viralvector,韩晓,Methodsofgenedelivery,ViralVectors:AdenovirusRetrovirusLentivirusAdeno-associatedvirus(AAV)Herpessimplexvirus(HSV)Non-viralvectorbasedNakedDNA(plasmidDNA):injectionorgenegunLiposomes(cationiclipids):mixwithgenesEx-vivoInvivo,Whyuseviralvectors,VirusareobligateintracellularparasitesVeryefficientattransferringviralDNAintohostcellsSpecifictargetcells:dependingontheviralattachmentproteins(capsidorglycoproteins)Genereplacement:non-essentialgenesofvirusaredeletedandexogenousgenesareinserted,Generationofviralvectorforgenetherapy,Replication-competentvirusReplication-defectivevirusAmplicon:doesntencodestructuralproteinsCantreplicatebeyondthefirstcycleofinfectionElementsneededtogenerateampliconTransferVector:plasmid(promoter,geneofinterest,ori,packagingsignal)Packagingvector(cosmidorcelllines):providetheviralstructuralproteinsforpackagingoftransfervectorHelpervirus(packagingoftransfervector):deletedPackagingsignalsequence,Adeno-associatedvirusvectors,Non-pathogenichumanparvovirus,non-envelopedssDNAvirus,4.6kilobasesDependentonahelpervirus(adenovirusorherpesvirus)forreplication(dependovirus)AAV-2mostlyusedforvector,Adeno-AssociatedVirus(AAV),SingleStrandedDNAVirusViralITRs&Rep/Cap5kbCapacityIntegrating/ConcatamericLongTermExpressionComplexProduction&PurificationMultipDNA&/orHelperVirusLowerTitreThanAdenovirusSerotypeDifferencesInReceptorsAAV2HeparanSulphateAAV5SialicAcid,Generationofadeno-associatedvirusvector,CharacteristicsofAAVvector,AdvantagesIntegrationandpersistentexpressionNoinsertionalmutagenesisInfectingdividingandnondividingcellsSafeDisadvantagesSizelimitation,4.9kbLowtiterofvirus,lowlevelofgeneexpression,Adenoviralvectors,Non-envelopeddsDNA,36kilobasesEarlyproteins(E1A,E1B,E2,E3andE4),lateproteins(L1-L5)CausesabenignrespiratoryinfectionsinhumanSerotypes2and5arecommonlyusedasvectors,Earlygenerationsofadenoviralvector(replicationdefective),GutlessAdenoviralvector(Amplicon),Modificationofthetropismofadenovirusvector,AdenovirusfiberbindstoCAR(coxsakieandadenovirusreceptor,CAR),receptorwhichisubiquitousModifythefiberprotein,Characteristicsofadenoviralvector,AdvantagesHightitersBothdividingandnon-dividingcellsWidetissuetropismEasilymodifytissuetropismDisadvantagesTransientexpression(notgoodforgeneticdiseases)HighlyimmunogenicHightitersofviruscanbetoxicMoresuitableforcancerimmunotherapy,Retroviralvector,Moloneymurineleukemiavirus(MuLV)GenerationofreplicationdefectiveretroviralvectorTransferplasmidvector:GeneofinterestLongterminalrepeats(LTR):promoter,polyA,integration,replicationandreversetranscriptionPrimerbindingsite(PBS)(originofreplication)RNApackagingsignalPolypurinetract(importantforreplication)PackagingvectorCelllinestablytransfectedwithplasmidconstructscontainingGag/polandEnv,Generationofretroviralvector,Pseudotypedretroviralvector,Characteristicsofretroviralvector,AdvantagesIntegration:permanentexpressionPseudotypedvirusDisadvantagesOnlyinfectingdividingcellsInsertionalmutagenesis(tumorformation)ActivateoncogenesInhibittumorsuppressorgenes,Lentiviralvectors,Infectionofnon-dividingcells(hepatocytes,neurons)HIV,ahumanlethalpathogenDeleteaccessorygenesProvideanenvelopefromanon-retrovirus(VSV)Developvectorsfromlentivirusesofnon-humanpathogensSIV,FIV,EIAVetc,Herpesvirusvectors,Herpessimplexvirus1,milddiseaseinhuman,noriskLineardsDNA,152kb,abouthalfofthetotal81genesarenon-essentialforvirusreplication40-50kbofforeignDNAcanbeaccommodatedNeurotropicvirus,targettonervoussystemReplicationdefectiveampliconparticles,Comparisonofdifferentviralvectors,Viralvectortitersmanupilationofimmunogenicityinfectingoftropismnon-dividingcellsAdenovirus1011terrificveryhighyesRetrovirus107goodlowonlylentivirusHerpesvirus107notsogoodlowyesAAV107notsogoodlowyes,Genetherapy,Genetherapy:tocorrectageneticdefectbytransferringofafunctionalnormalcopyofthegeneintocellsExamplesofdiseasescausedbygeneticdefectOrnithinetranscarbamylase(OTCdeficiency)Hemophilia(bloodcoagulationfactorsVIIIorIX)SCID(severecombinedimmunodeficiency)MusculardystrophyCysticfibrosisSicklecellanemia,Applicationofgenetherapy,Geneticdisorder(deficiency):OTCCancerGeneticpredispositionMutationinoncogeneortumorsuppressorgeneAutoimmunitydiseases:rheumatoidarthritisDeliveryofcounteractinggeneDiseasesinvolveseveralgenesandtheenvironmentalinteract:diabetes,FactorstobeconsideredinGenetherapy,Howtodelivergenestospecificcells,tissueandwholeanimals?(methodsofdelivery)Howmuchandhowlongtheintroducedgenewillbeexpressed?ThesiteanddoseofgenedeliveryIsthereanyadverseimmunologicalconsequenceofbothdeliveryvehicle(Virus)andthegeneinanimals?Isthereanytoxiceffects?,Deathof18-yearoldJesseGelsinger,Liverdisease:OTCdeficiency(geneticdisease)UniversityofPennsylvaniaHighdoseofadenoviralvector(E1andE4genesdeleted)carryingthenormalcopyofOTCgenewasadministeredSuspectedcauseofdeath-Toxicityofhightiteradenoviralvector-Highimmunogenicityofadenoviralvector(animmunerevolt),AcaseofleukemiainaSCIDchildtreatedwitharetroviralvector,SCIDdiseaseorBubbleboydisease(Tcelldeficiency)Overallqui