《分子病毒学 8病毒载体(共页)[共37页]》由会员分享，可在线阅读，更多相关《分子病毒学 8病毒载体(共页)[共37页]（37页珍藏版）》请在金锄头文库上搜索。
1、viralvector,韩晓,Methodsofgenedelivery,ViralVectors:AdenovirusRetrovirusLentivirusAdeno-associatedvirus(AAV)Herpessimplexvirus(HSV)Non-viralvectorbasedNakedDNA(plasmidDNA):injectionorgenegunLiposomes(cationiclipids):mixwithgenesEx-vivoInvivo,Whyuseviralvectors,VirusareobligateintracellularparasitesVer
2、yefficientattransferringviralDNAintohostcellsSpecifictargetcells:dependingontheviralattachmentproteins(capsidorglycoproteins)Genereplacement:non-essentialgenesofvirusaredeletedandexogenousgenesareinserted,Generationofviralvectorforgenetherapy,Replication-competentvirusReplication-defectivevirusAmpli
3、con:doesntencodestructuralproteinsCantreplicatebeyondthefirstcycleofinfectionElementsneededtogenerateampliconTransferVector:plasmid(promoter,geneofinterest,ori,packagingsignal)Packagingvector(cosmidorcelllines):providetheviralstructuralproteinsforpackagingoftransfervectorHelpervirus(packagingoftrans
4、fervector):deletedPackagingsignalsequence,Adeno-associatedvirusvectors,Non-pathogenichumanparvovirus,non-envelopedssDNAvirus,4.6kilobasesDependentonahelpervirus(adenovirusorherpesvirus)forreplication(dependovirus)AAV-2mostlyusedforvector,Adeno-AssociatedVirus(AAV),SingleStrandedDNAVirusViralITRs&Rep
5、/Cap5kbCapacityIntegrating/ConcatamericLongTermExpressionComplexProduction&PurificationMultipDNA&/orHelperVirusLowerTitreThanAdenovirusSerotypeDifferencesInReceptorsAAV2HeparanSulphateAAV5SialicAcid,Generationofadeno-associatedvirusvector,CharacteristicsofAAVvector,AdvantagesIntegrationandpersistent
6、expressionNoinsertionalmutagenesisInfectingdividingandnondividingcellsSafeDisadvantagesSizelimitation,4.9kbLowtiterofvirus,lowlevelofgeneexpression,Adenoviralvectors,Non-envelopeddsDNA,36kilobasesEarlyproteins(E1A,E1B,E2,E3andE4),lateproteins(L1-L5)CausesabenignrespiratoryinfectionsinhumanSerotypes2
7、and5arecommonlyusedasvectors,Earlygenerationsofadenoviralvector(replicationdefective),GutlessAdenoviralvector(Amplicon),Modificationofthetropismofadenovirusvector,AdenovirusfiberbindstoCAR(coxsakieandadenovirusreceptor,CAR),receptorwhichisubiquitousModifythefiberprotein,Characteristicsofadenoviralve
8、ctor,AdvantagesHightitersBothdividingandnon-dividingcellsWidetissuetropismEasilymodifytissuetropismDisadvantagesTransientexpression(notgoodforgeneticdiseases)HighlyimmunogenicHightitersofviruscanbetoxicMoresuitableforcancerimmunotherapy,Retroviralvector,Moloneymurineleukemiavirus(MuLV)Generationofre
9、plicationdefectiveretroviralvectorTransferplasmidvector:GeneofinterestLongterminalrepeats(LTR):promoter,polyA,integration,replicationandreversetranscriptionPrimerbindingsite(PBS)(originofreplication)RNApackagingsignalPolypurinetract(importantforreplication)PackagingvectorCelllinestablytransfectedwit
10、hplasmidconstructscontainingGag/polandEnv,Generationofretroviralvector,Pseudotypedretroviralvector,Characteristicsofretroviralvector,AdvantagesIntegration:permanentexpressionPseudotypedvirusDisadvantagesOnlyinfectingdividingcellsInsertionalmutagenesis(tumorformation)ActivateoncogenesInhibittumorsupp
11、ressorgenes,Lentiviralvectors,Infectionofnon-dividingcells(hepatocytes,neurons)HIV,ahumanlethalpathogenDeleteaccessorygenesProvideanenvelopefromanon-retrovirus(VSV)Developvectorsfromlentivirusesofnon-humanpathogensSIV,FIV,EIAVetc,Herpesvirusvectors,Herpessimplexvirus1,milddiseaseinhuman,noriskLinear
12、dsDNA,152kb,abouthalfofthetotal81genesarenon-essentialforvirusreplication40-50kbofforeignDNAcanbeaccommodatedNeurotropicvirus,targettonervoussystemReplicationdefectiveampliconparticles,Comparisonofdifferentviralvectors,Viralvectortitersmanupilationofimmunogenicityinfectingoftropismnon-dividingcellsA
13、denovirus1011terrificveryhighyesRetrovirus107goodlowonlylentivirusHerpesvirus107notsogoodlowyesAAV107notsogoodlowyes,Genetherapy,Genetherapy:tocorrectageneticdefectbytransferringofafunctionalnormalcopyofthegeneintocellsExamplesofdiseasescausedbygeneticdefectOrnithinetranscarbamylase(OTCdeficiency)He
14、mophilia(bloodcoagulationfactorsVIIIorIX)SCID(severecombinedimmunodeficiency)MusculardystrophyCysticfibrosisSicklecellanemia,Applicationofgenetherapy,Geneticdisorder(deficiency):OTCCancerGeneticpredispositionMutationinoncogeneortumorsuppressorgeneAutoimmunitydiseases:rheumatoidarthritisDeliveryofcou
15、nteractinggeneDiseasesinvolveseveralgenesandtheenvironmentalinteract:diabetes,FactorstobeconsideredinGenetherapy,Howtodelivergenestospecificcells,tissueandwholeanimals?(methodsofdelivery)Howmuchandhowlongtheintroducedgenewillbeexpressed?ThesiteanddoseofgenedeliveryIsthereanyadverseimmunologicalconse
16、quenceofbothdeliveryvehicle(Virus)andthegeneinanimals?Isthereanytoxiceffects?,Deathof18-yearoldJesseGelsinger,Liverdisease:OTCdeficiency(geneticdisease)UniversityofPennsylvaniaHighdoseofadenoviralvector(E1andE4genesdeleted)carryingthenormalcopyofOTCgenewasadministeredSuspectedcauseofdeath-Toxicityofhightiteradenoviralvector-Highimmunogenicityofadenoviralvector(animmunerevolt),AcaseofleukemiainaSCIDchildtreatedwitharetroviralvector,SCIDdiseaseorBubbleboydisease(Tcelldeficiency)Overallqui
