Fig1SchematicillustrationofthebiogenesisandfunctionofmiRNAFuetalExRNA 2019 124 Page2of14 AsmountingreportsdocumentthatmiRNAsworkasextensiveregulatorsofvarioustypesofphysiologicalactivi ID: 945648
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miRNAcanalsobindtothe5-UTRoftargetgenes[13].ThebindingofmiRISCtoanmRNAcanleadtoei-thertherepressionorpromotionoftranslation,althoughthelatterisquiterare[14].TissueandtemporalspecificityTheexpressionofmostmiRNAsoccursinatissue-andtemporal-specificmanner[3].Recently,miRNAexpres-sionprofilingstudiesofmultipletumortypeshaverevealedthataberrantlyexpressedmiRNAsarebeneficialfortheclassification,diagnosis,staging,andprognosisofdisease[15].Theanalysisofsequencingdatafrom27differentorgans/tissuesofArabidopsisalsoprovedthatmostmiRNAsarewidelyexpressed,whereasafractionofmiRNAsexhibittissue-specificexpressionpatterns[16].CirculatingmiRNARecentresearchhasreportedthatalargenumberofstablemiRNAsderivedfromvariousorgans/tissueexistinbodyfluids,andthesemiRNAsarepromisingasnovelbiomarkersforthediagnosisofcancerando
therimmune-relateddiseasesviaexpressionprofil-ing.miRNA-21wasthefirstmiRNAdiscoveredinserum[17].Inadditiontoworkinginsidecells,miR-NAsalsocommunicateremotelyintheformofcirculat-ingmiRNAs[18].EmergingevidencehasindicatedthatcirculatingmiRNAsarelocalizedinmicrovesiclesorbindtootherplasmacomponentssuchashigh-densitylipopro-tein(HDL)particlesandRNA-bindingproteins[19,20].ThesecirculatingmiRNAscanenterrecipientcellsanddecreasetheproteinlevelsoftargetgenes[21].Cross-kingdomregulationEmergingevidencehasuncoveredtheabilityofsmallnon-codingRNAstotransformfromonespeciestootherspecies.ProfessorZhangandhisteamrevealedanimport-antfunctionofmiRNA:cross-kingdomregulation[22].TheirresultsrevealedthatexogenousplantmiRNAscouldbedetectedinbothtissuesandserafromdifferentanimalsaftertheoralintakeofplants.Subsequently,miR
NA-2911,anatypicalhoneysuckle-encodedmiRNA,wasfoundtodirectlytargetvariousinfluenzaAviruses,inhibitingvirusreplicationandultimatelyrescuingweightlossinviralinfectedmice[23].In2017,ProfessorZhangandco-workersagainreportedthatplantmiRNAsenrichedinlarvalbeebreadregulatedthedevelopmentofhoneybeecaste[24].Interestingly,arecentreportbySaimaetal.suggestedthepotentialcross-kingdomregulationofplant-derivedmiRNAsandindicatedthatmiRNAsfromtheparasiticplantCuscutacampestriscouldtargetthemRNAsofhostArabidopsisthaliana,leadingtomRNAcleavage,whichultimatelyinhibitedmRNAaccumulation[25].Thesenewmodesofcross-speciesregulationmaybeinvolvedinsymbioticandpathogenicrelationshipsamongdifferentkindsoforganisms[26,27]. Fig.1SchematicillustrationofthebiogenesisandfunctionofmiRNAFuetal.ExRNA (2019) 1:24 Page2of14 As
mountingreportsdocumentthatmiRNAsworkasextensiveregulatorsofvarioustypesofphysiologicalactiv-ity(e.g.,hematopoiesis,tumorigenesis,tumormetastasis,fatmetabolismandintestinalmucosalhomeostasis),inter-estindevelopingmiRNA-basedmedicinehasdramaticallyincreased[28 31].However,thehalf-lifeofmiRNAsisshortbecauseofthepresenceofnucleases[32].Moreover,duetotheirpolarity,miRNAshavedifficultypassingthroughthephospholipidbilayercellmembrane;thus,miRNAcannotrapidlypenetratethevascularendothe-liumandisretainedinbloodstorageorgans,includingtheliverandspleen,anditisultimatelyexcretedbythekidneys.Tosolvethisproblem,alargenumberofvectorshavebeendevelopedtodelivermiRNAs.Here,weprovidenovelinsightintothepromiseofmiRNA-basedtherapeuticapproachesandthedevelopmentofviralandnonviralvectors,includingtherapeuticapplicationsfor
modifiedmiRNAsandthechallengesofvectorconstruction.TherapeuticapproachesinvolvingmiRNAUsually,nakedRNAishighlysusceptibletodegradationbyabundantribonucleasesinthebloodandtophagocytosisbythereticuloendothelialsystem(RES).Chemicalmodifi-cationscanincreasethestabilityofoligonucleotidesforinvivodelivery.Antisenseoligonucleotide(ASO)technologywasintroducedforfunctionallystudyingmiRNA,andtheASOsthatareusedtosilencemiRNAarecalledanti-miRNAoligonucleotides(AMOs)[33].Chemicalmodificationsincludephosphorothioate-containingoligonucleotides,methylphosphonate-containingoligonu-cleotides,boranophosphate-containingoligonucleotides,2-O-methyl-(2-O-Me)or2-O-methoxyethyloligonucleo-tides(2-O-MOE),2-fluorooligonucleotides(2-F),lockednucleicacid(LNA)oligonucleotides,peptidenucleicacids(PNAs),phos
phorodiamidatemorpholinooligomers(PMOs)andotherchemicalmodifications,suchasCy3-,cholesterol-,biotin-andamino-modifiedoligonucleotides(Fig.2).Phosphorothioate-,methylphosphonate-,orboranophosphate-containingoligonucleotidessubstituteasul-fur,methyl,orboranogroup,respectively,forthe-oxygenofthephosphate,inanattempttoovercomethestabilityissue[34].Theintroductionofa2-O-methylor2-O-methoxyethylgrouptotheribosemoietyofaphosphorothioateoligoribo-nucleotidedramaticallyenhancesbindingstabilityandprotectsoligonucleotidesfromnucleasedegradation.2-fluoro-oligoribonucleotidescontainafluorinemoleculeboundtothe2-oxygenoftheribose[35].LNAsareRNAanalogsthatintroducea2,4methy-lenebridgeintheribosetoformabicyclicnucleotide[36].PNAisanartificiallysynthesizedpolymersimilartoDNAorRNAthatiscompo
sedofrepeatingN-(2-aminoethyl)-glycineunitslinkedbypeptidebonds[37].PMOscontainmorpholineringsthatarelinkedthroughphosphorodiamidategroups[38].Terminalchemicalmodifications,includingCy3-,cholesterol-,biotin-andamino-modifiedoligonucleo-tides,canincreasethestabilityandtracerfunctionof Fig.2Chemicalmodificationsimprovestability,biodistribution,cellularuptakeanddeliveryefficiencyandincreasethetracerfunctionofoligonucleotides.(0)UnmodifiedRNA;(1)phosphorothioate-,(2)methylphosphonate-,or(3)boranophosphate-containingoligonucleotidescontainingasulfur,methyl,orboranogroup,respectively;(4)2-O-methyl,(5)2-O-methoxyethyl,(6)or2-fluorointroducedintothe2oxygenoftheribose;(7)LNAs;(8)PNAs;(9)PMOs;andterminalchemicalmodifications,including(10)Cy3-,(11)cholesterol-,(12)biotin-and(13)amino-modifiedoli
gonucleotidescouldincreasethestabilityandtracerfunctionofoligonucleotidesforinvivodeliveryFuetal.ExRNA (2019) 1:24 Page3of14 oligonucleotidesforinvivodelivery[39,40].Inpracticalapplications,multiplemodificationsareusedtogethertoincreasethestability,deliveryandcellularuptakeeffi-ciencyofoligonucleotidesinvivo.Tochangetheexpressionlevelsoftargetgenes,miRNA-basedtherapiesincludethefollowingtwotypes:(a)miRNAsuppressiontherapywhenthetargetgeneisdownregulatedand(b)miRNAreplacementtherapywhenthetargetgeneisdownregulated(Fig.3).miRNAsuppressiontherapymiRNAsuppressiontherapycanremovemiRNAsup-pressionofatargetmRNA,thusincreasingthemRNAexpressionlevel.AMOsbindtothemiRNAsensestrand,blockinteractionsbetweenmiRISCanditstargetmRNA,preventthedegradationofthemRNA,andthusallowthemRNAtobetranslated.Toimprovet
heinhib-itionefficiency,multiplechemicalmodificationsareap-pliedtoenhancetheaffinityandstabilityofAMOs,includingmiRNAinhibitorsandmiRNAantagomirs.miRNAinhibitors(alsodenotedasanti-miRNAs)aresingle-strandedRNAmolecules.Theseanti-miRNAscanspecificallybindtoendogenousmiRNAandabolishitsactivity.miRNAinhibitorsaremainlyusedinvitroincombinationwithLipofectaminetransfectionreagenttoinvestigatethebiologicalfunctionofmiRNAvialos-s-of-functionexperiments.Antagomirsaresingle-strandedRNAmoleculeswithspecificchemicalmodifications.2-Phosphorothioatesareintroducedatthe5endandthecholesterolgroup,and4-phosphorothioatesareintroducedatthe3end.More-over,2-methoxygroupsareintroducedintothefulllengtholigonucleotides[41].Thesechemicalmodifica-tionsenhancethestabilityandcellularuptakeefficiencyofantagomirs[42]
.Therefore,thesemiRNAantagomirscanbeusedinvivoviaeitherlocalorsystemicadminis-trationtodownregulatethecorrespondingendogenousmiRNAlevels.miRNAmasksare22-ntsingle-strandedoligoribonu-cleotideswith2-O-methyl-modifications[43].UnlikeAMOs,amiRNAmaskdoesnotdirectlybindtotargetmiRNA.Instead,themiRNAmaskcaninteractwithmiRNAbindingsiteslocalizedinthe3-UTRoftargetgenemRNAthroughafullycomplementarymechanism.ThemiRNAmaskapproachisanimportantsupplementtoAMOs,whichareusefulforinvestigatingthetotalbiologicalfunctionofaspecificmiRNA;however,miRNAmasksaremoresuitableforstudyingtheinflu-enceofmiRNAonspecificpathwayscontainingatargetgene.miRNAspongesareusuallyplasmid-encodingcopiesthatcontainbindingsitescomplementarytotheseedre-gionofthetargetmiRNA[44].Aftertransfectionintocells,theseplasmidscantranscribehighl
evelsofspongeRNAsthatbindtotheseedregion,whichallowsthemtoblockafamilyofmiRNAscontainingthesameseedsequence.Ascompetitiveinhibitors,miRNAspongesexhibitsimilarinhibitionefficiencywithshortnucleotidefragments.miRNAreplacementtherapymiRNAmimicsaresyntheticdouble-strandedmiRNA-likeRNAmoleculesthatcansimulateendogenousmiRNAsandbindtotargetgenemRNA,whichultimatelyleadstoposttranscriptionalrepression.miRNAagomirsareartificialdouble-strandedmiRNAmimicswithmorechemicalmodifications.Theantisensestrandofanagomirhasthesamemodificationasthatoftheantagomir.ComparedwithmiRNAmimics,thesechemicalmodificationsenhancethestabilityandactivityofmiRNAagomirs.Therefore,agomirscanalsobeusedtoupregulatetheircorrespondingmiRNAsinspecialtissuesandtoinvestigatethebiologicalfunctionofmiRNAinvivo.miRNAprecursors(alsoknownaspre-miR
NA)arechemicallymodifiedsingle-strandedRNAfragmentsthataresynthesizedtosimulatematuremiRNAs.ThesemiRNAprecursorsaretransfectedintocellsviaacom-mercialreagentorelectroporationsimilartosiRNAs.Afterenteringcells,miRNAprecursorsarecleavedbytheDicerenzymeandtransformedintomaturemiRNAs.Therefore,pre-miRNAscanbeusedtoinvestigatethebiologicalfunctionofmiRNAviagain-of-functionexperiments.miRNA-expressingplasmidscanalsoinducetheupregulationofmiRNAastheycarryafluorescentreporterthatcanhelpinvestigatorsverifytheexpressionandlocalizationofmiRNA.Forexample,TakaraBioconstructedthepmR-ZsGreen1andpmR-mCherryvectors,whichlinkaselectivemiRNAexpressioncas-settewithabrightgreenorredfluorescentreportergene,respectively.Althoughmanychemicalmodificationsincreasethesta-bilityofmiRNAs,thiseffectmaynotbesufficientforinvivoap
plications.AnefficientdeliverysystemisgenerallyacceptedtobeessentialfordevelopingmiRNA-basedther-apeutics.Inthisreview,wedividevectorsintotwotypes:viralvectors(1)andnonviralcarriers.Nonviralcarriersaredividedintosixcategories:(2)inorganicmaterial-basedde-liverysystems,(3)lipid-basednanocarriers,(4)polymericvectors/dendrimer-basedvectors,(5)cell-derivedmem-branevesiclesand(6)3Dscaffold-baseddeliverysystems(Fig.4).ViralvectorsformiRNAandanti-miRNAoligonucleotidedeliveryViralvectorscanefficientlytransfergenesintotargetcells.VariousviralvectorshavebeenconstructedtoFuetal.ExRNA (2019) 1:24 Page4of14 mediateRNAinterference(RNAi)becausetheycantransfergenesintodifferenttissues/organsandcauselong-termgeneexpression.Asviralvectorspossessdis-tinctcharacteristics,somevectorsaremoresuitableforcertainpur
posesthanothers.Here,weintroducefourwidelyusedviralvectorsformiRNAdeliveryincludingadenovirusvectors,adeno-associatedvirusvectors,retroviralvectors,andlentivirusvectors.AdenoviralvectorsAdenoviruses(Ad),whicharederivedfromtheAdeno-viridaefamily,arenonenvelopedvirusesthatcontainlin-eardouble-strandedDNAgenomesof~36kbinlengthwithtwoinvertedterminalrepeats(ITRs)atitstermini[45].Toupregulatetransgeneefficiencyandreduceim-munogenicityinvivo,alloftheviralprotein-coding Fig.3miRNA.(a)EndogenousmiRNAwithnormalfunction;(b)miRNAinhibitiontherapyusingmiRNAinhibitors,miRNAantagomirs,miRNAmasksandmiRNAsponges;(c)miRNAreplacementtherapyusingmiRNAmimics,miRNAagomirs,miRNAprecursorsandmiRNA-expressingplasmids.ThedottedlinesrepresentthemodifiedstructureofmiRNAantagomirsandmiRNAagomirsFuetal.ExRNA (2019) 1:2