viralvector韓 曉 viralvector韓 曉1MethodsofgenedeliveryViralVectors:AdenovirusRetrovirusLentivirusAdeno-associatedvirus(AAV)Herpessimplexvirus(HSV)Non-viralvectorbasedNakedDNA(plasmidDNA):injectionorgenegunLiposomes(cationiclipids):mixwithgenesEx-vivoInvivoMethodsofgenedeliveryViral2WhyuseviralvectorsVirusareobligateintracellularparasitesVeryefficientattransferringviralDNAintohostcellsSpecifictargetcells:dependingontheviralattachmentproteins(capsidorglycoproteins)Genereplacement:non-essentialgenesofvirusaredeletedandexogenousgenesareinsertedWhyuseviralvectorsVirusare3Generationofviralvector

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

Generationofviralvector

fo4Adeno-associatedvirusvectorsNon-pathogenichumanparvovirus,non-envelopedssDNAvirus,4.6kilobasesDependentonahelpervirus(adenovirusorherpesvirus)forreplication(dependovirus)AAV-2mostlyusedforvectorAdeno-associatedvirusvectors5

Adeno-AssociatedVirus(AAV)SingleStrandedDNAVirusViralITRs&Rep/Cap~5kbCapacityIntegrating/ConcatamericLongTermExpressionComplexProduction&PurificationMultipDNA&/orHelperVirusLowerTitreThanAdenovirusSerotypeDifferencesInReceptorsAAV2HeparanSulphateAAV5SialicAcid

Adeno-AssociatedVirus6分子病毒學8病毒載體課件7分子病毒學8病毒載體課件8分子病毒學8病毒載體課件9分子病毒學8病毒載體課件10分子病毒學8病毒載體課件11Generationofadeno-associatedvirusvectorGenerationofadeno-associated12分子病毒學8病毒載體課件13分子病毒學8病毒載體課件14分子病毒學8病毒載體課件15CharacteristicsofAAVvectorAdvantagesIntegrationandpersistentexpressionNoinsertionalmutagenesisInfectingdividingandnondividingcellsSafeDisadvantagesSizelimitation,4.9kbLowtiterofvirus,lowlevelofgeneexpressionCharacteristicsofAAVvectorA16AdenoviralvectorsNon-envelopeddsDNA,36kilobasesEarlyproteins(E1A,E1B,E2,E3andE4),lateproteins(L1-L5)CausesabenignrespiratoryinfectionsinhumanSerotypes2and5arecommonlyusedasvectorsAdenoviralvectorsNon-envelope17Earlygenerationsofadenoviralvector(replicationdefective)

Earlygenerationsofadenovira18GutlessAdenoviralvector(Amplicon)GutlessAdenoviralvector(Amp19ModificationofthetropismofadenovirusvectorAdenovirusfiberbindstoCAR(coxsakieandadenovirusreceptor,CAR),receptorwhichisubiquitousModifythefiberproteinModificationofthetropismof20CharacteristicsofadenoviralvectorAdvantagesHightitersBothdividingandnon-dividingcellsWidetissuetropismEasilymodifytissuetropismDisadvantagesTransientexpression(notgoodforgeneticdiseases)HighlyimmunogenicHightitersofviruscanbetoxicMoresuitableforcancerimmunotherapyCharacteristicsofadenoviral21RetroviralvectorMoloneymurineleukemiavirus(MuLV)GenerationofreplicationdefectiveretroviralvectorTransferplasmidvector:GeneofinterestLongterminalrepeats(LTR):promoter,polyA,integration,replicationandreversetranscriptionPrimerbindingsite(PBS)(originofreplication)RNApackagingsignalPolypurinetract(importantforreplication)PackagingvectorCelllinestablytransfectedwithplasmidconstructscontainingGag/polandEnvRetroviralvectorMoloneymurin22GenerationofretroviralvectorGenerationofretroviralvecto23PseudotypedretroviralvectorPseudotypedretroviralvector24分子病毒學8病毒載體課件25CharacteristicsofretroviralvectorAdvantagesIntegration:permanentexpressionPseudotypedvirusDisadvantagesOnlyinfectingdividingcellsInsertionalmutagenesis(tumorformation)ActivateoncogenesInhibittumorsuppressorgenesCharacteristicsofretroviral26LentiviralvectorsInfectionofnon-dividingcells(hepatocytes,neurons)HIV,ahumanlethalpathogenDeleteaccessorygenesProvideanenvelopefromanon-retrovirus(VSV)Developvectorsfromlentivirusesofnon-humanpathogensSIV,FIV,EIAVetcLentiviralvectorsInfectionof27分子病毒學8病毒載體課件28分子病毒學8病毒載體課件29HerpesvirusvectorsHerpessimplexvirus1,milddiseaseinhuman,noriskLineardsDNA,152kb,abouthalfofthetotal81genesarenon-essentialforvirusreplication40-50kbofforeignDNAcanbeaccommodatedNeurotropicvirus,targettonervoussystemReplicationdefectiveampliconparticlesHerpesvirusvectorsHerpessimp30ComparisonofdifferentviralvectorsViralvector titers manupilationof immunogenicity infectingof tropism non-dividingcellsAdenovirus 1011 terrific veryhigh yesRetrovirus 107 good low onlylentivirusHerpesvirus 107 notsogood low yesAAV 107 notsogood low yesComparisonofdifferentviral31Genetherapy

Genetherapy:tocorrectageneticdefectbytransferringofafunctionalnormalcopyofthegeneintocellsExamplesofdiseasescausedbygeneticdefectOrnithinetranscarbamylase(OTCdeficiency)Hemophilia(bloodcoagulationfactorsVIIIorIX)SCID(severecombinedimmunodeficiency)MusculardystrophyCysticfibrosisSicklecellanemia

GenetherapyGenetherapy:32ApplicationofgenetherapyGeneticdisorder(deficiency):OTCCancerGeneticpredispositionMutationinoncogeneortumorsuppressorgeneAutoimmunitydiseases:rheumatoidarthritisDeliveryofcounteractinggeneDiseasesinvolveseveralgenesandtheenvironmentalinteract:diabetesApplicationofgenetherapyGen33Factorstobeconsideredin

GenetherapyHowtodelivergenestospecificcells,tissueandwholeanimals?(methodsofdelivery)Howmuchandhowlongtheintroducedgenewillbeexpressed?Thesiteanddoseofgenedelivery Isthereanyadverseimmunologicalconsequenceofbothdeliveryvehicle(Virus)andthegeneinanimals?Isthereanytoxiceffects?Factorstobeconsideredin

G34

Deathof18-yearoldJesseGelsingerLiverdisease:OTCdeficiency(geneticdisease)UniversityofPennsylvaniaHighdoseofadenoviralvector(E1andE4genes deleted)carryingthenormalcopyofOTCgenewasadministeredSuspectedcauseofdeath-Toxicityofhightiteradenoviralvector-Highimmunogenicity