viralvector韓 曉MethodsofgenedeliveryViralVectors:AdenovirusRetrovirusLentivirusAdeno-associatedvirus(AAV)Herpessimplexvirus(HSV)Non-viralvectorbasedNakedDNA(plasmidDNA):injectionorgenegunLiposomes(cationiclipids):mixwithgenesEx-vivoInvivoWhyuseviralvectorsVirusareobligateintracellularparasitesVeryefficientattransferringviralDNAintohostcellsSpecifictargetcells:dependingontheviralattachmentproteins(capsidorglycoproteins)Genereplacement:non-essentialgenesofvirusaredeletedandexogenousgenesareinsertedGenerationofviralvector

forgenetherapyReplication-competentvirusReplication-defectivevirusAmplicon:doesn’tencodestructuralproteinsCan’treplicatebeyondthefirstcycleofinfectionElementsneededtogenerateampliconTransferVector:plasmid(promoter,geneofinterest,ori,packagingsignal)Packagingvector(cosmidorcelllines):providetheviralstructuralproteinsforpackagingoftransfervectorHelpervirus(packagingoftransfervector):deletedPackagingsignalsequence

Adeno-associatedvirusvectorsNon-pathogenichumanparvovirus,non-envelopedssDNAvirus,4.6kilobasesDependentonahelpervirus(adenovirusorherpesvirus)forreplication(dependovirus)AAV-2mostlyusedforvector

Adeno-AssociatedVirus(AAV)SingleStrandedDNAVirusViralITRs&Rep/Cap~5kbCapacityIntegrating/ConcatamericLongTermExpressionComplexProduction&PurificationMultipDNA&/orHelperVirusLowerTitreThanAdenovirusSerotypeDifferencesInReceptorsAAV2HeparanSulphateAAV5SialicAcidGenerationofadeno-associatedvirusvectorCharacteristicsofAAVvectorAdvantagesIntegrationandpersistentexpressionNoinsertionalmutagenesisInfectingdividingandnondividingcellsSafeDisadvantagesSizelimitation,4.9kbLowtiterofvirus,lowlevelofgeneexpressionAdenoviralvectorsNon-envelopeddsDNA,36kilobasesEarlyproteins(E1A,E1B,E2,E3andE4),lateproteins(L1-L5)CausesabenignrespiratoryinfectionsinhumanSerotypes2and5arecommonlyusedasvectorsEarlygenerationsofadenoviralvector(replicationdefective)

GutlessAdenoviralvector(Amplicon)ModificationofthetropismofadenovirusvectorAdenovirusfiberbindstoCAR(coxsakieandadenovirusreceptor,CAR),receptorwhichisubiquitousModifythefiberproteinCharacteristicsofadenoviralvectorAdvantagesHightitersBothdividingandnon-dividingcellsWidetissuetropismEasilymodifytissuetropismDisadvantagesTransientexpression(notgoodforgeneticdiseases)HighlyimmunogenicHightitersofviruscanbetoxicMoresuitableforcancerimmunotherapyRetroviralvectorMoloneymurineleukemiavirus(MuLV)GenerationofreplicationdefectiveretroviralvectorTransferplasmidvector:GeneofinterestLongterminalrepeats(LTR):promoter,polyA,integration,replicationandreversetranscriptionPrimerbindingsite(PBS)(originofreplication)RNApackagingsignalPolypurinetract(importantforreplication)PackagingvectorCelllinestablytransfectedwithplasmidconstructscontainingGag/polandEnvGenerationofretroviralvectorPseudotypedretroviralvectorCharacteristicsofretroviralvectorAdvantagesIntegration:permanentexpressionPseudotypedvirusDisadvantagesOnlyinfectingdividingcellsInsertionalmutagenesis(tumorformation)ActivateoncogenesInhibittumorsuppressorgenesLentiviralvectorsInfectionofnon-dividingcells(hepatocytes