分子病毒学病毒载体专家讲座

viralvector韩 晓第1页MethodsofgenedeliveryViralVectors:AdenovirusRetrovirusLentivirusAdeno-associatedvirus(AAV)Herpessimplexvirus(HSV)Non-viralvectorbasedNakedDNA(plasmidDNA):injectionorgenegunLiposomes(cationiclipids):mixwithgenesEx-vivoInvivo第2页WhyuseviralvectorsVirusareobligateintracellularparasitesVeryefficientattransferringviralDNAintohostcellsSpecifictargetcells:dependingontheviralattachmentproteins(capsidorglycoproteins)Genereplacement:non-essentialgenesofvirusaredeletedandexogenousgenesareinserted第3页Generationofviralvector

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

第4页Adeno-associatedvirusvectorsNon-pathogenichumanparvovirus,non-envelopedssDNAvirus,4.6kilobasesDependentonahelpervirus(adenovirusorherpesvirus)forreplication(dependovirus)AAV-2mostlyusedforvector第5页

Adeno-AssociatedVirus(AAV)SingleStrandedDNAVirusViralITRs&Rep/Cap~5kbCapacityIntegrating/ConcatamericLongTermExpressionComplexProduction&PurificationMultipDNA&/orHelperVirusLowerTitreThanAdenovirusSerotypeDifferencesInReceptorsAAV2HeparanSulphateAAV5SialicAcid第6页第7页第8页第9页第10页第11页Generationofadeno-associatedvirusvector第12页第13页第14页第15页CharacteristicsofAAVvectorAdvantagesIntegrationandpersistentexpressionNoinsertionalmutagenesisInfectingdividingandnondividingcellsSafeDisadvantagesSizelimitation,4.9kbLowtiterofvirus,lowlevelofgeneexpression第16页AdenoviralvectorsNon-envelopeddsDNA,36kilobasesEarlyproteins(E1A,E1B,E2,E3andE4),lateproteins(L1-L5)CausesabenignrespiratoryinfectionsinhumanSerotypes2and5arecommonlyusedasvectors第17页Earlygenerationsofadenoviralvector(replicationdefective)

第18页GutlessAdenoviralvector(Amplicon)第19页ModificationofthetropismofadenovirusvectorAdenovirusfiberbindstoCAR(coxsakieandadenovirusreceptor,CAR),receptorwhichisubiquitousModifythefiberprotein第20页CharacteristicsofadenoviralvectorAdvantagesHightitersBothdividingandnon-dividingcellsWidetissuetropismEasilymodifytissuetropismDisadvantagesTransientexpression(notgoodforgeneticdiseases)HighlyimmunogenicHightitersofviruscanbetoxicMoresuitableforcancerimmunotherapy第21页RetroviralvectorMoloneymurineleukemiavirus(MuLV)GenerationofreplicationdefectiveretroviralvectorTransferplasmidvector:GeneofinterestLongterminalrepeats(LTR):promoter,polyA,integration,replicationandreversetranscriptionPrimerbindingsite(PBS)(originofreplication)RNApackagingsignalPolypurinetract(importantforreplication)PackagingvectorCelllinestablytransfectedwithplasmidconstructscontainingGag/polandEnv第22页Generationofretroviralvector第23页Pseudotypedretroviralvector第24页第25页CharacteristicsofretroviralvectorAdvantagesIntegration:permanentexpressionPseudotypedvirusDisadvantagesOnlyinfectingdividingcellsInsertionalmutagenesis(tumorformation)ActivateoncogenesInhibittumorsuppressorgenes第26页LentiviralvectorsInfectionofnon-dividingcells(hepatocytes,neurons)HIV,ahumanlethalpathogenDeleteaccessorygenesProvideanenvelopefromanon-retrovirus(VSV)Developvectorsfromlentivirusesofnon-humanpathogensSIV,FIV,EIAVetc第27页第28页第29页HerpesvirusvectorsHerpessimplexvirus1,milddiseaseinhuman,noriskLineardsDNA,152kb,abouthalfofthetotal81genesarenon-essentialforvirusreplication40-50kbofforeignDNAcanbeaccommodatedNeurotropicvirus,targettonervoussystemReplicationdefectiveampliconparticles第30页ComparisonofdifferentviralvectorsViralvector titers manupilationof immunogenicity infectingof tropism non-dividingcellsAdenovirus 1011 terrific veryhigh yesRetrovirus 107 good low onlylentivirusHerpesvirus 107 notsogood low yesAAV 107 notsogood low yes第31页Genetherapy

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

第32页ApplicationofgenetherapyGeneticdisorder(deficiency):OTCCancerGeneticpredispositionMutationinoncogeneortumorsuppressorgeneAutoimmunitydiseases:rheumatoidarthritisDeliveryofcounteractinggeneDiseasesinvolveseveralgenesandtheenvironmentalinteract:diabetes第33页Factorstobeconsideredin

GenetherapyHowtodelivergenestospecificcells,tissueandwholeanimals?(methodsofdelivery)Howmuchandhowlongtheintroducedgenewillbeexpressed?Thesiteanddoseofgenedelivery Isthereanyadverseimmunologicalconsequenceofbothdeliveryvehicle(Virus)andthegeneinanimals?Isthereanytoxiceffects?第34页

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