分子病毒学 8病毒载体.ppt

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1、viralvector韩晓MethodsofgenedeliveryViralVectors:AdenovirusRetrovirusLentivirusAdeno-associatedvirus(AAV)Herpessimplexvirus(HSV)Non-viralvectorbasedNakedDNA(plasmidDNA):injectionorgenegunLiposomes(cationiclipids):mixwithgenesEx-vivoInvivoWhyuseviralvectorsVirusareobligateintracellularparasitesVer

2、yefficientattransferringviralDNAintohostcellsSpecifictargetcells:dependingontheviralattachmentproteins(capsidorglycoproteins)Genereplacement:non-essentialgenesofvirusaredeletedandexogenousgenesareinsertedGenerationofviralvectorforgenetherapyReplication-competentvirusReplication-defectivevirusA

3、mplicon:doesn’tencodestructuralproteinsCan’treplicatebeyondthefirstcycleofinfectionElementsneededtogenerateampliconTransferVector:plasmid(promoter,geneofinterest,ori,packagingsignal)Packagingvector(cosmidorcelllines):providetheviralstructuralproteinsforpackagingoftransfervectorHelpervirus(packa

4、gingoftransfervector):deletedPackagingsignalsequenceAdeno-associatedvirusvectorsNon-pathogenichumanparvovirus,non-envelopedssDNAvirus,4.6kilobasesDependentonahelpervirus(adenovirusorherpesvirus)forreplication(dependovirus)AAV-2mostlyusedforvectorAdeno-AssociatedVirus(AAV)SingleStrandedDNAVirusV

5、iralITRs&Rep/Cap~5kbCapacityIntegrating/ConcatamericLongTermExpressionComplexProduction&PurificationMultipDNA&/orHelperVirusLowerTitreThanAdenovirusSerotypeDifferencesInReceptorsAAV2HeparanSulphateAAV5SialicAcidGenerationofadeno-associatedvirusvectorCharacteristicsofAAVvectorAdvantagesIntegrati

6、onandpersistentexpressionNoinsertionalmutagenesisInfectingdividingandnondividingcellsSafeDisadvantagesSizelimitation,4.9kbLowtiterofvirus,lowlevelofgeneexpressionAdenoviralvectorsNon-envelopeddsDNA,36kilobasesEarlyproteins(E1A,E1B,E2,E3andE4),lateproteins(L1-L5)Causesabenignrespiratoryinfection

7、sinhumanSerotypes2and5arecommonlyusedasvectorsEarlygenerationsofadenoviralvector(replicationdefective)GutlessAdenoviralvector(Amplicon)ModificationofthetropismofadenovirusvectorAdenovirusfiberbindstoCAR(coxsakieandadenovirusrecept