor

curingHIV,promptingconsiderationofcombiningvaccineswith othertreatmentmodalities.155,160 Theextensivegeneticdiversity ofHIVanditscapacitytointegrateintothehostcellgenome undoubtedlyposesigniﬁcantchallengesforthedevelopmentof HIVvaccines.161,162

Monkeypoxvirus: Monkeypoxisacommunicabledisease causedbythemonkeypoxvirus(MPXV),whichcanbetransmitted betweenhumansandanimals.163,164 MPXVisatypeofdoublestrandedDNAvirus163,165 thatbelongstothe Orthopoxvirus genus.165,166 Currently,amultivalentmRNAvaccine(BNT166) encodingMPXVantigensA35,B6,H3,andM1hasenteredPhaseI clinicaltrial(NCT05988203)167.Severalpreclinicalstudiesare underwaytodevelopnovelmRNAvaccinestargetingvarious antigensofthevirus.Tianetal.168 designedanmRNA-based vaccineencodingtheMPXVA29Lantigen.Suetal.169 developeda quadrivalentmRNAvaccinethatencodedtheantigensA27,L1, A33,andB5ofvacciniavirus.Itisnoteworthythattheserafrom miceimmunizedwiththisvaccinedemonstratedreactivitywith theantigensofvariousorthopoxvirusesinvitro.Thissuggeststhat themRNAvaccineholdspromiseforsafeguardinghumansagainst MPXVandotherorthopoxvirusinfections.

TumorantigenmRNAvaccine:Cancerisamajorcauseofglobal mortality.170 TheinceptionofantitumormRNAvaccinescanbe tracedbackto1995,whenConryetal.171 constructedanmRNA encodingthetumorantigen–humancarcinogenicantigen(CEA) thatcouldbedirectlyinjectedintotheskintocombattumorsin mousemodels.In2004,aclinicaltrialwasinitiatedto intradermallyadministermRNA-encodingmelanoma-associated antigenstostageIII/IVmelanomapatients(NCT00204607).9 In 2009,Weideetal.10 reportedtheresultsofthePhaseI/IItrial, demonstratingthatthefeasibilityandsafetyofdirectinjectionof protamine-protectedmRNA,therebyencouragingfurtherclinical studiesonmRNAvaccines.

Acriticalstepinthedevelopmentofcancervaccinesinvolves theidentiﬁcationandselectionofappropriatetumorantigens, encompassingbothTAAsandtumor-speciﬁcantigens(TSAs).172

TAAsarenon-mutatedproteinsthatexhibithighexpressionin tumorswhileshowingno-to-lowexpressioninnormaltissues.173 TheRNA-LPXvaccine(BNT111),whichhassuccessfullycompleted PhaseIclinicaltrial(NCT02410733)andisnowprogressingto PhaseII(NCT04526899),isatumorvaccinedesignedtoencode TAAs(NewYorkesophagealsquamouscellcarcinoma1, melanoma-associatedantigen3,tyrosinase,andtransmembrane phosphatasewithtensinhomology)formelanoma.174,175 The resultsoftheclinicaltrialdemonstratedthattheintravenous administrationofBNT111vaccineinpatientscanelicitarobust andbroadT-cellresponseagainstTAAs.117 However,theuseof TAAsastumorantigensislimitedbytheirexpressioninnormal tissuestosomeextent,whichmayhinderthedevelopmentof effectiveantitumorimmuneresponsesowingtotheself-tolerance mechanismsandthepotentialforoff-targeteffectsthatmaylead toautoimmunetoxicity.173,176

TSAs,whichresultfrommutationsinsomaticcells,hold significantpotentialforinducingspecificT-cellresponsesagainst tumors.173 Forinstance,Moderna’smRNA-4157isanmRNA vaccinethatencodesupto34patient-specifictumorneoantigens.177 Itbecamethe firstmRNAcancervaccinetoenterPhaseIII clinicaltrial(NCT05933577).TheclinicalIIbtrialofmRNA-4157in combinationwithPembrolizumabtreatmentdemonstrateda49% reductionintheriskofmortalityamonghigh-riskmelanoma patients.176 TSAsarepromisingtargetsforcancervaccines.178 However,onlyasmallfractionofsomaticmutationsincancercells couldberecognizedbyspontaneouslyoccurringTcells,andthe efficacyoftheseneoepitopesinmediatingantitumoreffects varies,posingchallengesfortheirpreciseutilization.173

Toaddressthechallengeoflimitedavailabilityoftargetable neoantigensandantigentargetlossintumors,itisimperativeto

investigateandutilizethefullspectrumoftumorantigens,to developpersonalizedcancervaccines.Trivedietal.179 established animmunogenomicspipelinecalled “OpenReadingFramework AntigenNetwork(O.R.A.N.)” toeffectivelyidentifyimmunogenic antigenswithhighlikelihoodofbecomingtherapeutictargets.In addition,theydevelopedaplatformcalled “TumorOpenreading FramesthatareUnique(TOFU)” whichutilizesIVTmRNA technologyforencodingmultipletumorantigensintoasingle mRNAvaccine,therebyenablingthecustomizationofvirtually limitlessquantitiesofantigensspeciﬁctoeachtumortype.BenAkivaetal.180 developedaclassofbioreduciblenanocarriers utilizinglipophilicpoly(beta-aminoester)toencapsulateantigenencodingmRNAandToll-likereceptor(TLR)agonistadjuvantsfor thetreatmentofmurinemelanomaandcolonadenocarcinoma. DuetoitsmRNAsequence-independentencapsulationcapacity, thisplatformcanalsobeextendedtoaddressothercancer diseases.

ThestandalonemRNAcancervaccinemayserveasaneffective treatmentoptionforearly-stagecancer.However,inthecaseof advancedcancerpatients,thetumormicroenvironmentoften exhibitsahighlevelofimmunesuppression,therebydiminishing theefﬁcacyofmRNAmonotherapy.181 Instead,combining therapeuticcancermRNAvaccineswithotherimmunotherapies,182 suchasthecombinationofmRNA-4157andPembrolizumabtotreathigh-riskmelanoma,holdspromiseforsuccess.183 DespitethefactthatmRNAvaccinesencodingtumorantigens enteredPhaseItrialsearlierthanviralmRNAvaccines,itis disappointingthatfewmRNAvaccinesagainstcancerseemto haveadvancedtoPhaseIIIorIVtrials(Fig. 3b).184 Thereasonsare summarizedin “Conclusionsandprospects” section.Weanticipate aninﬂuxofapproveddrugsandmethodologiesforcancer treatment,instillingrenewedoptimisminpatients.

BacterialantigenmRNAvaccine:Theconventionalapproachto eradicatingbacteriainvolvestheadministrationofantibiotics. However,duetotheoveruseofthesedrugs,wehavebeen alarmedbytheemergenceofantibiotic-resistantbacteriaand eventhedevelopmentofhighlyresistantstrains.Nevertheless,itis importanttonotethattheresearchandapprovalprocessfor creatinganewantibioticisexceedinglytime-consumingand necessitatessubstantial financialresources.Regrettably,bacteria candevelopresistanceatamuchfasterratethannewdrugscan bedeveloped.Consequently,despiteourawarenessregardingthe imperativeneedforantibioticscontrolmeasures,wemay find ourselvesinasituationwherenoeffectivedrugexiststocombat bacterialinfections.Therefore,itisessentialtoproposean innovativeantimicrobialtherapy.GiventhatmammalianorganismsarecapableoftranslatingmRNA-encodingbacterialproteins, mRNAvaccinesofferapromisingandefficaciousavenuefor preventingbacterialdiseases.185,186 LymediseaseisaprevalentinfectiousailmentintheU.S.that currentlylacksaspecificvaccine.Amongvariouscandidates,outer surfaceproteinA(OspA)thatexpressedonthepathogenBorrelia burgdorferihasemergedasmostpromisingantigenfordevelopingaplatformtocombatLymedisease.Therefore,Pineetal.187 developedamRNALNPsencodingOspAandobservedthatits protectiveefficacysurpassedthatoftheOspAproteinsubunit vaccine.ThetreatmentregimenofmRNA-1975andmRNA-1982 developedbyModernaTX,Inc.forLymediseaseiscurrentlyin PhaseI/IIclinicaltrial(NCT05975099).188

Currently,theapplicationofantibacterialmRNAvaccinesin preclinicalandclinicalattemptsislimited,possiblyduetothe intricatenatureofbacterialinfectionscomparedtoviral infections.Bacterialinfectionsex hibitdistinctmolecularexpressioncharacteristicsatdifferentstagesofinfection,makingit virtuallyimpossibleforasingleantigentoeffectivelycoverall thesestages. 193 Moreover,bacterialpathogensdisplaydiversity andthevariabilityinantigenexpressionwhilealsoevolving multiplemechanismstoevadeorsuppressimmune responses. 185 Therefore,ifmRNAvaccinesareintendedfor furtherapplicationinpreventingandtreatingbacterialinfections,carefulconsiderationmustbegiventorationalantigen selectionalongwiththeincorporationofadjuvantsandother optimizationstrategies. 193 TheresearchconductedbyMeulewaeteretal. 194 demonstratedthattheincorporationofadjuvant α GCintothe Listeriamonocytogenes mRNALNPsresultedina synergisticprotectiveeffectagainstlisteriosis,underscoringthe therapeuticadvantagesofco-activatinginvariantnaturalkillerT (iNKT)cellsinantibacterialmRNAvaccines.Wearelooking forwardtothesuccessfuldevelopmentofthe ﬁ rstantibacterial mRNAvaccineproduct.

ParasiteantigenmRNAvaccine:Inadditiontoviralinfections, parasiticdiseasesarealsoinfectiousillnessesthatdebilitate individuals.Parasiticdiseasescanbegenerallycategorizedinto threegroups:ectoparasitic,protozoal,andhelminthicdiseases. Theseconditionscanbemanagedthroughproactivevector control,diseasemonitoring,theuseofeffectivevaccines/ medications,andtheimplementationofappropriatesanitation infrastructure.195 However,thelackofpublicawarenessandthe absenceofappropriatetreatmentoptionshaveresultedin widespreadparasiticinfections.196

Malariaisthemostdeadlyparasiticvector-bornedisease197 causedbyatleast fivespeciesof Plasmodium parasites.198–200 Overtheyears,researchershaveendeavoredtodevelopvaccines targetingdifferentstagesofthemalariaparasite’slifecycle. However,theonlyapprovedvaccinetodateisMosquirix,whichis basedonrecombinantproteins.Nevertheless,Mosquirixhas encounteredseverallimitations,includingsuboptimalefficacyin specificagegroupsandthenecessityformultipleboostershotsto achieveasatisfactorylevelofprotection.Fortunately,themRNA vaccine(BNT165b1)developedbyBioNTech,whichencodesa segmentofthe Plasmodiumfalciparum circumsporozoiteprotein (PfCSP),hasprogressedtoPhaseIclinicaltrial(NCT05581641).201 Kunkeawetal.202 developedtheLNPsplatformtodeliver nucleotide-modifiedmRNAencodingthePvs25antigenof Plasmodiumvivax,apotentialvaccineagainstmalariatransmission.Pvs25isanantigencandidateforblockingmalaria transmission.TheydiscoveredthatthemRNALNPsplatform couldelicitstrongerandmoreenduringfunctionalimmunity comparedtothePvs25recombinantproteinvaccine.Additional mRNAvaccinesagainstmalariaarecurrentlyundergoingpreclinicalresearch.203–205

Thelackofinterestinthecommercialdevelopmentof parasitevaccinesmaybeattributedtothefactthatparasitic diseasesprimarilyaffecteconomicallydisadvantagedpopulations,andmostparasitescausechronicillnessesthatarenonlethaltothehost,withtheexc eptionofthedeadlymalaria. Parasiteinfectionsareintricate,andthedevelopmentof conventionalvaccinesentailshighcosts. 206 Intheseregards, mRNA-basedvaccinespresentapromisingapproachtoaddress thesechallenges.

Asasignificantinfectiousdisease,Tuberculosis(TB),causedby MycobacteriumTuberculosis (M.TB),189 continuestopresenta graveglobalpublichealthmenace.Currently,theonlylicensed vaccineforTBisBacillusCalmette–Guerin(BCG),whichexhibits limitedefficacyinpreventingthedisease.190 Theabsenceof effectivevaccinestrategiesandtheemergenceanddissemination ofmultidrug-resistantTB(MDR-TB)bacteriahavefurthercomplicatedendeavorstopreventandtreatTB.SimilartoCOVID-19,TB alsorepresentsarespiratoryinfection.CanthemRNAvaccine strategysuccessfullyemployedforCOVID-19bereplicatedfor TB?191 Fortunately,in2023,BioNTechSEcommencedPhaseI/II clinicaltrial(NCT05547464)foranmRNAapproachtargetingTB (BNT164a1andBNT164b1),185,192 andweexpectasatisfactory therapeuticefficacyfromthisstrategy.

Autoimmunediseaseantigentherapy:Theimmunesystemplays acriticalroleindefendingthebodyagainstinfectionscausedby viruses,bacteria,fungi,andparasites.Additionally,itsurveilsand eliminatescancerouscellstomaintainthebody’shealth. Autoimmunedisordersarisewhentheimmunesystemerroneouslytargetshealthytissuesandcellsinthebody.207,208 An essentialcharacteristicofautoimmunediseasesisthepresenceof autoantibodiesthathavetheabilitytospeciﬁcallybindtothe antigenspresentinthepatient’shealthycellsortissues.207,209 Therearemorethan80autoimmunedisorders,includingmultiple sclerosis(MS),210 type1diabetes(T1D),211 systemiclupus erythematosus(SLE),212 inﬂammatoryboweldisease(IBD),213 andrheumatoidarthritis(RA).214 Theobjectiveoftreating autoimmunediseasesistoregulatetheactivityofautoreactive cellsinthebodywithoutinducingsystemicimmune suppression.215

Krienkeetal.210 utilizedanon-inflammatorylipidcomplex carriertoencapsulate N1-methyl-pseudouridine-modifiedmRNA (m1Ψ mRNA)encodingdisease-relatedautoantigens.Their findingsindicatedthattheuseofthism1Ψ mRNAvaccinecould stimulateantigen-specificregulatoryTcells(Tregs)andmitigate theseverityofmultiplesclerosisinamousemodel.216 This strategyseemscapableofinducingcross-toleranceinautoimmuneTcellswhilepreservingthenormalimmuneresponseofthe immunesystem.217 Thisalsoimpliedthatbyreducingunnecessary immunogenicity,mRNAvaccinesencodingautoantigenscanbea meanstoinduceandsustainnaturalperipheralimmune tolerance.218

Type1diabetesisanautoimmuneconditioninwhichthe immunesystemmistakenlyattacksanddestroysthepancreatic islet β-cells,resultingininsufﬁcientinsulinproduction.211,219 Fosteretal.220 discoveredthattheutilizationofanovelmRNA vaccineencodingproinsulinIIcouldtransientlydelaydiabetes symptomsinnonobesediabeticmice.Thiseffectmaybe attributedtotheabilityofthemRNAvaccinetorestoreimmune toleranceinnonobesediabeticmice.

ThemRNAtherapiesholdsigniﬁcantpromiseintherealmof autoimmunediseases.221,222 Forinstance,themRNAdrugscanbe tailoredtoencodeproteinsorpeptidescapableofbindingand neutralizingautoantibodies.Inaddition,mRNAcanbeengineered tospeciﬁcallytargetplasmacellsandexpresspro-apoptotic moleculeswithinthem,therebyselectivelydisruptingoreliminatingtheirfunctionandsurvival.Furthermore,giventhepivotalrole ofTregsinsuppressingimmuneresponses,mRNAcanbe designedtobolsterthefunctionalityofTregcells.207

Therapeuticantibodytherapy.Antibodies,alsoreferredtoas immunoglobulins,areeffectormoleculesgeneratedbythe humoralimmuneresponsesthatcanneutralizeantigens223 and playacrucialroleintheprevention,control,andeliminationof infections.224 Theemergenceofthetherapeuticantibodycanbe tracedbackto1975,whenKöhlerandMilsteinpioneeredthe hybridomatechnologyandlaidthefoundationfortheprogressof monoclonalantibodies(mAbs).225 Currently,therapeuticantibodieshaveemergedasapotentarmamentariumagainstavariety ofdiseases,223 demonstratingremarkableefﬁcacyintreatingviral illnesses,solidtumors,hematologicalmalignancies,autoimmune disorders,andotherindications.Inthelastfewdecades, antibodieshaveexperiencedunprecedentedgrowthasnew drugsfortheseindications.226

However,protein-baseddrugssuchasmAbsencounter challengesintermsofproductionandcost,includingintricate manufacturingprocess,highpurificationexpenses,anddifficulties inpost-translationalmodificationofproteins.Inaddition,the humanbodyharborsatotalof22distinctaminoacids,anddueto variationsintheirtypes,quantities,andarrangements,proteins canexhibitdiversephysicochemicalproperties.Toensureprotein storagestability,specializedbuffersolutionsoptimizedforthis

purposeareindispensable.227 Thenucleicacids,incontrast,carrya negativechargeandexhibitsimilarphysicochemicalcharacteristics.Therefore,theproductionandpuriﬁcationprocessofnucleic aciddrugsdoesnotnecessitatespeciﬁccustomization,anditis easytoobtainthenaturalstructureofthedesiredproteinowing tothetargetcell-endogenousexpressionmechanisms.227

DeliveringmRNAintothebodyfortranslationintoantibodies, ratherthandirectlyadministeringthemAbs,circumventsthe hasslesassociatedwithproteinproductionandpurification. Moreover,thisapproachenablestheencodingofproteinsthat arechallengingtosynthesizeinvitro.Dealetal.228 engineeredan mRNAsequenceencodingthepathogen-specificimmunoglobulin A(IgA)mAbs,whichwasencapsulatedinLNPsanddeliveredto mucosalsecretions.Intriguingly,thepharmacodynamicsof lgAmRNA encodedbythismRNAcloselyresemblethoseof endogenoushumanlgAratherthanrecombinantIgA(IgAR). These findingssuggestedthatthemRNAantibodytechnology heldpromiseforinterceptingpathogensonmucosalsurfaces, offeringanovelandeffectiveapproachforthepreventionand treatmentofdiseases.

Theintravenousadministrationofanti-vascularendothelial growthfactor(VEGF)antibodiestypicallyresultsintheirpredominantdistributioninthevasculatureandinterstitialspaces, withlimitedaccumulationinthetargetorgan thelungs.This limitedlocalizationimpairstheirtherapeuticefﬁcacyfornon-small celllungcancers(NSCLCs)andmayhaveadverseeffectson normaltissues.AteamledbyLeetal.229 developedapulmonarytargetingnanoparticlefordeliveringmRNA-encodingBevacizumab,ananti-VEGFantibodywidelyusedintheclinic.These nanoparticlesutilizedtheinherentpropertiesofpoly(beta-amino esters)(PBAEs)toachievetheirpulmonary-targetingfunction.This innovativeapproacheffectivelysuppressesangiogenesisin NSCLCs,offeringapromisingtherapeuticstrategyforthe treatmentofthisdisease.

The Orthopoxvirus genus,includingMPXVandvariolavirus (VARV),constitutesasignificantglobalhealththreatthatcauses severepoxdiseasesinbothhumansandanimals.Chietal.230 constructedmRNAcombinationsencodingfourmAbs(mAbs22, 283,26,and301)specificallydesignedtotargetandneutralize vacciniavirus(VACV)A33,VACVB5,MPXVM1andVACVA27 proteins.Thiswasthe firstapplicationofmRNAantibodiesagainst Orthopoxvirusinvivo.Theresultsindicatedthatasingleinjection ofLNPsencapsulatedwithmRNAinmicecouldrapidlyinducethe productionofcorrespondingneutralizingantibodies,which demonstratedsignificantprotectiveeffectsintheVACVlethal challengemousemodelandreducedmortalityrates.

Inthelastdecade,therehavebeensignificantadvancementsin thetreatmentofadvancedcancerswithimmunecheckpoint inhibitors(ICIs).Currently,clinicallyapprovedICIsconsistof innovativemedicationsthattargetprogrammedcelldeath protein-1(PD-1),programmeddeathligand-1(PD-L1),cytotoxic T-lymphocyte-associatedprotein4(CTLA-4),andlymphocyte activationgene-3(LAG-3).231,232 Nevertheless,ICIsstillpossess certainlimitations,suchasthepotentialforimmune-related adverseevents.233,234 Inordertoaddressthesesafetyconcerns, researchershaveexploredtheuseofmRNAtherapiesencoding ICIs.235 Pruittetal.236 transfectedDCswithmRNAencodingthe heavyandlightchainsofanti-CTLA-4andanti-GITRmAbs,aswell asmRNA-encodingtumorantigens.Their findingsdemonstrated thatthesemRNA-engineeredDCcellssignificantlyaugmented antitumorimmunityinmelanoma-bearingmicewithouteliciting anysignsofautoimmunity.Utilizingthistechnology,aPhaseI clinicaltrialforpatientswithmetastaticmelanomaispresently underassessment(NCT01216436).TheIVTmRNAplatformalso enablesthecontinuousendogenoussynthesisofbispecific antibodiesthatbindtoTcells.Zengetal.237 establishedand optimizedanmRNAsequencecalledZ15-0-2thatcodesfora bispecificnanobodyofanti-PD-1andanti-CTLA-4.Moreover,there

aremRNA-basedtherapeuticsencodinganti-PD-L1mAbfor cancertreatment238 andmRNA-6981encodingPD-L1forautoimmunediseases.239

Rituximabisahuman/murinechimericglycosylatedIgG1-κ mAbwithspecificaffinityforthetransmembraneproteinCD20on Blymphocytes.240 Thranetal.241 developedLNPsformulations containingmRNA-encodingRituximab(anti-CD20mAb)tocombat B-celllymphoma.TheyobservedthatthemRNALNPstreatment exhibitedapronouncedandpotentantitumoreffectcomparable tothatofrecombinantantibodytherapy.79 ThemRNAdrugs encodinganti-CD20mAbmayalsoofferpromisingtherapeutic optionsforautoimmuneconditionssuchasmultiplesclerosisand rheumatoidarthritis.207 Antibody-basedmRNAtherapieshold significantpotentialforthetreatmentoftumors,chronic inflammation,andautoimmunedisorders.242

Cytokine.Theoretically,mRNAhasthepotentialtoencodeany protein,includingcytokinesthatcanbeusedasstandalone therapiesorincombinationwithothertreatmentmodalitiesfor diseasemanagement.243

Cytokinesplayapivotalroleinregulatingvariousbiological processessuchascellsurvival,proliferation,differentiationand immunecellactivitybyfacilitatingcrucialintercellularcommunicationwithinshortdistances.244 Thepotentialofcytokinesinthe realmofantitumorresearchisexempliﬁedbytheircapacityto impedetheproliferationoftumorcells,facilitatetheinductionof apoptosis,andmodulatebothinnateandadaptiveimmunity.245 Furthermore,cytokinesmayexhibitimmunosuppressiveeffects, offeringapotentialtherapeuticapproachforconditionsderived fromhyperactiveimmuneresponsessuchasautoimmune diseases.ThecytokinesubgroupscommonlyencompassIL, interferons(IFNs),chemokines,granulocyte-macrophagecolonystimulatingfactor(GM-CSF)andsoon.244

Immune-potentiatingcytokine: IL-2,IL-21,andIL-15: TheFDA grantedapprovalfortheuseofrecombinanthumanIL-2in1992 forthetreatmentofmetastaticrenalcancer,andin1998forthe managementofmetastaticmelanoma.246 However,intravenous administrationofIL-2exhibitslimitedtumoraccumulation247 and possessesashorthalf-life(only5to7min).Inordertoachieve enhancedantitumoreffects,administrationofhighdosesofIL-2is imperative,leadingtoelevatedconcentrationsofIL-2inthe bloodstreamandsubsequentmanifestationofadversereactions inpatients,includingasthenia,pyrexia,andpotentiallylifethreateningtoxicitysuchascapillaryleaksyndrome.248 To mitigatethesystemictoxicityassociatedwithIL-2proteindelivery, Shinetal.249 proposedanovelapproachinvolvingintratumoral administrationofIL-2mRNANPs.TheseNPsarecomposedof polyethyleneimine-modifiedporoussilicaandeffectivelyminimize off-targettranslationofmRNA.Jiangetal.250 developedmRNA sequencesencodingfusionproteinsconsistingofIL-2,CD25(IL2Rα),andacleavablelinker,whichwereencapsulatedwithin ionizablelipidU-101–derivedNPs.Thedesignedlinkerwas susceptibletocleavagebytumor-specificmatrix metalloproteinase-14(MMP-14).Furthermore,theresearchers observedthatU-101–derivedNPsexhibitedsuperiortransfection efficacycomparedtoapprovedALC-0315–LNPs,therebycontributingtotheplatform’sabilitytoachieveenhancedantitumor efficacywithreducedtoxicity.Becketal.251 combinedlong-lasting mRNA-encodedIL-2withtumor-targetingmAbtherapy,thereby inducingahighlypro-inflammatoryTMEandovercomingtumor resistanceresultingfromtheabsenceofMHCI.

InadditiontoIL-2,othercytokinesthatbelongtothegammachainreceptorfamilyincludeIL-4,IL-7,IL-15,IL-21.252 Theroleof IL-21intheclearanceofhepatitisBvirus(HBV)issigniﬁcant.The mRNALNPsencodingIL-21,deliveredbyShenetal.253,demonstratedremarkableefﬁcacyininducingeffectiveclearanceofHBV anditscovalentlyclosedcircularDNA(cccDNA).

IL-15sharesastructuralresemblancewithIL-2254 andplaysa crucialroleintheregulationofbothinnateandadaptive immunity.255 IL-15iswidelyacknowledgedasaT-cellgrowth factorthatgovernstheactivation,proliferation,andcytotoxicityof Tcells,Bcells,andnaturalkiller(NK)cells,therebyenhancingthe productionofIFN-γ andTNF-α.Moreover,duetoitsunlikely inductionofTregcellactivityduringimmunestimulation,254 IL-15 hasbeenregardedasapromisingcandidateinoncology.255 Nevertheless,IL-15isassociatedwithsignificantdrawbackssuch asseveretoxicitiesandashorthalf-life.Thesechallengescanbe addressedthroughefficientdeliveryofmRNA-encodingIL-15to thetumor.Wangetal.256 designedananoplatformtospecifically targetPD-L1forthedeliveryofIL-15mRNA,andintegrateditwith anultrasound-targetedmicrobubbledestructiontherapy approach.Thistherapeuticapproachnotonlyeffectivelyfacilitates theexpressionofIL-15,butalsosignificantlyenhancesintracellular reactiveoxygenspecies(ROS)levels,inducesimmunogeniccell death(ICD),therebyeffectivelysuppressingtumorgrowthand recurrence.Furthermore,thisintegratedtherapyoffersreal-time ultrasoundimagingguidance.Gaviganetal.257 developeda combinationtherapycomprisingIFNα,IL-7,IL-15,andaTNF receptorsuperfamilyagonist.Their findingsdemonstratedthatcodeliveryofmRNAmixtureencodingIFNα,IL-7,aproteinfused withIL-15anditsreceptor α chain,andOX40Lcouldelicitpotent antitumorimmuneresponsesinmice.

IL-12andIL-27: Liuetal.258 endeavoredtosynergizeIL-12 mRNAwithOxaliplatinprodrugforthemanagementofcolorectal cancer(CRC).Hewittetal.259 discoveredinpreclinicalinvestigationsthatintratumoraladministrationofLNPsloadedwithmRNAencodingIL-12couldaugmenttheantitumoreffectsbyinducing thereleaseofIFN-γ andpromotingthetransformationofTh1cells withintheTME.ThePhaseItrialofhumanIL-12mRNA(MEDI1191) hasbeensuccessfullycompleted(NCT03946800).Tofurther enhancetumorinﬁltrationofIL-12andmitigatesystemictoxicity causedbyitsextratumoralpresence,Tranietal.260 engineered mRNAsencodingsingle-chainIL-12alongwithtwoadditional components:aratanti-mouseAFS98mAbvariableregionthat targetscolony-stimulatingfactor-1receptor(CSF1R)andthe variableregionofAvelumab(anti-PD-L1antibody),which signiﬁcantlyaugmentpost-transcriptionalbindingofIL-12to thetumor.

IL-27,belongingtotheIL-12family,exhibitscomparable antitumoreffectsasIL-12.TheactivationofTcellsandNKcells byIL-12andIL-27occursthroughdistinctmechanisms,whereby IL-12activatesTcellsviathesignaltransducerandactivatorof transcription(Stat)4pathway,whileIL-27engagesTcellsthrough boththeStat1andStat3pathways.Liuetal.261 developedLNPs loadedwithmRNAandobservedthatco-deliveringIL-12 + IL-27 mRNAsusingthesameLNPscarrierresultedinsuperiortumor suppressioncomparedtothesingledeliveryofIL-12mRNA. Furthermore,theincorporationofIL-27,ananti-inflammatory cytokine,couldmitigatepotentialtoxicinflammationassociated withmono-administrationofIL-12.Thiscombinationnotonly enhancedefficacybutalsoreducedsystemictoxicity.

GM-CSF: GM-CSFisacytokinethatpromotesthegenerationof variousmyeloidcellsubpopulations,suchasDCs,monocytes, macrophages,andneutrophils.Excessiveorinadequatelevelsof GM-CSFcouldenhancethemalignancyofcancer.262 ThemRNA mixture(SAR441000)encodingIL-12,IFNalpha-2b,GM-CSF,and IL-15wasadministeredintratumorallyasamonotherapyorin combinationwithCemiplimabduringthePhaseItrial (NCT03871348)conductedin2019forthetreatmentofadvanced solidtumors.Theresultsdemonstratedfavorabletolerabilityand discernibleimmunomodulatoryeffectsforbothSAR441000 monotherapyanditscombinationwithCemiplimab.263

Toextendtheserumhalf-lifeofGM-CSF,itcanbeconjugated withbiologicallysafeandstructurallystableproteins.Yeapuri etal.264 developedmRNALNPsencodingthealbumin-GM-CSF

fusionprotein,therebyprolongingthehalf-lifeofthetarget proteinandenhancingdrugbioavailabilitytooptimizeits therapeuticefﬁcacy.

Immune-suppressingcytokine: IL-2: AlthoughmRNA-based cytokinetherapiesarecommonlyemployedinthe fieldof antitumorresearch,theirpotentialapplicationsinthetreatment ofotherdiseasesshouldnotbeunderestimated.TheIL-2receptor existsintwoformswithvaryingaffinities,theformexpressedby TregcellsexhibitingahigheraffinityforIL-2.Consequently,atlow concentrations,IL-2preferentiallyactivatesTregs.118,119 However, athigherconcentrations,italsotriggerstheactivationofproinflammatoryTcellsandNKcells.Tregcellsrepresentasubsetof CD4+ Tlymphocyteswithsignificantimmunosuppressivecapabilities.265 ImpairmentsinthefunctionofTregcellsora deficiencyintheirnumberscanresultinthebreakdownof immunetoleranceandthedevelopmentofautoimmunediseases withinthebody.266 Consequently,inducedTregcellsholdpromise forthetreatmentofautoimmunediseasesandtherestorationof immunologicalself-tolerance. 267,268 Picciottoetal.118 engineered mRNAtoencodeafusionproteinofhumanserumalbuminIL-2 mutein(HSA-IL2m)withanextendedhalf-life.Their findings demonstratedthatdeliveringthislipid-encapsulatedmRNAcould selectivelyexpandTregsinvivowithoutactivatingNKcellsor conventionalTcells.Thisapproachoffersanovelstrategyfor suppressingautoreactiveTcellsandrestoringimmunebalancein autoimmunediseases.ThemRNA-6231,encodingHSA-IL2m,has alreadyprogressedtoPhaseIclinicaltrial(NCT04916431).221

IL-4: IL-4isinvolvedintheregulationoftheactivation, proliferation,differentiation,andsurvivalofvariousT-cellsubtypes.269 ItsupportsthedifferentiationofTh2cellsand antagonizesthefunctionofTh1cellsassociatedwithnumerous autoimmunedisorders.270 ThemodulationofTh-cellssubgroups, particularlythebalancebetweenTh1andTh2,exertsasignificant influenceonthepathogenesisoftype1diabetes.271,272 Creusot etal.270 reportedanoveltherapythatdeliveredtranslationally enhancedIL-4mRNAintoDCsusingelectroporation.Theyhave shownthatmodifiedDCscontainingmRNAcouldmodulate autoimmunediabetesinnonobesediabeticmicebyboostingthe functionalityofTregcellsandinducingashiftintheproportionof Thcellpopulations.

IL-10: IL-10isacytokinewithanti-inflammatoryproperties, capableofsuppressingtheexpressionofpro-inflammatory cytokinesandinhibitingthepresentationofalloantigensby APCs.273 Chenetal.274 developedanoveldeliverysystemwith self-protectionandactivetargetingcapabilitiesforencapsulating mRNAencodingIL-10.Nucleicacidswerecombinedwith polyphenolstomitigateenzymaticdegradation.Duetothe susceptibilityofhyaluronicacidtooxidationintheinflamedcolon andsubsequentinactivation,itisessentialtocombinebilirubin, knownforitspotentantioxidantactivity,withhyaluronicacidin ordertoensureeffectivetargetingofCD44.Theydemonstrated thatrectaladministrationoftheIL-10mRNAdeliverysystemcould effectivelyinducetherapeuticeffectsinamurinemodelofcolitis. Atherosclerosisisachronicinflammatorycondition.Buetal.275 developedanexosome-baseddeliveryplatformforloading engineeredIL-10mRNA.Theyobservedthatthisplatformcould effectivelymitigateatherosclerosisinApoE / (Apolipoprotein E-deficient)mice.

Ligand.ThemRNAcanalsoencodeimmune-stimulatingligands, therebyfacilitatinganimmunogenicresponse.TheTriMixtherapy consistsofacombinationofmRNAencodingthreedistinct immune-stimulatingproteins,namelyCD40ligand(CD40L),CD70, andconstitutivelyactiveTLR4.276 ThisinnovativemRNA-based vaccinecanbeadministeredviaintratumoralinjectionforpatients withearly-stage,resectablebreastcancer,whichiscurrently undergoingPhaseIclinicaltrial(NCT03788083).

TheTriMixcompoundnotonlyservesasadirectmRNAvaccine butalsofunctionsasanadjuvantinimmunizationresearchdueto itscapacitytoencodeDC-activatingproteins.TheECI-006vaccine consistsofacombinationofmRNA-encodingadjuvantproteins (TriMix)andmRNA-encodingmelanoma-speciﬁcTAAs(tyrosinase, gp100,MAGE-A3,MAGE-C2,andPRAME).APhaseIclinicaltrialhas beenconductedtoevaluatethismRNAvaccineinpatientswith stageIIc/III/IVmelanomawhohaveundergonesurgicalresection (NCT03394937).Theresultsdemonstratedfavorabletolerability proﬁleswhenadministeringECI-006atdosesof600or1800 μgto thepatients,andimmunogenicitywasobservedinasubsetof patients.277

Tumorsuppressorprotein.Theoptimalgoalofcancertreatment istoeradicatetumorcellswhileminimizingtheharmtohealthy tissuesandcells.Inpursuitofthisobjective,suicidegenetherapy, anovelapproachtocancertreatment,hasgarneredattention.278 Cancersuicidegenetherapiescanbecategorizedintothree groups:enzyme/prodrugsystems,pro-apoptoticgenes,and toxins.279 Theenzyme/prodrugsysteminvolvesthetransferof geneticmaterialencodingenzymestotumorcells,leadingtothe conversionofnontoxicprodrugsbytheseenzymesintocytotoxic metabolitesthatinduceapoptosisintumorcells.278,280 For instance,theinnovativehumanherpessimplexvirus–thymidine kinase(HSV-TK)/ganciclovirsuicidegenesystemshowspromise. However,theHSV-TKsystemishamperedbysigniﬁcantlimitationsassociatedwithitsimmunogenicity.281 Furthermore, enzyme/prodrugsystemsalsoencounterthechallengeofofftargeteffectsonnormaltissues,resultingintoxicity,aswellas suboptimalefﬁcacyagainstslow-dividingtumorcells.Toaddress thesechallenges,theapproachofdevelopinggenesencoding pro-apoptoticproteinsandbacterialtoxinsassuicidegeneswas employed.282

Enzyme-encodingmRNAforenzyme/prodrugsystems:The combinationofcytosinedeaminaseanduridinephosphoryltransferase(UPRT)/5-fluorocytosine(5-FC)formsanenzyme/prodrug system.Thecytosinedeaminaseiscapableofconvertingthe nontoxic5-FCintothetoxicmetabolite5-fluorouracil(5-FU).The geneencodingcytosinedeaminaseiscommonlyfusedwiththe UPRTgenetoenhanceitsefficacy.279 Mizraketal.283 obtained microvesicles(MVs)loadedwithcytosinedeaminase-UPRTmRNA/ proteinbytransfectingthecytosinedeaminase-UPRT-EGFP plasmidintoHEK-293Tcells.Subsequently,theyobservedthat intratumoralinjectionofcytosinedeaminase-UPRT-mRNA/proteincontainingMVsandtreatmentwith5-FCsignificantlysuppressed tumorgrowthintumor-bearingmice.

Induciblecaspase-9(iC9)isanengineeredversionofcaspase-9 thatcanbedimerizedandactivateduponexposuretothe chemicalinducersofdimerization(CID),suchastheAP1903 molecule,subsequentlytriggeringdownstreamapoptoticpathwaysandleadingtocelldeath.284 Nakashimaetal.285 developed LNPscarryingiC9mRNA.Theydiscoveredthatthecombinationof iC9mRNAandCIDexhibitedcytotoxiceffectsonthreebreast cancercelllinesinvitro.Ontheotherhand,Saitoetal.286 engineeredliposomesforthedeliveryofiC9mRNAandcombined themwithCIDforthetreatmentofT-cellmalignancies.Itwas foundthatthetreatmentregimenexhibitedanti-leukemia therapeuticeffectsbothinvitroandinvivo.

Pro-apoptoticprotein: p53andPTEN:Itiswidelyacknowledged thatcanceroriginatesfromthemutationofsomaticcells,andthat oncogenicmutationsorabnormalexpressionofcellularprotooncogenescantriggercarcinogenesis.Adistinctcategoryof tumorsuppressorgenecancounteractthesecarcinogeniceffects andimpedetumordevelopment.Notably,p53,retinoblastoma (Rb),andphosphataseandtensinhomologdeletedonchromosometen(PTEN)arethreepivotaltumorsuppressorproteinswith

interconnectedfunctions.287 Themajorityofhumancancers exhibitloss-of-functionmutationsinthep53protein.However, duetothefactthatp53isatranscriptionfactor,ithaslongbeen deemedundruggable.288 Nevertheless,theemergenceofmRNA therapieshasfacilitatedthedevelopmentofnumerousp53-based cancertreatmentsutilizingmRNA.

Toaugmentthetherapeuticefﬁcacyofp53mRNAtherapy,it canbesynergisticallycombinedwithotherwell-established antitumormodalities.Zhangetal.289 developedpaclitaxelamino fat-derivedNPsforencapsulatingp53mRNAandchemotherapy drugs,demonstratingremarkableantitumoreffectsinanorthotopictriple-negativebreastcancer(TNBC)mousemodel.Zhou etal.290 werethepioneersinreportingtheintegrationofmRNA therapywithphotodynamictherapy(PDT)fortumortreatment. TheydevelopedaROS-responsiveNPsplatformencapsulatingp53 mRNAandindocyaninegreen(ICG).TheseNPscanbetriggered byROStoreleasep53mRNA,therebyaugmentingp53expression andinducingapoptosisinlungcancercell.Simultaneously,laser irradiationcouldactivatethereleasedICGtofacilitatePDT.To enhancetheexpressionofintracellulartargetproteins,Cao etal.291 employedanovelstrategybydesigningtheCRISPR/ dCasRx-SINEB2platformtoaugmentthetranslationofendogenousmRNA-encodingtumorsuppressorproteins(includingPTEN andp53),therebyinducingsuppressedproliferationandincreased apoptosisinbladdercancercells.Liuetal.292 establisheda nanoparticleplatformtoencapsulatePTENmRNAforbrain delivery.TheNPsexhibitedenhancedcapabilitytocrossthe blood-brainbarrierandtargettumorcells,demonstratingthe potentialofusingmRNA-basedtechnologiesfortreatingbrain diseases.

BimandBax: Bimisasigniﬁcantpro-apoptoticmemberwithin theBcl-2proteinfamily.293 Gaoetal.294 developedadelivery systemthatcombinedcell-penetratingpeptides(CPPs),cRGD-R9, andcationicnano-sizedDOTAP-mPEG-PCLscaffoldstodeliver mRNA-encodingBim.TheBimmRNAdeliverysystemexhibited potentinhibitoryeffectsontumorsinvariouscoloncancermodels throughtheinductionofmitochondrialapoptosis.

Baxisapro-apoptoticmoleculethatcanserveasatumor suppressorprotein.Okumuraetal. 295 developedliposomesto delivermRNA-encodingBaxandcomparedtheantitumor effectswiththoseofaBaxplasmidintumor-bearingmice. Their ﬁ ndingsindicatedthattheuseofBaxmRNAtherapyvia liposomesexhibitedstronger antitumoreffectsthantheBax plasmid.

TRAIL: Tumornecrosisfactor-relatedapoptosis-inducingligand (TRAIL)cantriggerapoptoticcelldeathintumorcellswhile sparingnormalcells.Guetal.296 developedmRNA-basedLNPs encodingafusionproteinofTRAILandthereceptor-binding domain(RBD)fromtheSARS-CoV-2spikeprotein,whichwere administeredviaintratumoralinjectionincoloncancermice models.TheyobservedthattheRBD-TRAILmRNALNPseffectively inhibitedtumorgrowthinmice.Silvaetal.297 discoveredthatthe directintratumoralinjectionofLNPsloadedwithTRAILmRNA couldeffectivelyinducetumorcelldeath,particularlywhen combinedwithLosartanorangiotensin1–7.

Apoptin: Apoptinisaproteinencodedbythegeneofthe chickenanemiavirus.298 Itiscapableofinducingapoptosisin tumorcellsthroughap53-independentpathway.299 Itis noteworthythatApoptincantriggerapoptosisintumorcells whilesparingnormaldiploidcells.298 Therefore,asatumor suppressorprotein,Apoptinholdspromiseasasafeanticancer medication.300 However,thecollectionandpuriﬁcationprocessof thisproteinishighlyintricateandcostly.299 Therefore,thegene therapybeingdevelopedforantitumorApoptinprimarilyrelieson DNA301 orviralvectors.298 Tangetal.299 engineerednanospheres withself-assemblycapabilitiestoencapsulatemRNA-encoding Apoptin.ThecombinationofApoptinmRNAwithDoxorubicin exhibitedremarkablesynergisticantitumoreffects.

ThemRNA-basedtherapythatencodestumorsuppressor proteinsiscurrentlyinitsearlystagesofdevelopment.Ongoing explorationsarebeingconductedregardingnumerousoncogenes andtheircorrespondingtumorsuppressorproteins.Quetal.302 proposedthatHuntingtin-associatedprotein-1(HAP1)mayfunctionasapotentialtumorsuppressoringastriccancer.Zhengand Song303 believedthatSynaptopodin2(SYNPO2)functionsasboth astructuralproteininmuscletissueandanemergingtumor suppressorprotein,supportedbyitspositivecorrelationwith favorablecancerprognosis,therebyindicatingitscrucialrolein cancerpreventionandtreatment.Withtheidentiﬁcationof additionaltumorsuppressorproteins,itisanticipatedthat mRNA-basedtherapiesencodingtheseproteinswillgradually emergeasapromisingavenueincancerresearch.

Immunotoxin:Immunotoxinsaretypicallychimericproteins composedofaproteintoxinconjugatedtoanantibody fragment,304 enablingspeciﬁctargetingandeliminationof antigen-bearingcells.305 Thetoxiccomponentsinimmunotoxins typicallyoriginatefromavarietyofsources,includingbacteria, plants,andhumancells.Bacterial-derivedtoxinssuchas diphtheriatoxinandpseudomonasexotoxinA(PE),aswellas plant-derivedtoxinslikericinandgelonin,arecommonlyused. Human-derivedtoxinsincludegranzymesandRNases.Among these,theapplicationofbacterialandplanttoxinsislimiteddue totheirpotentialimmunogenicityinhumans.306 Variousstrategies havebeendevelopedtomitigatetheimmunogenicityof immunotoxins.Theseincludeemployingimmunosuppressive therapy,utilizinghuman-derivedtoxinproteins,andmodifying toxinmoietytoevadedetectionbythehumanimmunesystem.307

Theshorthalf-lifeoftheimmunotoxininthecirculatorysystem maylimititstherapeuticactivity.Toextendthehalf-lifeof immunotoxinincirculation,Guoetal.308 fuseditwiththe albumin-bindingdomain.Theproductionofrecombinantimmunotoxinsistypicallyconductedinbacteria.However,itisgenerally challengingtoproducesolubleproteinswithmolecularweights exceeding80kDainbacterialsystems.309 mRNA-basedimmunotoxintherapypresentsapromisingalternative.

Theapplicationofimmunotoxinsforthetreatmentofcanceris currentlyunderinvestigation,whichis,however,constrainedby theirlargemolecularweight,limitedtumorpenetration,andthe presenceofcellularresistance.Consequently,immunotoxinshave notyetreceivedapprovalforclinicaluseagainstsolidtumors.To addressthesechallenges,Granot-Matoketal.310 developedaLNPs systemthatdeliveredmodiﬁedmRNAencodingthePEdomain. Thisinnovativeapproachemployedtoxin-encodingmRNAto overcomethelimitationsassociatedwithimmunotoxintherapy, offeringimprovedsafetyandenhancedtherapeuticefﬁcacy.

PE38,aderivativeofthepotenttoxinPEproducedby Pseudomonasaeruginosa,hasbeenengineeredtoremoveits targetingdomainwhileretainingitstoxicity.ThetoxicityofPElies initsspecificinhibitionofelongationfactor2(EF-2)inthecytosol ofeukaryotes.Eggersetal.311 utilizedPE38inthedevelopmentof immunotoxinsbyfusingitwithhumanVEGFandananti-human epidermalgrowthfactorreceptor-2(HER2)/neusingle-chain variablefragment(scFv).TheytransfectedhumanprimaryTcells withmRNA-encodingimmunotoxinsanddemonstratedthatthe engineeredTcellsdisplayedsignificantlyenhancedcytotoxicity againstcancercellsinvitrowhencombinedwithbispecific antibodies.

Thesubtilasecytotoxin,diphtheriatoxin,andabrin-abelongto theABtoxinfamily.TheirB-subunitinitiallybindstothetargetcell, followedbytheentryoftheA-subunitintothecell,whereitexerts toxicitybyinhibitingproteinsynthesis,ultimatelyleadingtothe demiseofthetargetcell.Hirschbergeretal.312 developedthree chemicallymodiﬁedmRNAsencodingsubtilasecytotoxin, diphtheriatoxin,andabrin-a.TheydemonstratedthatthemRNA therapyencodingabrin-aexhibitedsuperiorefﬁcacyinreducing

theviabilityoftumorcellsinvitrocomparedtotheothertwo mRNAtherapies.

Furthermore,immunotoxinsofferapromisingtherapeutic approachforantiviraltreatment.Theirmechanisminvolves targetinganderadicatinginfectedcells,ratherthantheinhibition ofviralorcellularpathwaysessentialforvirusreplicationand spread.313 Thereisgreatpotentialforachievingsigniﬁcant advancementsinthe ﬁeldsofantitumorandantiviralresearches, amongothers,throughtheapplicationofimmunotoxins.

Adoptivecelltherapy.Adoptivecelltherapyinvolvestheextractionofthepatient’simmunecells,followedbyexvivoeditingand activation,andsubsequentreintroductionintothepatientfor diseasetreatment.Thisapproachaimstoenhancetheidentiﬁcationandeliminationoftumorcells.Additionally,thereisongoing developmentofinsituadoptivecelltherapy.314 Itisnoteworthy thatthesubsequentadoptivecelltherapiesarefoundedupon RNAmethods,asopposedtoconventionalDNAmethodologies. Speciﬁcally,CAR-TtherapyshouldbefurthersubdividedintoRNA chimericantigenreceptorTcells(rCAR-T).

CAR-T:Inthepastdecade,chimericantigenreceptor(CAR)expressingT-cell(CAR-T-cell)therapyhasmademajorbreakthroughsinthetreatmentofhematologicalcancers.Disappointingly,itseffectivenessintreatingsolidtumorsisstilllimiteddueto alackofcomprehensiveunderstandingoftumor-specificantigens,thesuppressionofT-cellinfiltrationandkillingabilitybythe TME,andlimitedexpansionpotentialofTcells.315 Therefore, Mackensenetal.designedCAR-Tcellstargetingthetumor-specific antigenClaudin6(CLDN6)anddemonstratedpotentialclinical activity.However,duetothelimiteddurabilityofasingledose, theyintegrateditwithaCAR-T-cellAmplifyingRNAVaccine (CARVac)foradministration.Thiscombinationapproachexhibited goodtolerabilityinsubjectsandshowedpromiseofenhancing CAR-T-cellexpansionandimprovingtumortreatmentefficacy. Currently,thestudyisinPhaseI/IIclinicaltrial (NCT04503278).120,316 Despitepromisingresultsobservedin clinicaltrial,the findingsfellshortofmeetingexpectations, underscoringtheurgentchallengesthatstillneedtobeaddressed intargetingCLDN6CAR-TtherapycombinedwithCARVac administration.315 Themethodologyemployedinthisstudyfor CAR-T-cellproductionalignswithclinicalpractice,encompassing exvivoculturingofCAR-Tcellsfollowedbytheirinfusioninto patients.Thisapproachentailssignificantexpendituresfor manufacturing.Hence,thereisanurgentimperativetodevelop aswift,robust,andeconomicallyefficientalternativestrategyfor manufacturingCAR-Tcells.Apromisingavenueinvolvesthedirect deliveryofmRNAintothebodytoinduceinsituformationofCARTcells.

Despitethechallengesassociatedwithtransfectingexogenous mRNAintoTcells,Tombáczetal.317 discoveredthatthe applicationofCD4antibody-conjugatedmRNALNPsencoding Crerecombinaseor fireflyluciferasesignificantlyaugmentedthe signalofmRNAby~30-foldinspleenTcellsandincreasedthe expressionofreportergenesinCD4+ Tcellsinvivowhen comparedtonon-targetedmRNALNPs.Zhouetal.318 usedCD3 antibody-modifiedLNPsloadedwithIL-6shorthairpinRNA(IL6shRNA)andCD19-CAR(basedonplasmidDNA)fortargeted deliverytoCD3+ Tcells,whichresultedinthedownregulationof IL-6withinCAR-Tcellsandareductionincytokinerelease syndrome(CRS),therebyenhancingthesafetyprofileofleukemia treatment.Ruriketal.319 developedCD5-targetedLNPsencapsulatingmRNAencodingaCARagainst fibroblastactivationprotein (FAP).ByadministeringtheseCD5-targetedLNPs,potentantifibroticCAR-Tcellscouldbegeneratedinvivo,whichmitigated cardiac fibrosisandrestoredcardiacfunction.320

GiventhepivotalroleplayedbyautoreactiveTandBcellsin autoimmunedisorders,CAR-T-celltherapyhasalsobecomea

therapeuticoptionwithintherealmofautoimmunediseases.321 Thepathogenesisofmyastheniagravis(MG)involvesthepresence ofpathogenicplasmacellsandautoantibodies,makingita prototypicalautoimmunedisorder.TheongoingPhaseIb/IIatrial (NCT04146051)involvingDescartes-08rCAR-Tcellsforthe treatmentofgeneralizedMGentailedtransfectingmRNAencodinganti-BCMA(B-cellmaturationantigen)intoCD8+ Tcells.The findingsdemonstratedthatDescartes-08,which expressedanti-BCMAtargetingCARproteinonautologousCD8+ Tcells,exhibitedfavorablesafetyandtolerabilityprofilesinMG patients.Furthermore,sustainedreductioninMGseveritywas observedwithcontinuedadministrationofDescartes-08infusions overafollow-upperiodofupto9months.Thesepromising outcomeswarrantedfurtherinvestigationintothepotential therapeuticefficacyofthisapproachinotherautoimmune disorders.121 Excitingly,theDescartes-08anti-BCMArCAR-T therapyforsystemiclupuserythematosus(SLE-001)hassuccessfullyprogressedintoPhaseIIasofFebruary2024 (NCT06038474).322 Ontheotherhand,Thatteetal.323 administeredFoxp3mRNALNPstoCD4+ Tcellsinordertogenerate immunosuppressiveFoxp3-Tcells.Thesecellshavethecapacityto suppresstheproliferationofeffectorTcells,highlightingthe potentialofmRNA-engineeredimmunosuppressiveTcellsinthe managementofautoimmunedisorders.

TCR-T:InadditiontoCAR-T-celltherapy,therealsoexisttargeted adoptivecelltherapiesemployingT-cellreceptor(TCR)engineeredTcell(TCR-Tcell).Toaddresshepatocellularcarcinoma (HCC)associatedwithHBVfollowinglivertransplantation,Yang etal.324 employedelectroporationtointroducemRNA-encoding HBV-speciﬁcTCRintoexvivoTcells,whichweresubsequently transferredtothepatients.CurrentlyinPhaseIclinicaltrial (NCT02719782),thestudydemonstratedfavorabletolerabilityof thistreatmentapproach.

Maggadottiretal.325 conductedastudyonthetreatmentof metastaticmicrosatelliteinstability-high(MSI-H)CRC,whereinthey employedanmRNATCR-modifiedT-celltherapyapproach.This involvedadministeringautologousTcellselectroporatedwithIVT Radium-1TCRmRNA,specificallytargetingthe-1Aframeshift mutationinthe TGFβRII gene,topatientswithadvancedMSI-H CRC.Theresultsdemonstratedafavorablesafetyprofileforthis TCR-T-celltherapy(NCT03431311).

Inaddition,alsoforthetreatmentofviral-associatedtumor diseases,suchasHPV-associatedcervicalcancer,Lingetal.326 designedapH-responsivenonviralnano-carriertodeliverCas9 mRNAandtheguideRNAs(gRNAs)ofoncogenesE6andE7that targetHPV,whichcaneffectivelyknockouttheE6/E7oncogenes, reversethetumor’simmunosuppressiveenvironment,andpromoteCD8+ T-cellsurvival.Therefore,genomeeditingtherapies co-deliveringCas9mRNAandgRNAsthattargetoncogenescan alsobecombinedwithadoptiveT-celltransfertoimprovecancer treatmentoutcomes.

CAR-M:ThetherapeuticefficacyofCAR-Ttherapyintreating solidtumorsislimited,whichisgreatlyderivedfromthelimited infiltrationcapacityofTcellswithintheTME.Conversely, macrophagespossesstheabilitytoinfiltratesolidtumortissue andengagewithalmostallcellsintheTME,includingtumorcells anddiverseimmunecells.Consequently,exploringthepotential ofCAR-expressingmacrophages(CAR-M)representsanovel avenueforimmunotherapy.327 TheinvestigationalagentCT0058,apro-inflammatorymacrophagecellproduct,iscurrently undergoingPhaseIclinicaltrialforthetreatmentofsolidtumors (NCT04660929).Theanti-HER2CARexpressedinCAR-Mcellswas obtainedviaadenoviralvectorsratherthanmRNAtransfection.328 Meanwhile,theMCY-M11,currentlyundergoingclinicalPhaseIfor advancedovariancancerandperitonealmesotheliomatreatment, isderivedfromthetransfectionofanti-mesothelinCAR(Meso-

CAR)mRNAintoperipheralbloodmononuclearcells(PBMCs) (NCT03608618).329

CAR-Mtherapynotonlyholdsimmensepotentialincombating cancer,butalsoexhibitspromisingapplicationsinconducting cutting-edgeresearchonbacterialeradication.Tangetal.330 developedLNPssystemencapsulatingmRNAthatencodedaCAR speciﬁcallytargetingmethicillin-resistant Staphylococcusaureus (MRSA).TheLNPssurfacewasmodiﬁedwiththeCRVpeptide (sequenceCRVLRSGSC)tofacilitatemacrophagestargeting.This antibacterialCAR-Mtherapydemonstratedpromisingpotentialin eradicatingMRSA.

mRNA-engineeredDCs:DCsareacrucialsubsetofAPCs responsibleforpresentingTAAstoTcells,andtheirfunctionality canbecompromisedbyTME.AlthoughengineeredDCstherapy hasgainedattention,itsstandaloneefficacyremainsunsatisfactory,necessitatingcombinationwithothertherapiestocounteract theinhibitoryeffectsoftheTME.331 Theyear2002witnesseda clinicaltrialevaluatingtheefficacyaIVTmRNADCvaccineagainst melanoma(NCT01278940).332 In2006,Kyteetal.333 reportedthe resultsofthisPhaseI/IItrial,indicatingthatthetreatmentof tumor-mRNAtransfectedDCswassafeandfeasible,andthatthe DCvaccinewasabletoinduceantigen-specificT-cellresponsesin ~50%ofpatients.

ToreprogramtheTMEandinitiatetumor-speciﬁcT-cell responses,Zhangetal. 331 designedtwomRNALNPs.Oneof thesemRNALNPsencodedCD40ligand,whichelicitedpotent ICDintumortissues,resultingintheexpressionofTAAsandCD40 ligandonthesurfaceoftumorcells.TheotherLNPscarriedCD40 mRNAwiththeaimtobeinternalizedbyDCsforgenerating insituengineeredDCs,leadingtoincreasedexpressionofCD40 proteinandactivationuponinteractionwithCD40ligandon tumorcells.

Themajorityofpatientsdiagnosedwithacutemyeloid leukemia(AML)exhibitapersistentandrecurrentmanifestation ofthediseaseattributedtoresidualleukemiccells,evenfollowing standardchemotherapy,resultinginasurvivalrateoflessthan5 years.Consequently,supplementarytherapeuticinterventionsare imperativeforeradicatingminimalresidualdisease(MRD).The significantoverexpressionofWilmstumor1(WT1)inAMLandits associationwiththepathogenesisofthediseaserenderita promisingtargetforTcells.Inlightofthis,aPhaseIIclinicaltrial wasconductedin2010toevaluatetheefficacyofloading autologousDCswithWT1antigenviamRNAelectroporationasan adjunctivetreatmentstrategyforAMLpatientswhohad completedchemotherapy(NCT00965224).The findingsreported byAnguilleetal.334 demonstratedthatthisvaccinationapproach effectivelyelicitsrobustT-cellimmuneresponsesinAMLpatients athighriskofrelapse,therebyestablishingboththesafetyand potentialutilityofautologous WT1 mRNA-electroporatedDC vaccinefollowingchemotherapy.

Non-immunotherapy

TheemergenceofmRNAtherapieshasinfusedconsiderablehope intothebattleagainstpreviouslyincurablediseases.Theoretically, optimallydesignedIVTmRNAcanbeintroducedintocellseither throughinvivoorexvivotransfectionandsubsequently translatedintobiologicallyactivepeptide/protein.335 ThenonimmunotherapydescribedinthisarticleentailsdirectlytranscriptionofmRNAtocomplementdeﬁcientoraberrantproteinswithin theorganism,aswellasforgeneeditingtechnology,while evadinganyimmuneresponsefromtherecipient(Fig. 4).

TheeffectivetreatmentofgeneticdefectdiseaseswithIVT mRNAreliesonmultiplefactors,encompassingthestabilityand translationalpotencyofthemRNA,theef ﬁ cacyandbiological activityoftheencodedprotein,as wellasitscirculationhalf-life. Importantly,itisworthnotingthatthetherapeuticthreshold necessitatesanactiveproteinquantityrangingfrommilligrams

tograms,whereasevenatthemilligramornanogramlevel, antigenscanprovokeanimmuneresponseinthebody. 336 To mitigatetheimmunogenicityofmRNAtherapies,several optimizationscanbeimplemen tedforIVTmRNA,including re ﬁ ningthenucleotidesequenc e,employingdiversemRNA nucleosidemodi ﬁ cations,optimizingreac tionconditionsofthe IVTconditionstominimizebyproductsgeneration,andpurifying thesynthesizedmRNA. 337

Therepeatedadministrationremainseffectiveinmaintaining highproteinexpressionandensuringinvivosafety,whichis crucialforthetransformationofmRNAvaccinesintomRNA therapeutics.Long-termorevenlifelongrepeatedadministration isnecessaryfortreatingchronicdiseases.338 However,delivery vectors,suchasLNPs,constituteacrucialcomponentinmRNAbasedtherapeutics,necessitatingcarefulconsiderationoftheir immunogenicpotential.

Thepattern-recognitionreceptorfamilyonthecellmembrane includesTLR4,whichisacrucialmemberknowntobeactivated bydamage-andpathogen-associatedmolecularpatterns(DAMPs andPAMPs).PAMPsencompasslipopolysaccharide(LPS)339 and monophosphoryllipidA(MPLA;anontoxicTLR4agonistapproved forclinicaluse).340 Kedmietal.341 discoveredthatthesystemic administrationofpositivelychargedNPscarryingsmallinterfering RNA(siRNA)couldtriggerTLR4-dependentimmuneresponses, leadingtotoxicity.Furthermore,theydemonstratedthatthe inﬂammatoryreactionwasprimarilycausedbycationiccomponentsratherthanthesiRNA.ThecationiclipidDiC14-amidine, synthesizedbyLonezetal.,339 wasshowntoactivateTLR4. Moreover,competitionexperimentsconﬁrmedthatthebinding siteofthiscationiclipidtoTLR4differedfromthatofLPS.Lonez etal.342 synthesizedacationiclipid,RPR206252,whicheffectively triggeredtheNF-κBpathwayandelicitedTNF-α,IL-1β,IL-6,and IFN-γ productioninhumanormousemacrophages.These cascadeswerereliantonTLR2/CD14andNOD-likereceptor protein3(NLRP3)signaling.

Therefore,itisimperativetodeveloplow-immunogenicLNPs withdiversecompositionsandenhancedorgan-selectivity,or explorealternativedeliveryvehiclestoamplifythetherapeutic efficacyofmRNAtherapiesfornon-immunediseases.343 In addition,thecombinationofmRNAtherapywithimmunosuppressantssuchasDexamethasone(asteroidalanti-inflammatory drug)notonlyholdsthepotentialtoenhancemRNAtransfection efficiency344 butalsoexhibitspromiseinaugmentingthesafety andefficacyofnon-immunotherapy.

InconsiderationofthesafetyofmRNAdrugs,itisrecommendedthatpreclinicalstudiesencompassthefollowingareas:(1) investigatingthecorrelationbetweenmRNAdosage,carrier properties,andinvivolevelsofcytokinesecretionaswellas complementactivation;(2)assessingthepotentialdevelopment ofanti-drugantibodiesuponrepeatedadministration;(3) evaluatingriskoforgandamage,particularlyfocusingontarget organsandconcernsrelatedtolivertoxicity,alongwithpotential accumulationrisksassociatedwithcarriers.337

Proteinreplacementtherapy.Proteinreplacementtherapyisa broadtermencompassingthesupplementationofproteinswith functionaldefectsorthesubstitutionofdeficientproteinsto achievetherapeuticefficacy.345 Thedeliveryoftherapeutic proteinsdirectlytothetargettissueissignificantlyimpededby factorssuchasproteinsizeandbiochemicalproperties.346 In contrast,mRNAtherapiesaremorelikelytobypassthese challenges.Atpresent,theuseofmRNAtranslationinvivoto replacethedirectdeliveryofproteinshasbecomeanewpillarof proteinreplacementtherapy,andhasbeenextensivelystudiedin variousdiseaseareas,includingblooddiseases(suchasHemophiliaA,HemA),andmetabolicdiseases(suchasornithine transcarbamylasedeficiency,OTCD;Fabrydisease,FD;Propionic acidaemia,PA).335

Fig.4 OverviewdiagramofmRNA-basednon-immunotherapy.BDNFbrain-derivedneurotrophicfactor,NEPneprilysin,CFTRcystic ﬁbrosis transmembraneconductanceregulator,HNF4Ahumanhepatocytenuclearfactor α,COL1A1extracellular-matrix α1type-Icollagen,VEGF-A vascularendothelialgrowthfactor-A,TEtropoelastin,BMP-2bonemorphogeneticprotein-2,RUNX1runt-relatedtranscriptionfactor1,OTC ornithinetranscarbamylase,G6Pase-α glucose-6-phosphatase-alpha,AGLamylo-α-1,6-glucosidase4-alpha-glucanotransferase,IGF-1insulinlikegrowthfactor1,ZFNsZinc ﬁngernucleases,CRISPR/Cas9clusteredregularlyinterspacedshortpalindromicrepeats/associatedprotein9. ThegraphiciscreatedwithBioRender.com

mRNAtherapyprovidesapromisingtreatmentoptionfor refractorygeneticdiseases.HemA,aformofhemophiliacaused byinsuf ﬁ cientexpressionorgenemu tationofcoagulation factorVIII(FVIII),resultsinimpairedbloodclottingabilityand canleadtolife-threateningbleedingepisodes,including intracranialhemorrhage.Currentproteinreplacementtherapies arecostlyandrequirefrequentadministrationduetotheshort half-lifeofFVIIIprotein. 347 In2023,theFDAapprovedRoctavian asthe ﬁ rstgenetherapydrugforsevereHemAinadults, utilizingadeno-associatedvirusvectors;however,concernsarise regardingpotentialriskssuchaslivercancerassociatedwiththe introductionofDNAsequence sfromRoctavianproducts. 348 mRNAtherapycanexpressthetargetproteininthecytoplasm withoutenteringthenucleus orinsertingthetargetcell genome,whichismuchsaferthanviralvectorandDNA therapy. 1 Chenetal. 347 designedanLNPsencapsulating

mRNA-encodingFVIIIprotein,whicheffectivelyprolongedthe durationofFVIIIproteinexpression.

Lowlevelsofkeyenzymeproteinsorlackofenzymeactivity resultingfromgeneticdefectscangiverisetoinheritedmetabolic disorders(IMDs).Amongthese,OTCDiscausedbythelossof crucialenzymesinvolvedintheureacyclewithintheliver,leading toimpairedammoniametabolism.Elevatedlevelsofammoniain thebloodstreamcanresultinnervedamageandevenfatality. Existingapproaches,includingdietarycontrolsandammonia scavengers,failtoaddresstherootcause.349 Basedonthis,Prieve etal.349 designedaHybridmRNATechnologydeliverysystem (HMT)thatprotectedOTC-encodingmRNAagainstdegradationby nucleaseswhileexhibitinghepatocellular-speciﬁctargetingcapabilities.HMTconsistsoftwotypesofnanoparticles:di-block polymermicellesandinertLNPs.Thepolymercomprisesthree functionaldomains:(1)GalNAc,whichservesasatargetingligand

forliver-speciﬁcuptakebybindingtotheasialoglycoprotein receptorabundantlyexpressedinlivercells;(2)hydrophilic polymersegmentsthatmaintainthesolubilityofthepolymer; (3)polymersegmentscontainingbutylmethacrylate(hydrophobic monomer),2-propylacrylicacid,and2-(dimethylamino)ethyl methacrylate.Thispolymericvesiclewasdesignedtoachieve liver-speciﬁctargetingandfurtherfacilitatethepH-dependent releaseofmRNAfromendosomalorganellestothecytosol.The inertLNPscanshieldmRNAfromnucleases.Thisplatformisnot onlysafebutalsoextendsthetherapeuticeffectsforOTCD treatment.Currently,ARCT-810(OTCmRNALNPs)hasentereda PhaseIIclinicaltrial(NCT05526066)inOCTpatientsaged12years andolder.350

FDisanX-linkedlysosomalstoragedisorder(LSD).351 Itsroot causeliesinthepathogenicmutationoftheGLAgene(Xq21.3q22),whichleadstoalossofenzymeactivityencodinglysosomal enzyme α-galactosidaseA.Thisresultsinabnormalmetabolismof relatedglycolipidsandthesubsequentaccumulationofglycolipidsthroughoutthebody,causingstructuraldamageand functionallossinvarioustissuesandorgans,particularlyaffecting theheart,kidney,andnervoussystem.Theresultingcardiac dysfunctionandcerebrovasculareventsposelife-threateningrisks. Althoughthereareclinicallyapprovedenzymereplacement therapies(ERTs),theyfailtomeetmedicalneedsduetotheir inabilitytopenetratetheblood-brainbarrier.352 Tothisend,in 2019,DeRosaetal.351 andZhuetal.353,respectively,reported systemicallydeliveryofmRNA-encodinghumanalphagalactosidaseA(H-α-galA)usingLNPs,bothofwhichindicated thatmRNALNPswasapotentialtreatmentmodalityforFD.

PAisalife-threateningIMDscausedbyapathogenicmutation inthepropionyl-coenzymeAcarboxylase α or β (PCCAorPCCB) subunitsgene,andtherearecurrentlynoapproveddrugsto addressthisenzymedeficiency.Aninnovativeapproachinvolving twomRNAsencodingnormalhumanPCCAandPCCBprotein subunitshasenteredPhaseI/IIclinicaltrials(NCT04159103). EncapsulatedwithinLNPs,thesemRNAsarespecificallydesigned fortargeteddeliverytothelivertorestorePCCenzymes. Promisinginterimresultsfromthistrialhavedemonstrateda favorablesafetyprofileofmRNA-3927inPApatients.Notably, dose-optimizationstudieshaverevealedaremarkable70% reductioninrelativeriskassociatedwithmetabolicdecompensationevents(MDEs),highlightingthetherapeuticefficacy.354

Hormonereplacementtherapy.Hormonesproducedbyvarious glandsinthebodyserveascrucialsignalingmoleculesthatare transportedthroughthebloodstreamtotargetorgans,tissues, andcells,playingapivotalroleinmaintainingphysiological balance.Disruptionsinhormonalregulationcangiveriseto diversediseases.355 Duringmenopause,hormonal ﬂuctuations leadtothemanifestationofmenopausalsymptomsandan increasedriskofcardiovasculardisorders.Toalleviatethese symptomsandslowdowntheprogressionofcardiovascular diseases,non-proteinhormoneslikeestrogencanbeadministered orallyortopicallyviagelapplication.Similarly,forconditionssuch asandrogendeﬁcitinagingmen(ADAM)ormalehypogonadism, supplementationthroughsimilarmeansmayalsobeconsidered.356 However,duetotheirsizeandspatialstructure,protein hormonescannotbeeffectivelyadministeredorallyastheywould undergodegradationwithinthedigestivesystem.Surprisingly,in 1992,Jirikowskietal.357 injectedmRNA-encodingvasopressininto Brattlebororatswithsuccessfulexpressionofvasopressininthe hypothalamusandshowedtherapeuticeffect(temporaryreversal ofdiabetesinsipidusforupto5days).

Insulin-likegrowthfactor1(IGF-1)isaversatilegrowthfactor primarilysynthesizedintheliver.358 Itcanregulatethe chondrogenesisofmesenchymalstemcells(MSCs)bystimulating theirproliferationandfacilitatingcartilagedifferentiation.Wu etal.359 usedmodiﬁedmRNA(modRNA)encodingIGF-1to

engineeradipose-derivedstemcells(ADSCs).Thetherapeutic effectofengineeredADSCsonOsteoarthritis(OA)issuperiorto thatofnaturalADSCs.

Althoughtherehavebeenlimitedpreclinicalinvestigationson mRNA-encodedprotein-likehormones,itisexcitingthatthat mRNA-0184(encodingtheRelaxin-2-variableLightChainKappa) developedbyModernaTX,Inc.forthetreatmentofchronicheart failurehasenteredPhaseIclinicaltrialsin2023(NCT05659264).

Regenerativemedicineapplication

Myocardialinfarction:Inhumans,myocardialinfarctionresultsin theocclusionofaportionoftheheart’sbloodsupplyandthe subsequentdeathofbillionsofcardiomyocytes,leadingtoahigh mortalityrateduetothechallengingregenerationoflost cardiomyocytes.Thisdifficultyhinderstherepairofdamaged heartfunction.Thevascularendothelialgrowthfactor(VEGF-A) hasbeenidentifiedashavingangiogenicproperties,360 makingit apotentialtherapeuticagentforpatientswithischemicheart disease.However,itssystemicadministrationisassociatedwith long-termsideeffectsandposeschallengesinspecifically targetingtheheart.361 In2013,Zangietal.360 designedmodRNA encodingVEGF-Aandfoundthatasingleintracardialinfusionof VEGF-AmodRNAcouldincreasethedensityofbloodvessels aroundinfarction,reduceinfarctsizeandcelldeath.Inaddition, comparedtomicetreatedwithVEGF-ADNA,thosereceiving VEGF-AmodRNAshowedimprovedsurvivalrates.Thisimprovementcanbeattributedtocarefulconsiderationregardingtiming anddosagesinceprolongedexposuretoVEGF-AintheDNA groupresultedinincreasedvascularpermeabilityandcardiac edema.Furthermore,theydiscussedhowmobilizationofepicardialprogenitorcellsplayedacrucialroleinenhancingcardiac functionthroughexpansionanddifferentiationintocardiovascular lineages.

TheVEGF-A165 mRNA(AZD8601),dissolvedinbiocompatible citratesaline,hascommencedclinicaltrials.Specifically,thePhase IIaclinicaltrial(NCT03370887)aimstoenhanceoutcomesin coronaryarterybypassgraftpatientsthroughendocardial injectionofAZD8601.362 Inaddition,AZD8601exhibitspromise fortreatingtype2diabetesmellitus(T2DM)andiscurrently undergoingPhaseIclinicaltrials(NCT02935712).363 Nawazetal.364 comparedVEGF-AproteinproductioninLNPscontainingthe sameamountofVEGF-AmRNAorinextracellularvesicles(EVs) secretedbythreedifferenttypesofcells(cardiacprogenitorcells, CPCs;humanlungepithelialHTB-177cells;humanumbilicalvein endothelialcells,HUVECs)treatedwithVEGF-AmRNALNPs.Their findingsrevealedthatwhendeliveredtoendothelialcellsinvitro, theLNPscarrierproducedthehighestlevelofVEGF-AwhileCPCEVsdemonstratedthelowestproteinyield.However,CPC-EVswas themosteffectiveinpromotingangiogenesispergivenamountof VEGF-Aproduced.Intriguingly,duringintramyocardialinjection, EVs-basedcarriersdisplayedthehighestexpressionofVEDF-A. ConsideringthatCPC-EVsinduceminimalinflammatorycytokine expressioncomparedtoothervectors,theyarebettersuitedfor deliveringmRNAtotheheart.

TheT-boxtranscriptionfactor18(TBX18)playsapivotalrolein theformationanddifferentiationofthesinoatrialnode(SAN). 365 Wolfsonetal. 366 discoveredthatdirectinjectionofnakedTBX18 mRNAintothemyocardiumofratscouldeffectivelytransfect cardiomyocytes,leadingtotransientexpressionofTBX18and subsequentreprogrammingofthecardiomyocytesintopacemakercells.Furthermore,theyobservedthatcomparedto adenoviralvectors,mRNAtransfectionexhibitedasigni ﬁ cant reductioninoff-targetgeneexpressionandlower immunogenicity.

Liver ﬁbrosisandcirrhosis:Inthecontextofliverdiseases, humanhepatocytenuclearfactor α (HNF4A)hasbeenrecognizedasapivotalregulatoroflivercellphenotype,exertingits

in fl uenceonhepaticstellatecellsandlivermacrophagesby targetingparaoxoase-1.Thistargetedregulationeffectively mitigatesliver fi brosis.Yangetal.367 designedHNF4AmRNAloadedLNPstoimproveliver fi brosisandcirrhosisinvivo.Itwas noteworthythattheuseofhumanHNF4AmRNAnotonly restoredtheexpressionof HNF4A inmouseliverbutalso avoidedadverseimmunereactions,therebyfurtherenhancing itsanti- fi broticef fi cacy.

Cystic fibrosis:Thecystic fibrosistransmembraneconductance regulator(CFTR)geneencodesepithelialionchannelsresponsible forthetransportofchlorideandbicarbonate,playingacrucialrole inmaintainingnormalphysiologicalfunctions.Cystic fibrosisisa monogenicdiseasecausedbymutationsintheCFTRgenethat causesthelungsandotherorganstoproducethickmucusthat blockstheairwaysofthelungsandmakesbreathingdifficult, leadingtolungdamage,respiratoryfailureandchroniclung infections.Inaddition,cystic fibrosisalsoaffectsotherorganssuch asthepancreasresultingindigestiveproblemsandmalnutrition. Furthermore,cystic fibrosiscangiverisetovariouscomplications includingdiabetes,liverdisease,andinfertility.Unfortunately, thereiscurrentlynocureforthislifelongcondition.276,368

In2018,TranslateBio,Inc.conductedaPhaseIItrial (NCT03375047)involvingadultsandseniorsaged18yearsor olderwithcystic ﬁbrosistoinvestigatethesafetyandtherapeutic efﬁcacyofMRT5005 anaerosolcontainingcodon-optimized CFTRmRNA.Itwasfoundthattheoverallsafetyofpatients receivingMRT5005wasgood,butfeverandallergicreactions occurred.Theetiologyandmechanismoffeverremainunclear, necessitatingfurtherresearch.369 Additionally,twomoremRNA therapies,namelyARCT-032(encodingCFTRprotein, NCT05712538)andVX-522mRNAtherapy(NCT05668741),have commencedclinicaltrialsin2023,withtheoutcomesyettobe disclosed.

Degenerativedisease:Thetreatmentofbonedefectsresulting fromnonunion,trauma,orcraniofacialmalformationsischallengingandaffectsmillionsofindividuals.Currently,thedeliveryof bonemorphogeneticprotein-2(BMP-2)showspromiseasa treatmentapproachtoaddressthesedefects.However,ithas beenobservedthatexcessiveadministrationofBMP-2canleadto severesideeffectssuchassignificantinflammationandswelling. Forheterotopicossification,localdeliveryandprecisetranslation ofBMP-2mRNAtoproducefunctionalproteinscanbeachieved. Wangetal.370 employedadualdeliverystrategybysimultaneouslyadministeringBMP-2mRNAandnon-structuralprotein-1 (NS1)mRNAinamassratioof3:1.Remarkably,their findings demonstratedthatthisapproachinducedhighexpressionlevels ofBMP-2surpassingpreviouslyreportedyields.Notably,theyare the firsttouseunmodifiedmRNAinthe fieldofregenerative medicinesincetheco-deliveredNS1mRNAencodesanimmune evasionproteinoriginallyexpressedby influenzaA viruses(A/ Texas/36/1991),whicheffectivelyinhibitsRNAsensorswhile reducingtheproductionofimmunefactorstopreventactivation ofharmfulimmuneresponses.

Wangetal.371 furtherexploredthedeliveryplatformofBMP-2/ NS1mRNAbyincorporatingalipopolyplex(Lip100/His-lPEI/RNA ternarycomplex,LPR)loadedwithBMP-2/NS1mRNAintoa collagen-nanohydroxyapatitescaffolds.Theresultingready-to-use mRNA-activatedmatrices(RAMs),obtainedthroughfreeze–drying, exhibitaremarkableenhancementinthereleasetimeofmRNA, extendingupto16days.Thisapproachenablesinsituexpression andcontinuousproductionofBMP-2protein,therebypromoting boneformation.Gengetal.372 foundthatcomparedwiththe deliveryofrecombinantproteinorsinglemodRNAalone,thecodeliveryofhumanBMP-2(hBMP-2)andVEGF-AmodRNA-modiﬁed bonemarrowstemcells(BMSCs)increasedtheexpressionlevelof osteogenic-relatedgeneandenhancedtheformationofnew

boneinratsundergoingskulldefectsurgery.These ﬁndingsoffer apromisingtherapeuticoptionforregeneratingbonetissue.

Runt-relatedtranscriptionfactor1(RUNX1)isacartilageanabolicfactor,whichpromotestheproliferationofchondrocytes andincreasestheexpressionofcartilage-anabolicmarkersof chondrocytes.Furthermore,RUNX1enhancescoccygealdisc hydrationcontentwhilemitigatingdiscdegenerationbyminimizinglossofdisccartilageandotherconstituents.Inthisregard, Linetal.373 developedRUNX1mRNAnanomicellesforthe treatmentofintervertebraldisc(IVD)degeneration.Comparedto thecontrolgroup,administrationofRUNX1mRNAnanomicelles signiﬁcantlymaintainedahigherdischeightinratswithcoccygeal discdegenerationandprevented ﬁbrosisofthedisctissue.

Alzheimer’sdiseaseisadegenerativedisorderresultingfroman imbalanceinthemetabolicprocessesofamyloid-beta(Aβ) synthesisandclearance.374 Neprilysin(NEP)isknowntoplayan importantroleintheclearanceprocessofAβ.Therefore,Lin etal.375 designedpolyplexnanomicellesbasedonNEP-encoding mRNA,whichsignificantlyreducedtheconcentrationofAβ inthe brain,demonstratingthepotentialofmRNA-basedtherapyin braintherapy.Inaddition,Lietal.376 engineeredanmRNA encodingbrain-derivedneurotrophicfactor(BDNF)forADthat waspackagedonapoly(β aminoesters)(PBAE)nanoplatformand delivereddirectlytothecentralnervoussystem.Toprevent excessiveneuronalactivationcausedbyhightranslationofBDNF mRNA,theymodifiedthe3’UTRofthismRNAwithneuron-specific miRNA-124targetsequences,ensuringdegradationbymiRNA upondeliverytoneurons.ByexpressingandreleasingBDNFin astrocytestosupportneurons,theBDNFmRNAeffectively enhancedmemoryfunctioninADmodelmice,thushighlighting thepotentialofmRNAtherapyinthetreatmentofneurological diseases.

Connectivetissuedisorder:Elastin,oneofthemostenduring proteins(withahalf-lifeofapproximately74years),ispredominantlyexpressedduringtheneonatalperiod.However,withage, elastinsynthesisgraduallydiminishesandeventuallyceasesin adults.Elastinplaysapivotalroleinendowingtissuesandorgans withelasticity,therebycontributingsignificantlytothenormal functioningofelasticconnectivetissue.Unfortunately,genetic disorderslikeWilliams–Beurensyndrome(WBS)canleadto mutationsinelastin-relatedgenes,resultinginareductionof over50%inelastinsynthesis.Homozygouselastinnullmutants (ELN / )succumbshortlyafterbirth.Inaddition,factorssuchas agingandsunburncanalsoleadtoelastinreduction.Lescan etal.377 synthesizedandmodifiedthemRNA-encodinghuman tropoelastin(TE).Their findingsdemonstratedthatdeliveryofTE mRNAincreasedthesynthesisofelastininpigskinby20%. Furthermore,Golombeketal.378 carriedoutcodonoptimization andnaturalnucleotidemodificationonTEmRNAtoimprovethe translationefficiencyandstabilityofmRNA,thusimprovingthe expressionlevelofTEproteininvivoandinvitro.Thisstudynot onlyprovidesvaluableinsightsforotherconditionsnecessitating denovoelastinsynthesisbutalsoholdspromisefordiseaseslike myocardialinfarction.

Anotherkeyproteininconnectivetissueiscollagen.379 You etal.380 designedanexosome-basedextracellular-matrix α1type-I collagen(COL1A1)mRNAforanti-agingandtreatmentof photoagingskinthatsuccessfullyalleviatedUV-inducedskin agingthroughintradermaldeliveryviamicroneedlearrays.

Targetedgenomeengineering.Targetedgenomeengineeringis theuseofgeneeditingtechnologytoknockout,insertand replacethetargetgenomesequencetoachievechangesin geneticinformation,381 whichbringsanewwayforthetreatment ofdiseases(especiallygeneticdiseasescausedbygene defects).382 Zinc ﬁngernucleases(ZFNs)andtranscriptional activator-likeeffectornucleases(TALENs)arewidelyemployedin

geneeditingtechniquesoveradecadeago.383 However,their applicationiscomplexedandnecessitatesthedesignofproteins thatspeciﬁcallybindtotargetgenes.384 Theadventofclustered regularlyinterspacedshortpalindromicrepeats(CRISPR)/associatedprotein9(CRISPR/Cas9)systemshassigniﬁcantlypropelled theadvancementofgeneeditingtechnology.CRISPR/Cas9 obviatestheneedforalteringtheproteinsequencesandonly requiresthedesignofalternativegRNAsforcustomization purposes.385 TherearethreetypicalformsoftheCRISPR/ Cas9system:plasmidDNA(pDNA),Cas9mRNA/sgRNA,and ribonucleoprotein(RNP,Cas9proteincomplexedwithgRNA). CRISPR/Cas9pDNAfacesthechallengeofnuclearlocalizationand hastherisksofoff-targetrepeatexpressionandgenome integration;Cas9RNPdeliveryhastosurmountobstaclesrelated tomacromolecularsizeandinstability;whileCas9mRNA/sgRNA needstoaddresstheinstabilityofsingle-strandednucleicacid.386

TheCRISPR/Cas9technologycantreatdiseasesbyknockingout abnormalgenes.Thehallmarkofwetage-relatedmacular degeneration(wAMD)isvasculardysplasia.Atpresent,anti-VEGF reagents,the ﬁrst-linedrugforthetreatmentofwAMD,are expensiveandrequirerepeatedorevenlifelonginjection.Inlight ofthis,Lingetal.387 designedaplatformthatusedalentiviral systemtopackageCas9mRNAandVEGF-A-targetinggRNA.The areaofchoroidalneovascularization(CNV)wasreducedby63% withnodetectableoff-targeteffect.Legumain,encodedbythe LGMNgenelocatedonhumanchromosome14,hasbeen identiﬁedinvarioustumorssuchasbreastandgastriccancers whereitisassociatedwithtumoraggressiveness,migratory behavior,andpoorprognosis.Wangetal.388 usedLNPstocodeliverCas9mRNAandLGMNgene-targetinggRNA,andfound thatthisplatformheldpromiseasatherapeuticapproachto inhibitbreasttumormetastasis.

TheCRISPR/Cas9technologycanalsoremoveviralDNAfrom thebody.Thereare296millionpatientswithchronicHBV infectionintheworld,andcurrentclinicaltreatmentcanslowthe replicationofthevirustoacertainextentanddelaythe progressionofthedisease,buttheviralDNAininfectedcells cannotbecleared,andHBVDNAintegratedintothehostbodyis consideredtobeasigniﬁcantsourceofcancerrisk.Moreover,the formationofcccDNAinthelivernucleusandcontinuous productionofprogenyvirusposedachallengetotheradical treatmentofHBV.Toaddressthisissue,Yietal.389 usedSM-102basedLNPstodeliverCas9mRNAandgRNAstogether,and employedthisCRISPR/Cas9geneeditingtechnologytoachieve targetedsite-speciﬁcediting.ThisapproachprovidedanopportunityforcontinuouseliminationofcccDNAandintegratedHBV DNA,therebyfacilitatingaradicaltreatmentstrategyagainstHBV. Inaddition,totreatHPV-relatedcervicalcancer,Lingetal.326 designedapH-responsivenonviralnanonucleusforco-deliveryof Cas9mRNAandgRNAstargetingHPVoncogenesE6andE7.This platformallowedforcombiningCas9mRNA/gRNAwithadoptive celltherapytoeffectivelytreatcancerdiseases.

AlthoughCRISPR/Cas9technologyholdsimmensepotentialfor thetreatmentofgeneticallyrelateddiseasesandvirusclearance, itsdevelopmenthasbeenhinderedbythechallengesassociated withdeliveringmRNA/gRNA.Currently,CRISPR/Cas9ispredominantlyusedintheformofCas9RNPorpDNAanddeliveredvia viralvectorsorelectroporationinclinicaltrials.386 However,there areactiveeffortstodevelopmRNA/gRNAdeliverystrategiessuch asLNPs,388–390 lentivirus,andretroviralvectors.387,391 Welook forwardtodevelopingvariousdeliveryvectorstoadvancethe clinicalapplicationofmRNA/gRNA.

ClinicaltrialsbasedonCas9mRNA/gRNAhavebeenconducted. Transthyretin(TTR)amyloidosis(abbreviatedATTRamyloidosis)is alife-threateningdiseasecausedbytheaccumulationof misfoldedTTRproteinsintissues,particularlyintheheartand nerves.NTLA-2001representsageneeditingtherapybasedon theCRISPR-Cas9systemthatincludesLNPsencapsulatedwith

mRNA-encodingCas9proteinandTTR-targetinggRNA.The interimtrialresultsfromPhaseIstudy(NCT04601051)demonstratedthatNTLA-2001effectivelyreducedserumTTRconcentrationwithonlymildadverseevents.392 Otherexamplescanbe foundinTables 2 and 3.Currently,mRNA-basedtherapiesfor encodingZFN,TALEN,andCas9aregenerallyinthedevelopmentalstageandhavethepotentialtoplayacrucialrolein diseasetreatment.393

THEIMPACTSFROMINDIVIDUALPHYSIOLOGICALAND PATHOLOGICALCHARACTERISTICS

Beginninginlate2019,patientsinfectedwithSARS-CoV-2virus willprogresstoCOVID-19disease,rangingfromasymptomatic infectiontolife-threateningdisease,withparticularlyhigh mortalityamongvulnerablepopulations(suchastheelderlywith compromisedimmunity,individualswithchronicdiseases,those useimmunosuppressants,andcancerpatients).394 TheU.S.FDA andtheEuropeanMedicinesAgency(EMA)approvedmRNA-1273 (Moderna)andBNT162b2(Pﬁzer/BioNTech)forelicitingpotent B-cellandT-cellimmuneresponsesinhealthypeopletoprevent SARS-CoV-2infection.395,396 However,theprotectiveeffectofthe vaccinewanesovertime,whichcanbeobservedinhealthy subjectsandismorepronouncedinimmunocompromised subjects.Therefore,itbecomesimperativetoconsideradministeringathirdorevenfourthdoseofthevaccine.

Specifically,Rizzietal.394 recruitedpatientsreceivingimmunosuppressivetherapyforafollow-uptrial.Theyfoundanabsenceof protectiveantibodyresponseaftertwoprimaryvaccinations,and detectableantibodytiterswerenotproduceduntilathird vaccination.Moreover,giventhattheinducedimmuneresponse diminishesgraduallyafterthethirdvaccination,dependingonthe patient’scondition,evenafourthimmunizationwasnecessary. Nugentetal.397 studiedtheantibodyresponseandpseudovirus neutralizationagainstSARS-CoV-2wild-typestrain,OmicronBA.1, andBA.5variantsinnursinghomeresidentsandhealthcare workersafterprimarymRNAvaccination, firstandsecond boosters.Theyfoundthatantibodytitersandneutralizingability wereprogressivelyincreasingwitheachbooster,butsubsequently diminishedover3–6months.Consideringthatindividualswith humanimmunodeficiencyvirus(HIV)havesignificantlycompromisedimmunity,manycountrieshaveprioritizedSARS-CoV-2 mRNAvaccinesforthispopulation.Ithasbeenobservedthatafter receivingtwodosesofBNT162b2vaccine,HIV-infectedones exhibitamorepronouncedweakeningintheantibodyresponses thantheirhealthycounterparts,therefore,athirdvaccinationis recommended.Heftdaletal.398 investigatedthepersistenceof cellularandhumoralimmunityinHIVpatientsandcontrolsafter thethirddoseofvaccineandfoundnosignificantdifferencesin antibodytitersandcellularresponsesbetweenHIVpatientsand controlsat4monthsafterthethirddoseofBNT162b2vaccine.In conclusion,repeatedvaccinationismoreimperativeinsusceptible individualsthanintheirhealthycounterpartsfortheestablishmentofenduringimmuneprotection.394

Theaboveresultsareconsistentwiththeresultsofepidemiologicalstudiesonhospitalizedpatientswithsevereandhigh-risk COVID-19(includingtheelderlyandpatientswithotherdiseases). However,therestillexistsignificantvariationsinclinicaloutcomes amongepidemiologicalpatients,suggestingthattheunderlying causeforsymptomdisparitiesbetweensevereCOVID-19patients andasymptomaticcarriersmaybemoreprofound.Through differentialgenescreening,Zhangetal.399 identifiedmutationsin type-IIFNgenesamongsevereCOVID-19patients,andtheyfound thatatleast3.5%ofpatientswithsevereCOVID-19hadgenetic defectsingenesassociatedwithTLR3-andregulatoryfactor7 (IRF7)-dependentinductionandamplificationoftype-IIFN.

Furthermore,Bastardetal.400 foundthatsomeindividualswho remainedseverelyaffectedbyCOVID-19evenafterreceivingtwo

Table2. Representativecompletedandongoingclinicalstudies(non-immunotherapy)

PhaseStatusSponsorNCTnumber

Study started

TherapyDiseasemRNAmRNA-encodedproteinDeliverysystemAdministration route

NCT05526066

2022-07-06PhaseIIRecruitingArcturus Therapeutics,Inc.

LNPsIntravenous infusion

ARCT-810Ornithine transcarbamylase(OTC)

2019-12PhaseI/IIWithdrawnTranslateBio,Inc.NCT03767270

MRT5201LNPsIntravenous administration

Ornithine transcarbamylase de ﬁ ciency(OTCD)

Protein replacement therapy

2021-04-15PhaseI/IIRecruitingModernaTX,Inc.NCT04159103

Intravenous infusion

LNPs(SM-86,DSPC, cholesterol,and PEGlipid)

mRNA-3927Propionyl-coenzymeA carboxylase α or β (PCCA orPCCB)subunits

Propionicacidaemia (PA)

2022-06-01PhaseI/IIRecruitingModernaTX,Inc.NCT05095727

LNPsIntravenous infusion

mRNA-3745Glucose-6-phosphatase- alpha(G6Paseα )

Glycogenstorage disease1a(GSD1a)

NCT04990388

2021-10-18PhaseI/IITerminatedUltragenyx PharmaceuticalInc

LNPsIntravenous infusion

UX053Amyloα -1,6-glucosidase 4-alpha- glucanotransferase(AGL)

Glycogenstorage diseasetypeIII

2019-05-28PhaseI/IIWithdrawnModernaTX,Inc.NCT03810690

mRNA-3704UndisclosedLNPsIntravenous infusion

Methylmalonic acidemia(MMA)

2021-08-06PhaseI/IIRecruitingModernaTX,Inc.NCT04899310

Intravenous injection

Injection2022-03-08PhaseI/IIRecruitingModernaTX,Inc.NCT05295433

LNPs(SM-86,DSPC, cholesterol,and PEGlipid)

mRNA-3705Methylmalonyl- coenzymeAmutase

2022-12-05PhaseIRecruitingModernaTX,Inc.NCT05659264

UndisclosedIntravenous infusion

ChronicheartfailuremRNA-0184Relaxin-2-variablelight chainkappa

2024-02-16PhaseIRecruitingModernaTX,Inc.NCT06243770

HealthyparticipantsUndisclosedIntravenous infusion

Hormone replacement therapy

2018-02-05PhaseIIaCompletedAstraZenecaNCT03370887

NoneEpicardial injection

AZD8601Vascularendothelial growthfactor-A(VEGF-A)

2016-12-16PhaseICompletedAstraZenecaNCT02935712

NoneIntradermal injection

TranslateBio,Inc.NCT03375047

LNPsInhalation2018-05-10PhaseI/IIUnknown status

NCT05712538

ARCT-032LNPsInhalation2023-02-15PhaseIRecruitingArcturus Therapeutics,Inc.

NCT05668741

Myocardial infarction

Type2diabetes mellitus(T2DM)

Regenerative medicine application

Cystic ﬁ brosisMRT5005Cystic ﬁ brosis transmembrane conductanceregulator (CFTR)protein

2023-02-27PhaseI/IIRecruitingVertex

Pharmaceuticals Incorporated

2023-12-13PhaseIIIRecruitingIntelliaTherapeuticsNCT06128629

UndisclosedLNPsOralinhalation usingnebulizer

VX-522mRNA therapy

Intravenous infusion

NTLA-2001Cas9proteinLNPs(ionizable lipid,DSPC, cholesterol, PEG2000-DMG)

Transthyretin amyloidosis(ATTR) with cardiomyopathy

Targeted genome engineering

IntelliaTherapeuticsNCT05120830

2021-12-10PhaseI/IIActive,not recruiting

NTLA-2002LNPsIntravenous injection

Hereditary angioedema(HAE)

NCT04560790

CompletedShanghaiBDgene Co.,Ltd.

2020-11-04Not applicable

NCT02500849

CityofHope MedicalCenter

NCT03432364

SpCas9LentiviralparticlesCorneal injection

BD111CRISPR/ Cas9mRNA

Infusion2016-03-10PhaseIActive,not recruiting

Autologous hematopoieticstem andprogenitorcells (HSPCs)

Infusion2018-03-29PhaseI/IICompletedSangamo Therapeutics

Refractoryviral keratitis

HIVSB-728mR-HSPCC-Cmotifchemokine receptor5(CCR5) -speci ﬁ czinc ﬁ nger nucleases

ZFNsAutologous hematopoieticstem andprogenitorcells (HSPCs)

ST-400 investigational product

Transfusion- dependentbeta- thalassemia(TDT)

clusteredregularlyinterspacedshortpalindromicrepeats/associatedprotein9, HIV humanimmunode ﬁ ciency virus, ZFNs Zinc ﬁ ngernucleases

LNPs lipidnanoparticles, DSPC distearoylphosphatidylcholine, PEG polyethyleneglycol, CRISPR/Cas9

Table3. Representativepreclinicalstudies(non-immunotherapy)

TherapyTargetorgan/tissueDiseasemRNA-encodedproteinDeliverysystemAdministrationrouteRef.

Intravenousinjection

BloodHemophiliaACoagulationfactorVIIILNPs(ionizablelipid:DSPC: cholesterol:PEGlipid,50:10:38.5:1.5)

Proteinreplacement therapy

Intrahepaticdelivery

LNPs(DOTAP,cholesteryl hemisuccinate,cholesterol,PEG lipid);di-blockpolymermicelle

Ornithinetranscarbamylase (OTC)

LiverOrnithinetranscarbamylase de ﬁ ciency(OTCD)

Intravenousinjection

Fabrydisease(FD) α -galactosidaseALNPs(C12-200,DOPE,cholesterol, dimyristoylglycerol-polyethylene glycolanalog)

Wholebody(especially heart,kidneys,andbrain)

Intravenousinjection

Wholebody(especially heart,kidneys,andbrain) FD α -galactosidaseALNPs(ionizable:helper:structural: PEG,50:10:38.5:1.5)

BrainDiabetesinsipidusVasopressinNoneInjectionintothehypothalamus

BoneOsteoarthritis(OA)Insulin-likegrowthfactor1 (IGF-1)

Hormone replacementtherapy

NoneIntramyocardialinjection

HeartMyocardialinfarctionVascularendothelialgrowth factor-A(VEGF-A)

Regenerative medicineapplication

LiverLiver ﬁ brosisandcirrhosisHepatocytenuclearfactor alpha(HNF4A) LNPsIntravenousinjection

LipopolyplexSubcutaneouslyimplant 371

BoneBonedefectsBonemorphogenetic protein-2(BMP-2)

Administeredintoratcoccygealdisks

Polyplexnanomicelles(PEG- polyaminoacidblockcopolymers)

Intervertebraldisk(IVD)IVDdegenerationRunt-relatedtranscription factor1(RUNX1)

375

Intracerebrovetricularadministration

BrainAlzheimer ’ sdiseaseNeprilysin(NEP)Polyplexnanomicelles(PEG- polyaminoacidblockcatiomer)

PBAEpolymersDeliverytothecentralnervoussystem bybrainventriclepumping

NervoussystemAlzheimer ’ sdiseaseBrain-derivedneurotrophic factor(BDNF)

EVsIntradermaldelivery(microneedle array)

SkinPhotoagedskinExtracellular-matrix α 1type-I collagen(COL1A1)

Tropoelastin(TE)NoneIntradermalinjection

ConnectivetissueWilliams –Beurensyndrome (WBS)

Cas9LentiviralsystemSubretinalinjection

EyeWetage-relatedmacular degeneration

Targetedgenome engineering

TumorBreastcancerCas9LNPsIntravenousinjection

Intravenousinjection

ViralcarcinogenesisHBVCas9LNPs(SM-102:DSPC:cholesterol: PEGlipid,50:10:38.5:1.5)

LNPs lipidnanoparticles, DSPC distearoylphosphatidylcholine, PEG polyethyleneglycol, DOTAP (2,3-dioleoyloxy-propyl)-trimethylammoniumchloride, DOPE 1,2-dioleyl-sn-glycero-3-phosphoethanolamine, ADSCs adipose-derivedstemcells, EVs extracellularvesicles, HBV hepatitisBvirus

Fig.5 Immunizationbackgroundmap.Thevariationsinindividuals’ immunologicalbackgroundsmayserveasapotentialmechanism forthediversityinvaccineefﬁcacyandadversereactionsfollowing mRNAvaccination.Anindividual’simmunebackgroundisshaped byamultitudeoffactors,encompassingbothphysiological conditionsandpathologicalconditions.Moreover,itiscrucialto considerthepotentialadverseeffectsofrepeatedorintensive vaccinationusingthesameormultiplevaccines.Thegraphicis createdwithBioRender.com

dosesofSARS-CoV-2mRNAvaccineexhibitednotonlyneutralizingantibodiestothevirusbutalsoautoantibodiestargetingtype-I IFN.Thelackoftype-IIFNmaybethecauseofseverehypoxemic pneumoniaofCOVID-19.Theaforementionedevidenceimplies thatitisimperativetofocusmoreonthedistinctgeneticimmune characteristicsofvulnerablepopulations(Fig. 5).

Age

Vaccination,asapowerfulweaponinthepreventionand treatmentofdeadlyinfectiousdiseases,cansavehundredsof millionsoflivesbyharnessingthehumanimmuneresponse.401 However,humanimmunesystemisnotstatic,rather,itundergoes profoundchangeswithage.402 Age-dependentimmunesystem alterationsintheelderlyincludeimmunesenescenceininnate immuneresponses(i.e.,changesinthenumber,function,and distributionofneutrophils,NKcells,monocytes,andDCs),403 and adaptiveimmuneresponses(e.g.,decreasedTCRdiversityofaging naïveTcells,shortenedtelomeresofmemoryTcells,decreased B-cellpooldiversity,anddecreasedresponsetoforeign antigens).404

However,thereasonsfordecreasedvaccineeffectivenessinthe elderlygofarbeyondthis,thatis,itisnotonlyassociatedwith impairedantibodyresponse,decreasedT-cellresponse,and alteredantigenpresentationoftheimmunesystem,butalso relatedtothechronicandsystemicasepticinflammatorystate accompaniesaging.405 ResearchconductedbyPuzianowskaKuźnickaetal.406 hasshownthattwopro-inflammatorycytokines IL-6andC-reactiveprotein(CRP),increaseintheelderlyin anage-dependentmanner,highlightingtheconnectionbetween chronicinflammationandaging.Nakayaetal.407 conducted studiesthatrevealedanage-relateddisparityinantibody responsefollowinginfluenzavaccination,irrespectiveofraceor gender,andfoundthatolderadults(>65years)hadarelatively

reducedB-cellresponseaftervaccinationcomparedtothatof youngeradults(<65years).However,theseolderadultsdisplayed increasedmonocytesandactivatedcytotoxicNKcells.Notably, monocyteswerealsoincreasedinolderadultspriortovaccination, suggestingthatthebaselinestateoftheimmunesysteminolder adults(characterizedbyage-relatedlowinflammation)isa potentialmechanismforthereducedvaccineresponse. Age-dependentlow-gradechronicin fl ammationcanarise fromvariouspotentialsources.First,withage,theaccumulation ofdamagedcellsandthereleaseofmacromoleculessuchas advancedglycationend-products(AGE)continuetoactivate theinnateimmunesystemandtriggerthereleaseofdiverse pro-in fl ammatoryfactors.Second,immuneagingintheelderly mayalsoincreasetheburdenofantigenexposure.Forinstance, chronicCMVinfectionnotonlycontinuouslystimulatethe immunesystem,butalsoexacerbatebothimmuneagingand in fl ammation. 408 Comparedtoyoungerindividuals,theelderly andthepatientswithchronicdiseaseshaveapro-in fl ammatory statusandaremorepronetotriggeringcytokinestorms followingCOVID-19infection,leadingtosevereCOVID-19 clinicalsymptoms. 408 Immuneagingisaninevitablemajorrisk factorinvariouschronicdisea ses,particularlychronicin fl ammatoryconditionsincludingneurodegenerativediseases,cardiovasculardiseases,andchron ickidneydiseases.Inaddition, chronicin fl ammationplaysacrucialroleinthepathogenesisof variouscancersthatshareacommonfactor immuneaging. Theincidenceofthesepathologicalconditionssigni fi cantly increaseswithage. 409 Moreover,immuneagingcanexplainthe highermortalityrateobservedinolderCOVID-19patientsdue toineffectiveT-cellresponsesandtheinabilitytoproduce antibodiesagainstSARS-CoV-2.Therefore,identifyingmarkers thatreshapeimmuneresponseinolderadultscouldserveas potentialtargetsforpatientswithCOVID-19andotheragerelatedailments. 410 Immunesenescenceleadstothedisruption ofimmunecheckpointmolecules,suchasPD-1andCTLA-4,and cytokinessuchasIL-6andIL-1 β 411 , 412 Furthermore,immune senescenceisalsocloselyass ociatedwithmanyautoimmune diseases.Theincidenceofautoimmunediseasesappearstorise withage.Inturn,autoimmunediseasessuchasrheumatoid arthritismayalsoacceleratetheprocessofaging.Ononehand, aginginducesastateofage-dependentlow-gradechronic in fl ammation,whichinturntrigge rsimmunesuppression.This exacerbatesimmunesenescence andincreasestheprevalence ofvariousailmentsamongolderin dividuals,includingchronic in fl ammatoryconditionsthatfurtherhastenhumanaging. 409 Age-relatedimmunesenescenceandthepresenceofchronic inflammationinolderindividualspresentsignificantchallengesfor diseasetreatment.Despitetheavailabilityofvaccinesspecifically designedforthispopulation,theiroveralleffectivenessremains inadequate,particularlyininfectiousdiseasessuchasinfluenza. Researchersareexploringvarioustherapeuticapproachestargetingthepre-vaccinationagingcharacteristicssuchasinflammation, immunesenescence,andmitochondrialdysfunction.Notably, mRNAvaccineshavedemonstratedpromisingbenefitsduringthe COVID-19pandemic,includingenhancedefficacyamongelderly individuals.413

Duringearlychildhood,thethymuspredominantlyharborsa substantialpopulationofnaiveTcells,whichsubsequentlyundergo maturationprocesses.Duringthisperiod,whenencountering pathogens,memoryTcellsgraduallyaccumulateandovertake theoriginalTcells.404 Throughouttheprocessofmaturationfrom infancytoadulthood,agradualdeclineisobservedintheoverall percentageoflymphocytes,aswellasintheabsolutecountofT andBcells.Inaddition,thereisadecreaseinthenumberofNK cells.Interestingly,elderlyindividualsexhibitsigniﬁcantlyhigher levelsofNKcells,pro-inﬂammatorycytokines(INF-α,IL-6),and monocytechemoattractantprotein-1(MCP-1),whiledemonstrating lowerlevelsofepidermalgrowthfactor(EGF).414

TheclinicaltrialsofmRNAvaccinesaretypicallyconducted acrossdiverseagecohorts,includingpediatric,adult,andgeriatric populations(Table 4).Thismaybeattributedtovariationsintheir distinctimmunologicalproﬁles.Forinstance,mRNA-1273developedbyModernaTX,Inc.underwentclinicaltrialsindifferent phases.Initially,itwastestedinPhaseI(NCT04283461)andPhase IIIclinicaltrials(NCT04470427)forindividualsaged18yearsand older.Subsequently,itunderwentPhaseIIandIIItrialsfor adolescentsaged12to18years(NCT04649151),aswellas childrenaged6monthsto11years(NCT04796896).Encouragingly,olderadultsincludingparticipantsaged71yearsandolder exhibitedcomparableimmuneresponsestoyoungeradultsupon receivingtheseconddoseofthemRNA-1273vaccine (NCT04283461).415 BNT162b2,developedbyBioNTechSE,has encompassedagegroupsandhascompletedseveralclinicaltrials (NCT04368728,NCT04754594,NCT04816643).The ﬁndings revealedadecreaseinimmunogenicityofBNT162b1orBNT162b2 withadvancingage,resultinginacomparativelyattenuated overallhumoralresponseamongolderadults(aged65–85years) comparedtotheiryoungercounterparts(aged8to55years). Encouragingly,theadministrationoftheseconddoseofthese vaccinescouldenhanceantibodyresponsesinbothyoungand elderlyadults(NCT04368728).416

Epidemiologicaldataindicatesthatindividualsaged60years andaboveexhibitadisproportionatelyhigherpropensityfor severeclinicalsymptomsandincreasedratesofhospitalization followingSARS-CoV-2infectionduringtheCOVID-19pandemic. Whilecurrentvaccineshavedemonstratedfavorableefficacyin youngercohorts,theyfailtoelicitsustainedimmunityinthe elderlypopulation.Despitepreviousdiscussionsonpotential reasonsforthisphenomenon,furtherexplorationiswarrantedto uncoverdeeperunderlyingfactors.Theobjectiveofsuch investigationistoultimatelydevelophighlyeffectivevaccines withminimaladversereactionsspecificallytailoredforolder adults.Tofurtherenhancetheefficacyofthevaccine,inaddition toexploringnovelstrategiesagainstimmunosenescenceand inflammation,itisalsoimperativetodevelopandemploymore potentadjuvants.405 DuringtheCOVID-19pandemic,mRNA vaccineshaveaccumulatedvaluableinsightsintoage-related efficacyandadversereactionsofvaccination,thuspavingtheway fortheirextensiveapplicationinotherinfectiousdiseases,tumors, andbeyond.

Gender

Genderdimorphismsarepervasiveinhumandevelopmentand physiology.Thephenotypicdiscrepanciesofgenderderivefroma complexedmixtureofendogenousandexogenousfactors, particularlyhormonessuchasestrogenandandrogen.Inthe eraofprecisionmedicine,itbecomescrucialtoconsiderthe relationshipbetweengenderandimmuneresponsesfordisease preventionandtreatment.417 Theexistingliteraturesuggeststhat malepatientsdemonstrateahigherpropensityfordeveloping severeclinicalsymptomsofSARS-CoV-2comparedtotheirfemale counterparts.418 Thisphenomenonmaybeattributedtothe immunomodulatoryeffectsofestrogen,whereasandrogenand progesteroneexertimmunosuppressiveactions.404 However,the relationshipbetweengenderanddiseaseseverityisintricate, encompassingtheinﬂuenceofsexhormones,expressionof X-linkedgenes,andotherimmuneregulatoryfactors.Therefore,a comprehensiveunderstandingoftheCOVID-19infectionand symptomseveritycanbeachievedbyinvestigatinggenetic, hormonal,andspeciﬁcbehavioralfactorsassociatedwithgender andage.Moreover,suchresearchescancontributetothefuture developmentofmRNAvaccines.418

GenderplaysapivotalroleindeterminingtheriskofHIV infectionanditspathogenicprogression.Reproductivedisparities betweengenderscontributetoanaugmentedsusceptibilityof womentowardHIVinfection.However,duringtheearlystagesof

HIVinfection,womenexhibitheightenedactivationofCD8+ T cellsandincreasedexpressionofinterferon-stimulatedgenes. Nonetheless,post-HIVinfection,womenfaceanelevatedriskof non-acquiredimmunodeﬁciencysyndrome(AIDS)morbiditysuch ascerebrovascularevents.419

Comparedtofemalemice,malemicedemonstrateahigher susceptibilitytoinfluenzaBvirus(FLUBV),mirroringtheepidemiologicalfeaturesobservedinhumans.Cardenas-Garciaetal.420 conductedastudyencompassingvariousvaccinedesignsand foundthatfemalemiceexhibitedmorerobustlgGandlgA responsesandincreasedCD4+ Tcellscomparedtotheirmale counterparts.Furthermore,theyreportedthatboththequality andquantityofimmuneresponsewereinfluencedbyfactorssuch asvaccineplatform,gender,andinclusionofadjuvants,thereby impactingthevaccineefficacyagainstFLUBV.The findings suggestthat,ingeneral,malesdemonstratehighermorbidity andmortalityratesfromviralinfectiousdiseases.421 However,itis noteworthythatincertaininfectiousdiseasessuchasmeaslesand dengue,femalesactuallyexhibithighermorbidityandmortality rates.404

Forcardiovasculardisease,therearewell-establishedmodifiable riskfactorssuchassmoking,obesity,diabetes,hypertension,and hyperlipidemia.Inadditiontothesefactors,non-modifiablerisk factorssuchasgenetics,age,andgenderplayasignificantrolein cardiovasculardiseasedevelopment.Theriskofcardiovascular diseasesignificantlyincreasesformalesaged45yearsandabove, aswellasforfemaleswhoundergooophorectomyorexperience naturalmenopause.422

Itiswidelyacknowledgedthatsexhormonesplayapivotalrole inhormone-dependentorgandiseases,suchasbreastandovarian cancersinfemales,aswellasprostateandtesticularcancersin males.Theincidenceratesofvariousnon-reproductiveorgan cancersmayalsoexhibitasigniﬁcantassociationwithgender.423 Lietal.417 conductedananalysisutilizingdatafromtheUK Biobankandidentiﬁed119diseasesexhibitingagenderbias(P adjusted<0.05).Notably,stomach,kidneyandlungmalignancies demonstratedhigherincidenceratesinmen,whileasthma displayedahigherincidencerateinwomen.

Theimmunesystemisintricatelyinterconnectedwithcancer treatment,andvariationsinhormonalandXchromosomegene expressioncanleadtodiffe rentimmuneresponsesamong distinctpopulationsofinnateandadaptiveimmunecells. 424 For example,inpatientswithKRASmutationsinlungadenocarcinoma,femalesdemonstrateagreaterabundanceofCD4 + Tand CD8 + Tcellscomparedtomales. 425 Thestudyconductedby Thompsonetal. 426 revealedvariationsinthefunctionalityofthe transcriptionfactorFOXO3,whichisassociatedwithtumorin fi ltratingDCstolerance,betweengenders.Thesedisparities mayserveascrucialconsidera tionsforaugmentingcancer immunotherapy.Observationsfromclinicaltrialindicatethat immunecheckpointblockadeconfersgreaterbene fi tsinthe treatmentofmalecancerpatients. 427 However,certainstudies havereportednostatisticallysigni fi cantgender-baseddifferencesintheef fi cacyofICIs. 428 Additionally,clinicalinvestigationshavedemonstratedthatICIsampli fi edtherapeutic outcomesinfemalepatientswhencombinedwithCisplatin and/orradiotherapy. 429 Thesecon fl ictingexperimental fi ndings suggestthattheunderlyingmechanismin fl uencinggender disparitiesincancerincidenceratesandtreatmentoptionsare multifacetedandcomplex.

Insummary,womenusuallyexhibitstrongerimmune responsesagainstinfectiousdiseasesandmalignanttumors thanman.Inaddition,followingvaccination,womendemonstratesuperiorantibodyresponses,therebyreducingtheir susceptibilitytoinfectionsandloweringthemortalityrates. However,thebetterimmuneresponseofwomenalsopredisposesthemtoafourfoldincreasedriskofdevelopingautoimmunediseases. 430, 431 Forexample,theincidenceratiosof

Table4. Clinicaltrialswereconductedaccordingtodifferentagegroups

StatusSponsor

PhaseNCTnumberStudystarted (actual)

AgesSex/ gender

mRNADiseasemRNAencodedproteinEnrollment (actual)

AllPhaseIIINCT044704272020-07-27CompletedModernaTX,Inc.

mRNA-1273COVID-19SARS-CoV-2spikeprotein3041518yearsandolder(adult, olderadult)

AllPhaseII/IIINCT046491512020-12-09Active,not recruiting

433112yearsto18years(child, adult)

119506monthsto11years(child)AllPhaseII/IIINCT047968962021-03-15Completed

AllPhaseII/IIINCT043687282020-04-29CompletedBioNTechSE

4707912yearsandolder(child, adult,olderadult)

COVID-19SARS-CoV-2receptor- bindingdomain,SARS-CoV- 2spikeprotein

BNT162b1, BNT162b2

AllPhaseIIINCT049556262021-07-01Completed

BNT162b2SARS-CoV-2spikeprotein1638512yearsandolder(child, adult,olderadult)

FemalePhaseIIINCT047545942021-02-16Completed

68318yearsandolder(adult, olderadult,healthy pregnantwomen)

118376monthsto15years(child)AllPhaseII/IIINCT048166432021-03-24Completed 072daysto102days(child)AllPhaseINCT056303522025-01-06 (estimated) Withdrawn

AllPhaseII/IIINCT051274342021-11-17Active,not recruiting ModernaTX,Inc.

3655760yearsandolder(adult, olderadult)

mRNA-1345RSVRSVprefusionstabilizedF (preF)glycoprotein

AllPhaseIIINCT053309752022-04-01Active,not recruiting

380050yearsandolder(adult, olderadult)

AllPhaseIIINCT060672302023-10-06Recruiting

210(estimated)5monthsto24months (child)

18yearsandolder(adult, olderadult)

AllPhaseINCT057438812023-02-15Active,not recruiting 1150 (estimated)

340(estimated)2yearsto17years(child)AllPhaseIINCT060972992023-10-24Active,not recruiting

360(estimated)18yearsto40years(adult, pregnantwomenand infants)

AllPhaseIINCT061430462023-11-15Recruiting RSV respiratorysyncytialvirus

Note:Vaccinationsafetyandimmunogenicityarecloselyrelatedtoage,andclinicaltrialsatdifferentagesareneededtodetermineef ﬁ cacy

multiplesclerosis,scleroderma,andrheumatoidarthritisin femalescomparedtomalesrangefrom2:1to3:1.However, thegenderdistributionofsystemiclupuserythematosusis remarkablyskewedwitharatioof9:1,representingthemost pronouncedgender-baseddisparityamongautoimmunediseases.432 Theetiologyofthegenderdisparityinautoimmune diseaseincidenceismultifacetedandintricate.Forinstance,the potentialexacerbatingeffectofestrogenonsystemiclupus erythematosus,doesnotprecludeitspotentialimmuneprotectiveeffectonrheumatoidarthritis. 433 Itisnoteworthythat thegender-speci ﬁ cvariationsinintestinalmicrobiotamay underpincertaingender-relatedphysiologicalandpathological conditions,thusprovidingabiologicalbasisforsuch disparities. 434

Despitethehigherprevalenceofautoimmunediseasesin women,thereisnosigniﬁcantgenderdisparityintheincidenceof immune-relatedadverseevents(IrAEs)amongpatientsreceiving ICIsforskin,gastrointestinal,andlacrimalgland-relatedconditions.However,notablegenderdifferencesexistinspeciﬁc endocrine-relatedIrAEs.Forexample,thyroiddysfunctionismore commonlyseeninwomen,whereashypophysitisismore frequentlyobservedinmen.432

Previously,theroleofgenderinthedevelopmentand treatmentofdiseaseswasoverlooked.Forinstance,in1977,the U.S.FDArecommendedexcludingwomenofreproductiveage fromPhaseIandearlyPhaseIItrialsundertheguiseof safeguardingwomenandchildren.Nevertheless,itledtoalack ofresearchandinsufﬁcientawareness.432 Fortunately,in2015,the NationalInstitutesofHealth(NIH)issuedadirectiveurging researcherstoincorporategenderasabiologicalvariableinto theirresearchdesigns,datacollectionprotocols,andoutcome analysis.435

Currently,therearerelativelyfewmRNAvaccinesdesigned speciﬁcallyforgenderdifferences.However,thedisparitiesin immuneresponsesresultingfromdifferencesinX-linkedgenes andsex-basedhormonalvariationsdoindeedexist,leadingto variationsindiseasesusceptibilityandclinicalmanifestationsas wellasvaccineefﬁcacy.Thesedifferencesareincreasinglybeing recognized.436 Researchintotheinteractionsofageandgender mayaidinthedesignofpersonalizedmRNAvaccinetreatment strategiestailoredtoindividualpatient.422

Pathologicalconditionandvulnerablepopulation

Inadditiontoageandgender,thereareotherintricatefactors affecttheefficacyofvaccination.Levinetal.437 foundthat individualsagedover65years,males,andgroupswith immunosuppression-relatedconditionsexhibitedsignificantly lowerlevelsofhumoralimmunity6monthsafterreceivingthe seconddoseofBNT162b2.Fureretal.438 revealedthatpatients withautoimmuneinflammatoryrheumaticdisordersdisplayeda lowerhumoralresponsetothetwo-doseBNT162b2mRNAvaccine regimencomparedtoimmunocompetentindividuals.Although seropositivelevelswererestoredinimmunocompetentindividuals afterreceivingthethirdvaccinedose,only80.47%ofpatients experiencedrestorationoftheirhumoralresponse.Notably,all patientswhoreceivedanti-cytokinebiologics,Methotrexate monotherapy,AbataceptandJanuskinaseinhibitorshadtheir humoralimmunityrestored.However,onlyone-thirdofpatients whoreceivedRituximabwereabletoregainimmuneprotection. Theclinicaltrialsregardingvaccineadministrationinpatients receivingimmunosuppressantsarelistedinTable 5. Lestonetal. 439 classi fi edimmunosuppressionandcorrelated itwiththemortalityrateofCOVID-19.Their fi ndingsrevealed thatpatientswithsolidorgantra nsplantsandmalignanttumors exhibitedasigni fi cantlyhighermortalityriskfollowingCOVID19infectioncomparedtothatofimmunocompetentindividuals.Furthermore,incomparisontoimmunocompetent individuals,patientswithrheu matologicalconditionsandHIV

demonstratedaslightlyelevatedmortalityrisk.Thestudy establishedacloseassociationbetweenspeci fi csubgroupsof immunosuppressionandthemo rtalityriskassociatedwith COVID-19infection.Thiscategorizationmethodholdssubstantialreferencevaluefortargetedtreatmentstrategiesand focusedvaccinationprevention.Meerausetal. 440 discovered thatindividualswithcomorbidi tiesandvulnerablepopulations exhibitedreducedvaccineeffectivenessfollowingadministrationofAZD1222(ChAdOx1nCov-19)incomparisontothose withnormalimmunesystems,whileimmunosuppressed individualsdemonstratedthelowestvaccineeffectivenessafter theinitialdoseofAZD1222.Obeidetal. 441 conducteda comparativeanalysisonthepersistenceofhumoralresponses againstSARS-CoV-2anditsvariantsofconcern(VOCs)in immunocompromisedpatientswhoreceivedtwodosesof BNT162b2ormRNA-1273vaccines,aswellasinhealthy individuals.The fi ndingsrevealedthatapproximately50%of patientswithsolidtumorsandhematologicalmalignancies, about70%ofautoimmunediseasepatientsandsolidorgan transplantrecipients,and40%ofhealthyindividualshadlost protectivenAbs6monthsaftervaccination.Moreover,itwas observedthatthedurabilityof bindingIgGanti-spikeantibodiesagainstSARS-CoV-2wasfourtoninetimesgreaterthan thatofnAbs.Furthermore,var iationswerenotedintheimmune ef fi cacybetweendifferentmRNAvaccines,withBNT162b2 inducinglowermagnitudeandshorterdurationofnAbs comparedtomRNA-1273.Therefore,itisimperativetoselect appropriatemRNAvaccinesfordiversevulnerablepopulations inordertoensureeffectiveadministrationofvaccinations. InadditiontoCOVID-19virusinfection,similarscenariosseem toariseinotherinfectiousvirusesaswell.Astudyconductedby Mbondeetal.442 revealedthatimmunosuppressedindividuals withneuroinvasiveWestNilevirus(NWNV)exhibitedmoresevere clinicalmanifestationscomparedtoimmunocompetentpopulations,therebyindicatingaworseprognosisandhigherriskof adversereactions.Thesurvivalrateofsepsissurvivorsis significantlydiminishedfollowingsecondaryinfection,primarily duetotheprolongedstateofimmunosuppressionthatensues afteracuteinfection.AcomprehensivestudyconductedbyLiao etal.443 revealedacloseassociationbetweenIL-10productionby Siglec-F+ neutrophilsandthesuppressionofT-lymphocyte activityinthisphenomenon.Furthermore,their findingsindicated thatdepletionofneutrophilscouldenhanceT-lymphocyte proliferationandimproveT-lymphocyteactivity,therebyamelioratingthesurvivalrateamongimmunosuppressedmicesubjected tosecondaryinfection.However,furtherinvestigationisrequired toassessthesystemiceffectsassociatedwithneutrophils depletion.

PersistentimmunesuppressionisobservedinpeoplewithHIV, whichiscloselyassociatedwithlowCD4countsorabnormalCD4/ CD8ratios.ThepreventionandtreatmentofcancerinHIVinfectedindividualsfacechallengesduetodisparitiesinhealth status.444 EvidencessuggestedthatindividualsinfectedwithHIV mightundergoprematureaging.Theresearchconductedby Gianesinetal.445 furtherdemonstratedthatthisphenomenon extendedtoHIV-infectedchildren,resultinginaccelerated biologicalandimmunologicalsenescence,particularlyaffecting theCD8+ cellsubpopulation.Vergorietal.446 discoveredthatthe administrationofathirddoseoftheCOVID-19mRNAvaccineto individualswithHIVcouldelicitarobustimmuneresponse. However,themagnitudeoftheSARS-CoV-2-speciﬁcT-cell responseinHIV-positivesubjectswascomparativelylowerthan thatobservedintheHIV-negativecontrolgroup.Inaddition,they providedquantitativeanalysisregardingthepotentialaugmentationofimmunelevelsthroughextraboosterdosesforindividuals withHIV.

Pregnantwomenandinfantsarealsoconsideredvulnerable populations.AntibodiesderivedfromCOVID-19vaccinescanoffer

Table5. OngoingandcompletedclinicaltrialsofmRNAvaccinesforthetreatmentofimmunosuppressantpatients/patientswithautoimmunediseases

BackgrounddiseaseTrialpopulationmRNAvaccineImmunosuppressantPhaseNCTnumberSponsorLastupdate posted

OmicronXBB.1.5vaccineEverolimusPhaseIVNCT05924685UniversityMedical CenterGroningen 2024-03-04

KidneytransplantRecipientswhohadakidneytransplant atleast6monthsago

OfatumumabPhaseIVNCT04878211Novartis Pharmaceuticals 2024-02-08

OfatumumabtreatedparticipantsP ﬁ zerorModernamRNA COVID-19vaccine

Relapsingmultiplesclerosis (MS)

SiponimodPhaseIVNCT04792567Novartis Pharmaceuticals 2022-12-14

BasiliximabPhaseINCT00626483GaryArcherPh.D.2021-03-09

SecondaryprogressiveMSPatientswithsecondaryprogressiveMSCOVID-19modRNA vaccine

ObservationalNCT04818892Universityof Wisconsin,Madison 2022-12-07

2023-06-08

2024-03-26

Cytomegalovirus(CMV) pp65-LAMPmRNA-loaded dendriticcell(DC)vaccine

MalignantneoplasmsbrainGlioblastomamultiformepatients treatedwithTemozolomide

PatientswithIBDCOVID-19vaccineNon-systemicimmunosuppressive therapy(Mesalamine/Vedolizumab/ Vedolizumabcombinationtherapy withMethotrexateorAzathioprine); systemicimmunosuppression (Azathioprine/In ﬂ iximab/ Golimumab/Adalimumab/ Certolizumab/Ustekinumab/ Tofacitinib/Corticosteroid)

In ﬂ ammatoryboweldisease (IBD)

cDCvaccineBasiliximabPhaseIINCT02366728MustafaKhasraw, MBChB,MD,FRCP, FRACP

ﬁ

CMV-speci

Glioblastoma(GBM)GBMpatientswhounderwent resection,Temozolomide(TMZ)therapy, andradiationtherapywerealsopre- treatedwithtetanusandgiven Basiliximab

COVID-19vaccine/PhaseIINCT05000216NationalInstituteof AllergyandInfectious Diseases(NIAID)

Autoimmunedisease5autoimmunediseasesinadults [systemiclupuserythematosus(SLE)/ rheumatoidarthritis(RA)/MS/systemic sclerosis/pemphigus],4autoimmune diseasesinpediatricparticipants [systemiclupuserythematosus(SLE)/ juvenileidiopathicarthritis(JIA)/ pediatric-onsetmultiplesclerosis (POMS)/juveniledermatomyositis (JDM)]

PhaseIIINCT05415267KirbyInstitute2024-03-15

e.g.,Rituximab,Ocrelizumab, Ofatumumab

COVID-19vaccine, diphtheria/tetanus toxoidsvaccine

Hematologicalmalignancy + immunosuppressant/systematic autoimmunity

Immunosuppress/systematic autoimmunity

COVID-19vaccineTacrolimusPhaseIINCT05077254NIAID2024-03-29

SolidorgantransplantKidneytransplantrecipients,liver transplantrecipients

ObservationalNCT05060354BrighamandWomen ’s Hospital 2024-01-25

PhaseINCT00978913IngeMarieSvane2015-08-19

MSMSpatientsonimmunotherapyCOVID-19vaccineOfatumumab,Ocrelizumab, Fingolimod,Siponimod

DCvaccineCyclophosphamide(background drugs,asvaccineadjuvants, commonlyusedasanticancerdrugs)

Patientswithmetastaticbreastcancer ormalignantmelanoma

Breastcancer,malignant melanoma

/ObservationalNCT05020145P ﬁ zer2024-02-12

COVID-19vaccine (BNT162b2)

Patientswithimmunocompromising conditionsintheUnitedStates

Immunocompromised, immunosuppressed

DCvaccine/PhaseINCT03396575UniversityofFlorida2023-11-18

BrainstemgliomaBrainstemgliomapatientstreatedwith radiotherapyandTemozolomide

Note:Followallentriesfoundunder “ Searchfor:mRNAvaccine,immunosuppressant|CardResults|ClinicalTrials.gov ”

protectionagainstseverevirusinfectionsinnewbornsthroughthe transferofplacentalantibodies.Thereislimiteddataonthe antibodylevelsofinfants,particularlypreterminfants,following maternalvaccination.Therefore,Kachikisetal.447 conducteda studywhichrevealedthattherewasnodisparityinmaternally derivedSARS-CoV-2anti-spikeIgGlevelsbetweenfull-termand preterminfants.However,theselevelswerecloselyassociated withtheconcentrationofthematernalanti-spikeantibodies.In addition,itwasobservedthatreceivingtwoorfewerdosesofthe COVID-19vaccinemightnotprovideoptimalimmuneprotection forpregnantwomen,norforinfantsviacordblood.Consequently, carefulconsiderationshouldbegiventothetimingofvaccination forpregnantwomen.

Adversereaction

Johnstonetal.448 discoveredanadversereactionmanifestedasa delayedhypersensitivityattheinjectionsitesubsequentto administrationoftheModernaCOVID-19vaccine.CappellettiMontanoetal.449 discoveredthatthemajorityofsevereadverse reactionsreportedafterCOVID-19vaccinationwereassociated withcardiaccomplications,particularlyamongmaleadolescents. Inaddition,thrombosisanddyspneaemergedasprevalentserious symptomsfollowingCOVID-19vaccination.Incontrast,the adversereactionsaftervaccinationwithHPVandinﬂuenza seemedtobemorediverse.Dizziness,lossofconsciousness, dyspnea,andconvulsionsoccurredafterHPVvaccination,and Guillain-Barrésyndromeoccurredafterinﬂuenzavaccination. VaccinationwithCOVID-19vaccine(BNT162b2mRNAormRNA1273)canalsobeassociatedwiththedevelopmentofvarious autoimmunediseases,suchasautoimmunehepatitisornephritis, rheumatoidarthritis,andnew-onsetsystemiclupuserythematosus.Heil450 endeavoredtoinvestigatetheunderlyingfactors contributingtoadiverserangeoflife-threateningcomplicationsin patientsinfectedwithSARS-CoV-2.AlthoughtheCOVID-19 pandemicseemstobereceding,thelessonslearnedfromthe developmentofCOVID-19mRNAvaccinesareexpectedtohave implicationsfornon-COVID-19diseases.

ThestudyconductedbyPettinietal.451 demonstratedthat multipleadministrationofmRNA-1273vaccineeffectively enhancedthespecificmemoryB-cellandantibodyresponses againstSARS-CoV-2inallogeneichematopoieticcelltransplantationpatients.Furthermore,theyemphasizedthesignificanceof repeatedvaccination.Thephenomenonofextensiveandconsecutiveimmunizationhasbecomeapparentowingtorepetitive vaccinationsintendedtoaugmentvaccineefficacy,alongwith advancementsinvaccinesdesignedfordiversediseases.Nonetheless,thisintricacypresentsdifficultiesinevaluatingvaccine safetyaccurately.Interactionsbetweenvaccineshavethe potentialtoproducefavorableordetrimentaloutcomesalike. MawsonandCroft452 postulatedthatmultiplevaccinationsmight induceactivationoftheretinoidcascade,resultinginasequence ofunfavorablereactions.Theuncertaintiesoftherisksassociated withvaccinationnecessitatefurtherresearchtooptimizevaccine safety.

Thesafetyandimmunogenicitypro ﬁ lesfollowingvaccination wereassessedinclinicaltrialsthroughtheadministrationoftwo distinctvaccineformulationsinapredeterminedsequence.For example,thePhaseItrialsoftheinvestigationalvaccines BNT164a1andBNT164b1haver ecentlycommencedwith volunteerswhohavepreviouslyreceivedtheBCGvaccine (NCT05547464). 453 Severalclinicaltrialsarecurrentlyunderway toassessthesafetyandimmunogenicityofco-administering twovaccinessimultaneously(Table 6 ).Moreover,mRNA vaccinescanbeengineeredtoencodeseveralepitopesfrom multiplepathogenssimultaneously. 454 Currently,thereis ongoingdevelopmentofmulti- componentvaccinesaimedat achievingprogressivelymorecomprehensivediseaseprevention(Table 7 ).

SignalTransductionandTargetedTherapy(2024)9:322

Table6. Concurrentlyadministeredvaccines

VaccineDiseaseStudystarted(actual)PhaseStatusSponsorNCTnumber 9vHPV + mRNA-1273PapillomavirusinfectionandCOVID-192022-03-28PhaseIIICompletedMerckSharp&DohmeLLCNCT05119855 mRNA-1345 + in fl uenzavaccineRSVandin fl uenza2023-09-25PhaseIIIActive,notrecruitingModernaTX,Inc.NCT06060457 V110/V114 + mRNA-1273PneumococcalinfectionandCOVID-192022-01-12PhaseIIICompletedMerckSharp&DohmeLLCNCT05158140 ARCT-2303 + in fl uenzavaccineCOVID-19andin fl uenza2024-03(estimated)PhaseIIINotyetrecruitingArcturusTherapeutics,Inc.NCT06279871 BNT162b2(OmiXBB.1.5) + RIVCOVID-19andin fl uenza2024-01-31PhaseIIActive,notrecruitingP fi zerNCT06237049 HPV humanpapillomavirus, RSV respiratorysyncytialvirus

Table7. Multi-componentvaccines

VaccineDiseaseStudystarted(actual)PhaseStatusSponsorNCTnumber

mRNA-1045InfluenzaandRSV2022-10-14PhaseICompletedModernaTX,Inc.NCT05585632 mRNA-1230Influenza,RSVandCOVID-192022-10-14PhaseIICompletedModernaTX,Inc.NCT05585632 mRNA-1083InfluenzaandCOVID-192023-04-14PhaseI/IIActive,notrecruitingModernaTX,Inc.NCT05827926 mRNA-1073InfluenzaandCOVID-192022-05-13PhaseI/IICompletedModernaTX,Inc.NCT05375838 RSV respiratorysyncytialvirus

CONCLUSIONSANDPROSPECTS

ChallengesinmRNAdrugapplication

Despitesubstantialadvancementsinthe field,effectivelyand safelydeliveringmRNAtherapeuticstothetargetsiteremainsa significantchallenge.Thehalf-lifeofdirectintravenousadministrationofmRNAisveryshortduetotheirsusceptibilityto degradationbynucleases.Thenegativechargeandhigh molecularweightofmRNAhindercellulartranscytosisacrossthe biologicalmembrane.Additionalbarriersincludehighrenal clearance,non-specifictissuedistribution,andoff-targeteffects. Inaddition,mRNAthatentercellsfacethechallengeofescaping fromendosomes.455 Furthermore,exogenouslyintroducedmRNA canberecognizedbytheTLRsandcytosolicnucleicacid receptors,triggeringinnateimmuneresponsesandaccelerating thedeactivationofIVTmRNAwithinthebody.181

AlthoughtheapplicationofmRNAdeliverysystems,LNPsin particular,mitigatescertainoftheaboveobstacles,itisimportant toremaincautiousofthepotentialhazardsassociatedwith deliveryvehicles,suchasinﬂammation,immunogenicity,and cytotoxicity.105 Inadditiontothetoxicitycausedbythedelivery system,ithasbeendiscoveredthattheproductionmethods, routesofadministration,andevenproteinsgeneratedbythe complexedmRNAdrugspresenttoxicityconcerns.456

MainreasonsforfailedmRNAapplication Followingintravenousorintramuscularadministration,mRNA LNPsareextensivelydistributedintheliverandspleen,potentially resultinginpathologicalalterationswithintheseorgans,suchas thedisruptionofhepaticcellregulationoffattyacidmetabolism. Inaddition,mRNAnanomedicinesmayelicitadverseimmune reactions,suchassystemiccomplementactivation,hypersensitivityresponses,andcytokine-mediatedeffectspostvaccination.98,456 TheadverseimmunogenicitycausedbyLNPs, besidesthelipidcomponents,iscloselyassociatedwiththeirsize, charge,andaggregationcharacteristics.SmallerLNPsaremore advantageousinmitigatingtheactivationofundesiredimmune responses.Ontheotherhand,itisimportanttoinvestigatethe translationaccuracyofnucleotide-modifiedmRNAtherapeutics,as unintendedproteinsynthesismayresultinunforeseenoutcomes.457 Mulroneyetal.458 discoveredthattheuseofm1ΨmodifiedIVTmRNAledtoanotableincreaseinthe +1ribosomal frameshiftduringtranslation.Understandingthetranslation processofnucleotide-modifiedmRNAisessentialforfuturemRNA designandoptimizationtomitigatepotentialreductionsin efficacyandincreasesintoxicity.

ThesuccessapplicationofmRNAtherapiesisintricatelylinked tothecomplexityandpublicunderstandingoftargeteddisease. TaketheextensivelystudiedcancermRNAvaccineasanexample. Todate,thereisfewPhaseIIImRNAvaccineavailablefortumors, whichmaybeduetothefollowingreasons:(1)ThemRNA encodingasingletumorantigenmayinadvertentlyinduce mutationsintumorstoevadeimmuneresponse,necessitating theneedfordevelopingmRNAvaccinescapableofsimultaneouslytargetingmultipleTAAsorTSAs;(2)Thesurfaceantigens aredistinctacrossdifferenttumortypes,callingforpersonalized tumor-mRNAvaccinesforindividualpatient.However,the

productionandimplementationofpersonalizedvaccinesare oftenassociatedwithhighcostsandposechallengestotheir widespreadclinicalapplication;(3)Tumorsexertimmunosuppressiveeffectsbyimpedingtherecognitionofneoantigensand suppressingtheviabilityofantitumorimmunecells,emphasizing theimportanceofmeticulousselectionandapplicationof adjuvantsforpotentiatedimmuneresponses;(4)Theefficacyof mRNAvaccinesislargelylimitedbyitsinstability,inefficient delivery,andpoortransfectionefficiency.Inthesecases,iterative optimizationofthemRNAmoleculeanddeliveryvehicleis needed;(5)Tumorpatientsoftenexhibitcompromisedimmune responsesduetoamultitudeoffactors,includingadvancedage, concurrentmedication(e.g.,chemotherapy),andcomorbidity (e.g.,HIV).Hence,inadditiontobolsteringtheimmuneresponse withadjuvants,vigilanceagainstpotentialadversereactions shouldbetakenintoseriousconsideration;(6)Theformidable challengeoferadicatingadvancedtumorsusingmRNAvaccines alonenecessitatestheintegrationofmultipletherapiesfor synergistictherapeuticeffects,suchasadoptivecelltherapy.459

Effortsandimprovementsmadetoaddressthesechallenges ThecurrentlandscapeofmRNAvaccineswithpromisingclinical prospectsoftencomprisesthreecomponents:themRNA sequenceencodingtargetprotein,deliveryvector,andadjuvant.460 Incertaincases,themRNAcarrieritselffunctionsas vaccineadjuvant.460 Notably,althoughtheincreasedimmunogenicityofmRNAdrugsboostsimmuneresponse,ithampers mRNAtranslationandsuppressestargetproteinproduction.In thisconsideration,thetechnologyofimmune-silencingIVTmRNA byreplacingnaturallyoccurringnucleosidesintothemRNA sequencehasbeendeveloped.461 EmployingmodifiedmRNA representsastrategicapproachtobothenhanceprotein expressionandmitigateaberrantimmunogenicity.462 Inaddition, byoptimizingthemRNAsequence,suchasintroducingmultiple adeninesatthePoly(A)tail,mRNAstabilitycanbefurther enhancedwiththeproteintranslationefficiencyimproved. Moreover,purificationmethodsincludingcelluloseandfast proteinliquidchromatographycanbeemployedtoeliminate theimmunogenicdouble-strandedRNAgeneratedduringmRNA manufacturing,therebyreducingtheinflammatoryresponses unfavorabletomRNAstability.463

Inthecontextofimmunotherapy,toavoidtheinsufficient activationofAPCs,especiallyDCs,withnucleoside-modified immune-silencedmRNAfollowingvaccination,115 adjuvantscan beincorporatedintothedeliverysystem.Anidealadjuvantcan lowerthedoseofantigen-encodingmRNApervaccinedose, reducethefrequencyofvaccination,enhancetherobustnessand specificityofimmuneresponses,andincertaincases,assumethe roleofacarrierfortheefficientdeliveryofmRNAandcontribute tothestabilizationofthevaccineformulation.464,465 Guetal.466 usedTLR2/6agonistPam2Cys(asimplesyntheticmetabolizable lipoaminoacid)asanadjuvanttofacilitatemRNAencodingCT26 neoantigenpeptideorSARS-CoV-2spikeproteinantigenfor inductionofeffectiveimmuneresponsesagainstcanceror infectiousdiseases.Ontheotherhand,theadjuvantcanbe incorporateddirectlyintothemRNAsequence.Lietal.395

developedamultiplyadjuvantedmRNAvaccinebyfusing antigen-encodingmRNAwithC3d(theterminaldegradation productofmammaliancomplementcomponentC3)-basedmRNA adjuvant,whichevokedaprotectiveimmuneresponseagainst SARS-CoV-2atadosagetentimeslower.Ofnote,anincreasing numberofstudieshaverevealedthatadjuvantnotonlyenhances themagnitudeofimmuneresponsetovaccines,butguidethe typeofadaptiveimmuneresponse.467 Zhuetal.468 fabricated mRNALNPsandinvestigatedthedifferentialimmuneresponses elicitedbymRNALNPssupplementedwiththenaturalSTING agonist2'3’-cyclicGMP-AMP(cGAMP)and/orthemucosal adjuvantalpha-galactosylceramide(αGC).Theresultsindicated thatmRNALNPscontainingcGAMPaloneinducedastronger cellularimmuneresponsethanmRNALNPscontainingboth cGAMPand αGC,whichwasconsistentwithhigherlevelsofIgG andIgG2aantibodyresponses,aswellasbetterprotectionagainst homologousviralchallenge.Thestudyalsodemonstratedthat mRNALNPscontainingcGAMPcouldaccumulateCD4+ Tcellsin thelung,whilemRNALNPscontaining αGCcouldenlargethe iNKTcellsinthespleen.ActivatediNKTcellsmayexhibitpotent cytotoxicity,indicatingtheparamountimportanceofthejudicious selectionandapplicationofadjuvants.Atthecellularlevel,itis well-establishedthatActinidiaerianthapolysaccharide(AEPS) elicitsamixedCD4+ Th1andCD4+ Th2immuneresponse,while AluminducesastrictTh2response.Nevertheless,manyintricate molecularmechanismsremainunclear.Duetal.469 conducteda comparativeanalysisoftheinnateimmuneresponseselicitedby thetwoadjuvants,andfoundthatAEPSinducedhighermRNA expressionlevelsofC-X-Cmotifchemokineligand(CCL)2,CCL3, CCL4,CXCL10,IL-12β,IL-23α inimmuneeffectorprocess,and rapidlyrecruitedneutrophilsandmonocytes.Incomparison,Alum inducedIL-7A,IL-17F,andIL-17RA,andrecruitedneutrophilsand eosinophils.

ThedeliveryandstoragechallengesofmRNAcanberesolved throughoptimizationofthedeliveryvehicle.115 Todate,aplethora ofdeliverysystemsformRNAhavebeendeveloped.Amongthem, LNPshasprogressedinclinicalpracticemostrapidly.LNPsnot onlypossessesthecapacitytoencapsulateandtransportmRNA, butalsoshieldsthemRNAfromnucleasedegradation.However, theuseofLNPsstillpresentscertainissues,suchasthepotential damagetocellmembranesandorganellesduetothecationic characteristicsoftheionizablelipid.Thedevelopmentof biodegradableionizablelipidsappearstooffersomesolutions tothisproblem.102 105 Itiscrucialtorecognizethatnoionizable lipidisuniversallyoptimalforallnucleicacidformats,and variationsinnucleicacidmodification,size,andstructurecan impacttheeffectivenessofLNPsdelivery.470 Ontheotherhand, theoff-targetaccumulationofmRNAdrugsmayleadto suboptimaloutcomes.Inthiscase,variousstimulus-responsive nanotechnologieshavebeendevelopedtoutilizespecificinternal stimuluswithinthecellularmicroenvironment,includingthe expressionofspecificenzymes,increasedlevelsofROS,hypoxic environments,andchangesinthepHortemperature,aswellas externalstimulussuchaslightirradiation,magnetic fields,and ultrasound.Thesestimuli-responsivenanoplatformshavethe potentialtoenhancetissuetargeting,promoteexosomeescape ofmRNA,improvethetherapeuticeffectsofmRNAtherapy,and reducetoxicityandsideeffects.471 TheactivetargetingofmRNA drugstodesiredbiologicalsitesthroughLNPssurfacemodificationisalsowidelyused.Severalpreclinicalstudieshavealready demonstratedthesuccessfuldeliveryofmRNALNPstotarget tissuesandcells.472–474 Forinstance,Kimetal.475 utilizedLNPs modifiedwithtumor-targetingpeptidestoencapsulatemRNAencodingPETN.ThisPETNmRNALNPsspecificallyinducesICDin mice4T1breasttumor,therebyelicitingeffectiveantitumor immuneresponses.Tangetal.476 engineeredLNPswithsialicacid forDCstargetingandefficientendosomalescape.Furthermore,it isanticipatedthatthenext-generationLNPswiththeabilityto

targetdrugsspeciﬁcallytodistinctintracellularorganelles,suchas mitochondriaandthenucleus,willserveasaninnovative paradigm.472 Inadditiontotheoptimizationstrategiesmentioned above,thedeliverysystemformRNAcanbeoptimizedtoenhance theprecisionofmRNA-basedtherapiesfordiseasesbyadjusting factorssuchasdrugdissolution,diffusionrate,pharmacokinetics, toxicity,andhalf-life.477

PotentialofmRNAdrugstotreatvariousdiseases

ThemRNAvaccinehasemergedasaforefrontlineindisease preventionandtreatment.276 TherapiddevelopmentandregulatoryapprovalofmRNAvaccinesagainstSARS-CoV-2duringthe COVID-19pandemichavedemonstratedtheadvantagesofmRNA technology,478 exertingaprofoundinfluenceonitsapplicationin infectiousdiseasesandcancerimmunotherapy.ThePhaseIII clinicaltrialofSARS-CoV-2mRNAvaccineiscurrentlythemost clinicallyavailable.Additionally,aseriesofmRNAvaccinesagainst otherinfectiousdiseasesareunderdevelopment.Forexample,the PhaseIstudy(NCT04528719)ofmRNA-1345encodingthe prefusionstabilizedF(preF)glycoproteinofRSVdemonstrates favorabletolerabilityprofilesinbothyoungandelderlyindividuals, providingthefoundationforfurtherinvestigationswherehigh-risk adultsandsolidorgantransplantpatientsareenrolled(PhaseIII, NCT06067230).479 Meanwhile,comparedtomRNA-1345,the developmentofmRNA-1010forseasonalinfluenzaisprogressing atafasterpace,whichhassuccessfullycompletedaPhaseIIItrial with22,510adultsagedover50years(NCT05566639).mRNAbasedimmunotherapieshavealsobeenextensivelyresearchedfor antibacterialandanti-parasiteapplications.Ofnote,aggressive fungalinfectionsactuallyresultinahigherannualmortalitythan malaria,yetthereareveryfewmRNA-basedtreatmentsavailable forfungalinfections.Weaimtoincreaseawarenessofthepotential ofmRNA-basedtherapiesfortreatingfungaldiseases.480

Inthecontextofantitumorimmunotherapy,increasingmRNA vaccineshasenteredtheclinic.Forexample,mRNA-4157encodes upto34patient-specifictumorneoantigensisusedincombinationwithPembrolizumabforthetreatmentofmelanoma,which hasenteredPhaseIIbclinicaltrial(NCT03897881).481 Presently,the primaryfocusofresearchliesintheidentificationanddesignof mRNAsequencesthattargetspecificcancerantigensforthe purposeofachievingpersonalizedcancermRNAvaccinetherapy. Furthermore,whilethemajorityofexistingcancermRNAvaccines aredesignedfortherapeuticpurposesratherthanpreventive measures,thereisalsosignificantpotentialforprophylacticcancer mRNAvaccines.181 Giventhedesign flexibilityandadaptabilityof mRNA,thedevelopmentofpersonalizedcancermRNAvaccinesto optimizeantitumoreffectsbasedondiversetumortypesand individualpatientvariationsrepresentsacrucialavenueforfuture advancement.482

ItshouldbementionedthatmRNA-basedimmunotherapynot onlyhasthepotentialtoenhanceimmuneresponsesagainsta widerangeofinfectiousdiseasesandcancers,butalsooffersthe possibilityofmodulatingtheimmuneresponseforthetreatment ofautoimmuneconditions.mRNAtherapeuticsdesignedfor autoimmunediseasescanbetailoredtoencodedisease-speciﬁc antigensorimmunosuppressivecytokines.Forinstance,the mRNA-6231encodingHSA-IL2m,intendedforthetreatmentof autoimmunedisorders,hasenteredPhaseIclinicaltrial (NCT04916431).221

Incontrasttoimmunotherapywheretheincorporationof adjuvantsisimperative,itisofvitalimportancefornonimmunotherapytomitigatetheimmunogenicityofmRNAdrugs andtoamplifytheexpressionofencodedtargetproteins.483 mRNA-basedtherapeuticsfunctiontoreplenishdeﬁcientproteins orsubstituteabnormalproteinsthatexhibitdiminishedfunctionality/activity.Forexample,mRNAtherapiesforOTCD(ARCT-810, PhaseII,NCT05526066;MRT5201,PhaseI/II,NCT03767270),PA (mRNA-3927,PhaseI/II,NCT04159103),MMA(mRNA-3705,Phase

I/II,NCT05295433),andmyocardialinfarction(AZD8601,PhaseIIa, NCT03370887)haveenteredclinicaltrials(Table 2).

Inthisreview,wesummarizethepreclinicalandclinical progressesofmRNAdrugsinbothimmunotherapyandnonimmunotherapy,andhighlighttheimportanceoffocusingon host-specificvariationsformaximizedefficacyandsafety. Althoughintheearlystagesofdevelopment,thetherapeutic useofmRNAhasdemonstratedgreatpotentialincombating multiplediseases.484 ThemRNAdrugsdescribedinthisreview primarilyencompasstraditionalIVTmRNA.However,asmRNA technologyconstantlyinnovatingandadvancing,otherformsof mRNAdrugs,suchassaRNA,trans-amplifyingmRNA(taRNA),and circRNA,holdimmenseapplicability.Itisworthnotingthat successfulclinicalimplementationofthesenext-generationmRNA drugsrequirestheinvolvementofsuitabledeliveryvehiclesand furthermodificationstothemRNAmoleculeitselfforbetter tolerabilityandefficacy.485

ACKNOWLEDGEMENTS

ThisworkwassupportedbytheNationalKeyR&DProgramofChina (2023YFC340200),theNationalNaturalScienceFoundationofChina(No.82404520, 82273862),theChinaPostdoctoralScienceFoundation(2024M752834),andthe ChinaNationalPostdoctoralProgramforInnovativeTalents(BX20230321).

AUTHORCONTRIBUTIONS

Conceptualization:YingyingShi.Literaturecollection:YingyingShiandMeixingShi. Resources:YiWang.Writing originaldraft:YingyingShiandMeixingShi.Writing reviewandediting:YingyingShiandMeixingShi.Visualization:YingyingShiand MeixingShi.Supervision:YiWangandJianYou.Fundingacquisition:JianYouand YingyingShi.Allauthorshavereadandapprovedthearticle.

ADDITIONALINFORMATION

Competinginterests: Theauthorsdeclarenocompetinginterests.

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