分子遺傳學(xué)課件：Gene Therapy

GeneTherapyHumanHaveThreeGenomesInheritedDiseasesAcquiredDiseasesDiseasesInteractedbyGeneandEnvironmentChromosomeDiseasesMultipleGeneDiseasesSingleGeneDiseasesMitochondrialDiseasesEpigeneticDiseasesTraumaSuffocateRadiationStarvationClassificationofDiseasesChromosomalDisorders

Morethan100types，mentalretardationandgrowingdevelopmentdelayingarecommoncharacteristics.

Numericalabnormalitiesandstructureaberration3000types

Abnormalchromosomeinliveinfantis5%

Abnormaltypes:aneuploid(Trisomy21,18,13)，deletion,duplication,circularchoromosome,translocation,inversion,isochromosomeMonogeneticDisordersHereditarydeafnessα&βThalassemiaSickle-cellanemiaGlucose-6-phosphatedehydrogenasedeficiencyHemophiliaA,B,CFactorVdeficiencyFamilialhypercholesterolemia

morethan8000types

Pointmutation:missense,nonsense

Shiftingmutation:deletion,insertion,Region:

Coding,regulation,splicingFactorVIIIandHemophiliaAGaucher`sDiseaseGaucherdiseaseisthemostcommonoftheinheritedmetabolicdisorderknownaslipidstoragediseases.

Gaucherdiseaseiscausedbyadeficiencyoftheenzyme

glucocerebrosidase.

MitochondriaDiseases

Maternalinheritance，morethan100typesdiseases1/6500Atleast350,000patientsinEuropeUSA：1/4,000lessthan10ys.4,000newbornpatients.

Deafness,diabetes,Leberoptic-neuropathyMultipleGeneticDiseases

Autism(90%),Schizophrenia(80%),Congenitalasthma(80%),CleftlipsandPalate(76%),JuvenileDiabetes(75%),Congenitaldislocationofthehip(70%),Coronaryheartdisease(65%),Hypertension(62%),Idiopathicepilepsy(57.4%),Congenitalheartdisease(55%).Cancers(Geneticandepigeneticschanges)Hydrocephalus(49.6%),Peptic(37%),Senilediabetes(35%).CancerAutismSpectrumDisorders，ASDs

1%populationAutismGenomeProject，AGP：2002，morethan50institutesand120scientistsallovertheworld.

Susceptibilitygenes：124,NRXN1，MeCP2，CHD2,HDAC4,GDI1，SETD5,HDAC9，miR137,etc.Susceptibilityloci:55，15q11.2–q13and16p11.2duplication,16p11.2andX-linkedregion（PTCHD1-PTCHD1AS）deletions.EpigeneticsDiseasesBeckwith-WiedemannSyndrome:hemihypertrophyandnephromegaly,tumors.H19/IGF2imprintingdefects.Prader-WilliSyndrome:Autism,overweight,compactbodybuild,underdevelopedsexualcharacteristics,andpoormuscletone.SNRPN(smallnuclearribonucleoproteinpolypeptideN),MaternalImprinting.

AngelmanSyndrome:Neurologicaldisorderinwhichseverelearningdifficultiesareassociatedwithacharacteristicfacialappearanceandbehaviour.UBE3A(ubiquitinproteinligaseE3A),PaternalImprintingPrader-WilliSyndromeAngelmanSyndromeAIDSper100,000inhabitantsTypesofTreatment

AntibioticsNutritionDepressurizationReplacementBringdownafeverStoppainOperationEtiologyTreatmentSymptomTreatmentTypesofDrug

TherapeuticEffects？Firstunapprovedgenetherapy–1980,Dr.MartinCline,bonemarrowcellsoftwopatientswithbeta-thalassemia.UCLA1989:MichaelBlaese,FrenchAndersonandcolleaguesattheNIHperformthefirstapprovedgenetherapytrialinpatients.Itinvolvedretroviral-mediatedtransferofthegeneencodingADAintotheTcellsoftwochildrenwithseverecombinedimmunodeficiency(SCID).Onepatient,AshantiDeSilva,exhibitedatemporaryresponse,althoughshecontinuedonenzymereplacementtherapy.Theresponsewasfarmorelimitedinthesecondpatient.1980s:FailureandDawnADA:adenosinedeaminase1990s:SuccessandStrikeSicklecelldiseaseissuccessfullytreatedinmice.ClaudioBordignonworkingattheVita-SaluteSanRaffaeleUniversity,Milan,Italyperformedthefirstprocedureofgenetherapyusinghematopoieticstemcellsasvectorstodelivergenesintendedtocorrecthereditarydiseases.1999:JesseGelsinger,an18yearoldwitharelativelymildformofthenitrogenmetabolismdisorderornithinetranscarbamylase(鳥(niǎo)氨酸轉(zhuǎn)氨甲酰酶,OT)deficiency,isthefirstpersontodieonagenetherapytrial.HeexperiencedasevereinflammatoryresponseafterundergoinganinfusiontotheliverofanadenoviralvectorcarryingthegeneencodingOT.Hethensufferedlungfailurefollowedbymultipleorganfailure.2000s:KeeponGoing

2000.AlainFisheretal.reportedadramaticclinicalimprovementintwochildrenwithX-linkedSCID(SCID-X1),ageneticdisordercharacterizedbythefailureofT-cellsandnaturalkillercellstodifferentiate.Thepatients'bonemarrowcellsweremodifiedbytransferofthegeneencodingtheIL-2receptorgammachain,encodedbyamurineretroviralvector.Twenty

children

inallreceivedthistreatment,butfivesubsequentlydevelopedleukemia,oneofwhomdied,aftertheactivationofproto-oncogenespromotingT-cellproliferationbyanenhancersequenceencodedbythevector.

2001:ResearchersatSt.BarnabasHospitalinNewJerseyhadusedooplasmictransfertoenableseveralwomenwithimpairedfertilitytobearchildren.

2003:ResearchersinUCLAinsertedgenesintothebrainusingliposomescoatedinapolymer.Thetransferofgenesintothebrainisasignificantachievementbecauseviralvectorsaretoobigtogetacrosstheblood-brainbarrier.ThismethodhaspotentialfortreatingParkinson'sdisease.ScientistsattheNIHhavesuccessfullytreatedmetastaticmelanomausingkillerTcellsgeneticallyretargetedtoattackthecancercells.

2006:Aninternationalgroupofscientistsannouncedthesuccessfuluseofgenetherapytotreattwoadultpatientsforadiseaseaffectingmyeloidcells.

2006:AteamofscientistsinMilan,ItalyreportedabreakthroughforgenetherapyinwhichtheydevelopedmiRNAtopreventtheimmunesystemfromrejectinganewlydeliveredgene.

2006:PrestonNixfromtheUniversityofPennsylvaniareportedthetreatmentofHIVthatusesalentiviralvectorfordeliveryofanantisensegeneagainsttheHIVenvelope.2007:MoorfieldsEyeHospitalandUniversityCollegeLondon'sInstituteofOphthalmologyannouncedtheworld'sfirstgenetherapytrialforinheritedretinaldisease.TheyresearchedthesafetyofthesubretinaldeliveryofrecombinantAAVcarryingRPE65gene,andfoundityieldedpositiveresults,noapparentside-effects.

2008：amanwascuredofHIVbyrepeatedHematopoieticstemcelltransplantationwithdouble-delta-32mutationwhichdisablestheCCR5receptor2009:Sciencereportedthatresearcherssucceededathaltingafatalbraindisease,adrenoleukodystrophy(腎上腺腦白質(zhì)營(yíng)養(yǎng)不良)usinglentiviralvector.2011：twoofthreesubjectshavebeencuredfromchroniclymphocyticleukemiausedgeneticallymodifiedTcellstofightthedisease.HumanHGFplasmidDNAtherapyofcardiomyocytesisbeingexaminedasapotentialtreatmentforcoronaryarterydiseaseaswellastreatmentforthedamagethatoccurstotheheartaftermyocardialinfarction.

2012：Thetreatment,calledGlybera,compensatesforlipoproteinlipasedeficiency,whichcancauseseverepancreatitis.2013:LentiviralHematopoieticStemCellGeneTherapyBenefitsMetachromaticLeukodystrophy(異染性腦白質(zhì)營(yíng)養(yǎng)不良)byItalyScientists.2010s:Hoping

2014Rejuvenation:Thyroid

HIV:Deletionininfectedcells.

2015

Sicklecellanemia:Blood

Prostatecancer:Target,OncotargetCardiacpacemaker:NatureBiotechnology

Cysticfibrosis:LancetRespiratoryMedicine

Micedeafness:ScienceTranslationalMedicineBlind:ScienceTranslationalMedicineMitochondrialDiseases:Nature

1987:FudanUniversity,JinglunXue,fourhemophiliaBpatientstreatedin1991andremissionfor17yearswithAdenovirusvector.Secondclinicaltrialin2003,recombinant-AAV-2humanFIX.2004:ShenzhenSiBionoGenTech(Shenzhen,China)gainsapprovalinChinafortreatingheadandneckcancerwithGendicine,amodifiedadenovirusvectorencodingthep53tumorsuppressorgene.SunwayBiotech(Shanghai)gainedapprovaltwoyearslaterforH101,whichisbasedonOnyx-15,arecombinantoncolyticadenovirusoriginallydevelopedbyOnyxPharmaceuticals(Emeryville,CA,USA),whichtargetsp53-deficienttumorcells.GeneTherapyinChinaClinicalTrialWorldWideWhatisGeneTherapy?Genetherapyisanexperimentaltreatmentthatinvolvesintroducinggeneticmaterialintoaperson’scellstofightorpreventdisease.WhyGeneTherapy?HalftimeofFVIII:6-14hRecombinantFactorFVIII:250U/Bottle.Price:￥1320ExonSignalpeptide,locationsequenceEnzymeactivitycenter,disulfidebond,modificationsite,antigenicdeterminant,proteininteractionsiteIntronSplicingsite，parasiticgene,regulationsiteRegulationareaPromoter,enhancer,silencer,terminatorRNAbindingsiteGeneVariationandItsSignificantBasicMethodsofGeneTherapyReplacingorrepairingamutatedgenethatlosefunctionwithahealthycopyBasicMethodsofGeneTherapyInactivating,orknockout,amutatedgenehavingenhancefunctionThreedifferentwaystomutateProto-oncogenescanbecomeoncogenesBasicMethodsofGeneTherapyIntroducinganewgeneintothebodytohelpfightadiseaseTypesofGeneTherapySomaticGeneTherapyMoreconservative,saferapproachShort-lived

GermlineGeneTherapy

PermanentchangesPotentialforunforeseennegativeEffectsonfuturegenerationsPlayingGodWhatAreCriteriaForGeneTherapy?Incurable,life-threateningdiseaseDiseasehavebeenidentifiedNormalcounterpartofthedefectivegenehasbeenisolatedandclonedNormalgenecanbeintroducedintoasubstantialsubfractionofthecellsfromtheaffectedtissueGenecanbeexpressedadequatelyTechniquesareavailabletoverifythesafetyMorebeauty?Moresmart?KeyPointsofGeneTherapyNormalorCorrectedGeneGeneCarrierTargetCellHowtoGetANormalGene?PCRorRT-PCRDNAorcDNAlibraryArtificialsynthesizingHowtoSelectControlElements?HousekeepinggeneTissuespecificgeneInduciblegeneAntisenceGeneTherapy

DNARNAantisenceRNABlockExpressionRNAinterference,siRNAknockdownbyRNAiisincomplete,variesbetweenexperimentsandlaboratories,hasunpredictableoff-targeteffects,andprovidesonlytemporaryinhibitionofgenefunction.Eachzinc-fingerconsistsofapproximately30aminoacidsinanββαarrangement,andcontacts3or4bpinthemajorgrooveofDNA.ZFNtargetsitesconsistoftwozinc-fingerbindingsitesseparatedbya5–7-bpspacersequencerecognizedbytheFokIcleavagedomain.Off-targetingZincFingerNuclease(ZFN)TranscriptionActivator-likeEffectorNucleases(TALENs)IndividualTALErepeatscontain33–35aminoacidsthatrecognizeasinglebasepairviatwohypervariableresidues(repeat-variablediresidues,RVDs).TALENtargetsitesconsistoftwoTALEbindingsitesseparatedbyaspacersequenceofvaryinglength(12–20bp).TALEscanbedesignedtorecognizeuniqueleftandrighthalf-sites.RVDcompositionsareindicated.CRISPR–Cas9SystemCRISPR:ClusteredregulatoryinterspacedshortpalindromicrepeatsCas9servesasanRNA-guidedDNAendonucleaseThesystemrequirestheco-expressionofaCas9proteinwithaguideRNAvectorexpressedfromthehumanU6polymeraseIIIpromoter.Withtheprotospacer-adjacentmotif(PAM-thesequenceNGG)presentatthe3′end,Cas9willunwindtheDNAduplexandcleavebothstrandsuponrecognitionofatargetsequencebytheguideRNA.Cpf1IsaSingleRNA-GuidedEndonucleaseofaClass2CRISPR-CasSystemHighlightsCRISPR-Cpf1isaclass2CRISPRsystemCpf1isaCRISPR-associatedtwo-componentRNA-programmableDNAnucleasewhichsmallerthancas9TargetedDNAiscleavedasa5-ntstaggeredcutdistaltoa5′T-richPAMTwoCpf1orthologsexhibitrobustnucleaseactivityinhumancellsAbbreviatedlistofexamplesofZFN,TALEN,andCRISPR/Cas-mediatedgenomeeditinginhumancellsandmodelorganismsGeneorRNATransferPhysicalChemicalBiologicalPhysicalTransferofNakedDNATheexpressionislowElectroporation:150kbDNABallistictransfer(genegun)MicroinjectionSonoporationSonoporationusesultrasonicfrequenciestodeliverDNAintocells.TheprocessofacousticcavitationisthoughttodisruptthecellmembraneandallowDNAtomoveintocells.MagnetofectionDNAiscomplexedtomagneticparticles,andamagnetisplacedunderneaththetissueculturedishtobringDNAcomplexesintocontactwithacellmonolayer.

MagnetofectionisgenerallyapplicableforadherentcellsandhasbeentestedwithavarietyofimmortalizedcelllinesandprimarycellsNakedDNA-calciumphosphateco-precipitationLipoplexes:Liposome-DNAPolyplexes:PLA(poly-lacticacid),PLGA(poly-lactide-co-glycolide)ChemicalTransferViralVectorsFourBarriersToSuccessfulGeneTherapyRetroviralVectorGroup-specificantigen(gag):codesforcoreandstructuralproteinsPolymerase(pol)codesforreversetranscriptase,proteaseandintegraseEnvelope(env)codesfortheretroviralcoatproteinsRNAvirusTransduceavarietyofcelltypesIntegrateintothegenomicDNAofthedividingcellsExpressthetransducedgeneathighlevelsLong-termpersistenceandstabletransmissionUpto6.5kbofforeigngenebepackagedAbilitytobemanufacturedinlargequantitiesCharacteristicsofRetroviralVectorsHIV,Simianimmunodeficiencyvirus,andFelineinterfusionVirusLongincubationperiod2005,clinicaltrialHemophillia,diabeticwithPDGF(platelet-derivedgrowthfactor)LentiviralVectorsBelongtotheretrovirusfamilybutcaninfectbothdividingandnon-dividingcells.Theyaremorecomplicatedthanretroviruses,containinganadditionalsixproteins,tat,rev,vpr,vpu,nefandvif.

Lowcellularimmuneresponse,thusgoodpossibilityforinvivogenedeliverywithsustainedexpressionoversixmonths.Nopotentantibodyresponse.CharacteristicsofLentiviralVectorsAdenovirusVectorsDouble-strandedlinearDNAVirusResponsiblefor5–10%ofupperrespiratoryinfectionsinchildrenDNAmoleculeisleftfreeinthenucleusofthehostcellThedescendantsofthecellwillnothavetheextrageneGenerationofAdenovirusVectorsgenerationDeletedregionInsertedsizeTime(days)Hostimmuneresponse1stE1/E37.5kb7~10strongest2ndE1/E3,E210kb20~403rd(gutlessvector)All,expectITR&packagingseq36kb84leastHasshownrealpromiseintreatingcancerGendicine:licensedasanadenovirustotreatcancerSafety—lackofassociationwithoncogenicityWellcharacterizedandeasilymanipulatedStabilityandhightitersofrecombinantvectorsHighefficiencyofcellularuptakegeneupto36kbLittleriskofchromosomalintegration(notlong)Infectdividingandnon-dividingcells(nospecificity)CharacteristicsofAdenovirusVectorsAdeno-AssociatedViralVectors(AAV)SinglestrandedDNAInsertgeneticmaterialataspecificsiteonchromosome19withnear100%certaintyNon-pathogenicNoimmunityNoassociatedwithdiseaseReplicationofAAVdependsoncoinfectionbyahelpervirusIntegratesataspecificsiteinthehumangenome(19q13.4)StableexpressionTransduceavarietyofcelltypesLowcapacity:Upto4.4kbofforeigngenebepackagedDifficultyinproducingit

Mainlytryingtotreatmuscleandeyediseases;neuronsFeaturesofAAVVectorsHerpesvirusViralVectorsLargelinearDNAgenomeofdouble-strandedDNA150kbinlengthPersistinaquiescentbutpersistentformknownaslatentinfection,notablyinneuralgangliaCancarrylongsequencesofforeignDNAestablishlifelongnon-toxiclatentinfectionsinneuronsGlioma,melanomaandovariancancerpatients,PseudotypingofViralVectorsVirusesinwhichtheenvelopeproteinshavebeenreplacedasdescribedarereferredtoaspseudotypedviruses.Limitthetropismofviralvectorstooneorafewhostcellpopulations.CombiningStemCellsandGeneTherapyProtocolforExVivoHaematopoieticStemCellGeneTherapyRiskFactorsofVirusVectorsInfectmorethanonetypeofcellNewgenemightbeinsertedinthewronglocationintheDNAThereisaslightchancethatthisDNAcouldunintentionallybeintroducedintothepatient’sreproductivecellsTransferredgenescouldbeoverexpressed,producingsomuchofthemissingpro