遗传学遗传病的治疗

TheTreatmentofGeneticDiseases

Pompedisease-庞贝氏症GlycogenstoragediseasetypeII二型糖原储积症溶酶体贮积症常染色体隐性方式遗传。由于位于第17号染色体上编码(acidα-glucosidase)的基因突变，造成体内酸性α-葡糖苷酶缺乏，糖原不能正常代谢而贮积在肌肉细胞的溶酶体中，导致严重的神经肌肉病变。人群患病率为1/40,000-1/300,000。进行性肌无力，运动不耐受，逐渐出现呼吸肌受累并致呼吸功能衰竭。Acidα-glucosidase治疗策略

Thevariouslevelsoftreatmentthatarerelevanttogeneticdisease,withthecorrespondingstrategiesusedateachlevel.Foreachlevel,adiseasediscussedinthetextisgivenasanexample基因治疗GeneTherapy定义：Thedeliberateintroductionofgeneticmaterialintohumansomaticcellsfortherapeutic,prophylacticordiagnosticpurposesAdditionofEXTRAgenesAimistocuredisease(oratleasthelpthepatient)Firstintroductionofgene-modifiedcellsintoapatientwasin1989Firstgenetherapyproductapprovedformarketin2004 StillveryexperimentalandearlyinitsdevelopmentGeneTherapy-Background1953–WatsonandCrickproposedthatDNAisadoublehelix,suggestinghowthisstructurecouldbeusedtoreplicateandinheritgeneticinformation

1961–Nirenbergdecipheredtripletsinthegeneticcode

1978–Arber,NathansandSmithdiscoveredrestrictionenzymesandappliedittoproblemsofmoleculargeneticsJamesWatsonandFrancisCrickGeneTherapy-Background1990–首本关于基因治疗专业刊物出版《HumanGeneTherapy》

1990–首例人体临床试验：AshanthiDeSilva,a4yearoldgirlwithSevereCombinedImmunodeficiency,wasgivenherownTcellsengineeredwitharetroviralvectorcarryinganormalADAgene

2000–首个通过基因治疗成功痊愈的病例：AlainFischer(Paris)succeededintotallycorrectingchildrenwithSCID-X1,or“bubbleboy”syndrome“BubbleBoy”PTQAApril20088GeneTherapyMust:明确疾病的分子基础具有完整功能的基因疾病的病生理基础可接受的：危险、获益比(risk-to-benefitratio)待导入及基因的调控机制筛选目标细胞群/组织足够的安全性证明管理部门的许可GeneTherapy目的:Abletocorrectareversiblephenotype.Maybeundertakentoreplaceortoinactivateadominantmutantallelewhoseabnormalproductcausesthegenerallydominantdisease.Mayeventuallybemostwidelyusedtoachieveapharmacologicaleffect.GeneTherapyInvolvesdeliveryoftherapeuticgenesintothehumanbodytocorrectdiseaseconditionscreatedbyfaultygenesTwoprimarystrategies:

Exvivogenetherapy

InvivogenetherapyExvivo（自体）genetherapy

取患者细胞

实验室操作(对变异基因进行更正等)重新植入患者体内Moreeffectivethaninvivo

TransfectionistheintroductionofDNAintoanimalorplantcellsInvivogenetherapyIntroducinggenesdirectlyintotissuesororganswithoutremovingbodycellsChallengeisdeliveryonlytointendedtissuesVirusesactasvectorsforgenedelivery,butsomeinjecteddirectlyintotissueDeliveryoftherapeuticgenesTherapeuticgenesoftencalledpayloadMayrequirelong-termexpressionofcorrectivegeneOthersrequirerapidexpressionforshortperiodsoftime病毒载体Viralvectors（病毒载体）：useviralgenometocarrytherapeuticgene(s)andtoinfecthumanbodycellsAdenovirus(commoncold)Adeno-AssociatedVirusRetrovirus(HIV)Herpessimplexvirus(coldsores)

Virusesmustbeengineeredsothattheycanneitherproducediseasenorspread(extremelyeffectiveatinfectinghumancells)VectorTransfection(转染)TargetedgenetherapymayresultsincesomevirusesinfectcertainbodycellsAdenoviruses（腺病毒）infectbothdividing&non-dividingcellseffectivelyAdeno-Associatedviruses

donotcauseillnessinhumans,caninfectawidevarietyofcells,&integrate95%oftimeinsamelocationRetroviruses

（逆转录病毒）areofinterestbecausetheyinsertDNAintothegenomeofhostwhereitremainspermanently(integration),butoften,randomlyVectorTransfectionHerpesvirus(HSV-1)strainprimarilyaffectscentralnervoussystem(CNS)MayhelpdeveloptreatmentsforAlzheimer’s,Parkinson’s,andothergeneticneurodegenerativediseasesHowever,althoughviralvectorsmayhelp,mosthumancellsarenoteasilytransfected其他方法

Liposomes（脂质体）aresmalldiameter,hollowparticlesmadeoflipidmoleculesPackagedwithgenesandinjectedintotissuesAgeneguncouldalsobeused

“Naked”DNAinjecteddirectlyintobodytissues(ex.effectiveinliver/muscle),butnotenoughcellsexpressgenetohaveaffect

ArtificialchromosomesmayalsodelivertherapeuticgeneNon-proteincodingDNAwiththerapeuticgeneSimilarconstructiontonormalchromosomes(designedforpermanentincorporation)CuringGeneticDisease

单基因遗传病>3000种

有希望在将来治愈的疾病：CysticFibrosisHuntington’sdiseaseTay-SachsHemophiliaSicklecelldiseasePhenylketonuria(PKU)RisksofGeneTherapyDiscussionsofsafetyintensifiedwhen18-year-oldJesseGelsingerdiedduringaclinicaltrial@Upennin1999ComplicationsrelatedtoadenovirusvectorthatwasusedOrnithinetranscarbamylasedeficiency鸟氨酸氨甲酰基转移酶缺乏症(affectsabilitytobreakdowndietaryaminoacids)1stpersontodieasaresultofgenetherapySuccessofGeneTherapySuccessinRhysEvans,achildbornwithX-linkedSevereCombinedImmunodeficiencySyndrome(SCIDS-akabubbleboy),in2002Theteamtookstemcellsthatgiverisetoimmunecellsfromtheboy’sbonemarrowTheyusedamodifiedformofaretrovirusasavectorTheengineeredstemcellswerethenreturnedtotheboy’sbodyNow,hehasnormallevelsofTcellsUnresolvedQuestions

Cangeneexpressionbecontrolledinthepatient?Whathappensifnormalgeneisoverexpressed?Howlongwillthetherapylast?Whatisthebestvectortouse?Whatistheminimumnumberofcellsneededtoinfecttoachievesuccess?治疗策略

Thevariouslevelsoftreatmentthatarerelevanttogeneticdisease,withthecorrespondingstrategiesusedateachlevel.Foreachlevel,adiseasediscussedinthetextisgivenasanexample代谢系统遗传病的治疗Themostsuccessfulapproach.Inhibition（抑制）Depletion（清除）TypeofmetabolicinterventionSubstanceDiseaseDietaryrestriction（饮食控制）PhenylalanineGalactosePhenylketonuriaGalactosemiaReplacement（替代）BiotineEnzymeReplacementtherapyBiotinidasedeficiencyLysosomalstoragediseasesDiversion（转化）SodiumBenzoateUreacycledefectActiveenzymeEnzymereplacementtherapy

替代疗法LysosomeGolgiEndocytosisReplacement（

替代治疗）Theprovinsionofessentialmetabolites,cofactors,orhormoneswhosedeficiencyisduetoageneticdiseaseissimpleinconceptandoftensimpleinapplication.Someofthemostsuccessfullytreatedsingle-genedefectsbelongtothiscategory.Ex.Congenitalhypothyroidism.ReplacementCongenitalhypothyroidism（甲状腺功能低下）thyroid

hormone

(甲状腺激素)

deficiencypresentatbirth1in4000

Ifuntreatedforseveralmonthsafterbirth,canleadtogrowthfailureandpermanentmentalretardation.dailydoseofthyroidhormone(thyroxine)bymouthReplacementThegoalofnewbornscreeningprogramsistodetectandstarttreatmentwithinthefirst1–2weeksoflife.

Treatmentconsistsofadailydoseof

thyroxine,availableasasmalltablet(levothyroxine-左旋甲状腺素).Themostcommonlyrecommendeddoserangeis10-15μg/kg

daily.hedoseincreasesasthechildgrows.戈谢氏病的替代治疗GaucherDisease-MostprevalentlysosomaldisorderGlucocerebrosidease(葡糖脑苷脂酶)deficiency葡萄糖脑苷脂在单核巨噬细胞系统中大量沉积中枢神经系统受累RichsourceofWTenzymesEasytotargetmacrophageGaucherCellHematologicmanifestationsVisceralinvolvementBonediseaseNeurologicmanifestationsFourmainmanifestations:GaucherDisease

ActiveenzymeEnzymereplacementtherapy

替代疗法LysosomeGolgiEndocytosisM6P-ReceptorERTforGaucherAchievedbyeditingthesugarresidueoftheglycoprotein.Themodifiedproteindirectlytargetingtomacrophagethroughmannosereceptor(甘露糖受体）ofcellsurface.Benefits：Liver,spleensizereducedEnrichedhemoglobinImprovesskeletalconditionAcceleratesgrowthEnzymereplacementExtracellularaugmentationofanintracellularenzyme（细胞外补充）Example:adenosinedeaminase(腺苷脱氨酶)deficiencyanaccumulationofdeoxyadenosinetoxictoimmaturelymphocytescausesabuildupofdATPinallcells,whichinhibitsribonucleotidereductaseandpreventsDNAsynthesistheimmunesystemisseverelycompromisedorcompletelylackingAdenosinedeaminasedeficiencyTreatmenttoADAChemicallymodifiedADA:PEG-ADA(聚乙二醇-ADA)injectionAdvantages:lessantibodyresponseRemainsextracellularLongerhafl-life代谢系统遗传病的治疗Themostsuccessfulapproach.Inhibition（抑制）Depletion（清除）TypeofmetabolicinterventionSubstanceDiseaseDietaryrestriction（饮食控制）PhenylalanineGalactosePhenylketonuriaGalactosemiaReplacement（替代）BiotineEnzymeReplacementtherapyBiotinidasedeficiencyLysosomalstoragediseasesDiversion（转化）SodiumBenzoateUreacycledefectPhenylketonuria（PKU）

amodelfor

dieteticandpharmacologictherapyanditsconsequences苯丙酮尿症的治疗Hyperphenylalaninemia高苯丙氨酸血症ClassificationHyperphenylalaninemiaclassification.ClassicalPKU:PAH(苯丙氨酸羟化酶)-PAH活性:0-1%, PHE耐受:200-350mg.AtypicPKU:-PAH活性:1-3%, PHE耐受:350-850mg.ModerateHyperphenylalaninemia-PAH活性:3-5%, PHE耐受:>850mg~400mutationsworldwidehttp://www.pahdb.mcgill.caRationaleforaPhe-restricteddietControlofnaturalproteinintakeaccordingtoPHEtolerance-Avoidanceofhighproteinfood(milk,dairyproducts,meat,fish,chicken,eggs,beansandnuts,...Phenylalaninefreeformula,vitamins,oligoelementsLowproteinfood(bread,pasta,...)NocontroloffoodwithoutproteinPhe-restricteddietlimited+aminoacidsmixtureGalactosemia半乳糖血症Autosomalrecessive1/40000birthGalactose-1-phosphate-uridyl-transferase(GALT半乳糖1磷酸转尿苷酰酶)deficiency新生儿期发病Urgentdiagnostic-UrgenttreatmentPronosticreservedClassicalgalactosemia半乳糖醇GalactosemiaFirstsymptoms,firstweeksoflifeGastrointestinalproblemsHepaticinsufficiency,hepatomegaly-E.ColiinfectionDiagnostic:spottest-enzymologyonerythrocytesUrgenttreatment:removaloflactoseandgalactosefromdiet-rapidrecoveryDiversion转化治疗Ureacycledefect尿素循环障碍Anureacycledisorderorureacycledefectisageneticdisordercausedbyadeficiencyofoneoftheenzymesintheureacyclewhichisresponsibleforremovingammoniafromthebloodstream.Normally,theureaistransferredintotheurineandremovedfromthebody.nitrogenaccumulatesintheformofammonia,ahighlytoxicsubstance,andisnotremovedfromthebody.Ureacycledefect苯甲酸钠马尿酸盐Depletion（清除）Example–Hemochromatosis血色素沉着causesthebodytoabsorbandstoretoomuchiron.

Theextraironbuildsupinthebody’sorgansanddamagesthem.Withouttreatment,thediseasecancausetheliver,heart,andpancreastofail.Healthypeopleusuallyabsorbabout10percentoftheironcontainedinthefoodtheyeat,whichmeetsnormaldietaryrequirements.Peoplewithhemochromatosisabsorbupto30percentofiron.Overtime,theyabsorbandretainbetweenfiveto20timesmoreironthanthebodyneeds.Depletion（清除）Phlebotomy(放血)isaprocedurethatremovesbloodfromthebodyinaprocesssimilartodonatingblood.Phlebotomyisthepreferredmethodoftreatingmostformsofhemochromatosis.Mostpeoplewithhemochromatosisneedregularphlebotomythroughouttheirlives.DoctorswillmonitortheserumferritinlevelstomakesuretreatmentisloweringironstoresDepletionEx.2familialhypercholesterolemia家族性高胆固醇血症FamilialhypercholesterolemiaFamilialhypercholesterolemiaWhenthecholesterolloadisdecreasedbydivertingittoothercompoundsorbyremovingitwithphysicalmethods,thelivertriestocompensateforthedecreasedcholesterolintakebyup-regulatingcholesterolsynthesis.Cholesterolsynthesisinliver甲羟戊酸Inhibition（抑制）Thepharmacologicalinhibitionofenzymesissometimesusedtomodifythemetabolicabnormalitiesofinbornerrors.Example:familialhypercholesterolemiaDrug:statin(抑制素)Inhibits3-hydroxy-3-methylgutarylcoenzymeA(HMG-CoA,3-羟基-3-甲基戊二烯辅酶A)

reductase.TreatingFamilialhypercholesterolemiaThemoleculartreatmentofdisease蛋白质层面EnhancementProteinaugmentationEnzymereplacement基因表达层面Increase/reducegeneexpressionModificationcellsbytransplantationStemcell/organtransplantationGenetherapyEnhancementofmutantproteinfunctionwithsmallmoleculeThebiochemicalabnormalitiesofanumberofmetabolicdiseasesmayrespond,sometimesdramatically,totheadministrationoflargeamountsofthevitamincofactoroftheenzymeimpairedbythemutation.Vitamin-responsiveInborn

ErrorsofMetabolismHomocystinuria(高胱氨酸尿)AfamilyhistoryofhomocystinuriaFlush

acrossthecheeksMusculoskeletalTall,thinbuild(resembling

Marfanoidhabitus)Longlimbs(dolichostenomelia)High-archedfeet(pescavus)Knock-knees(genuvalgum)Pectusexcavatum

and

PectuscarinatumMentalretardationSeizuresPsychiatricdiseaseEyeanomalies:ectopialentis

(downwarddislocation)or

subluxation

of

lensMyopia

(Nearsightedness)GlaucomaOpticatrophyVasculardiseaseextensive

atheroma

formationatyoungagewhichaffectsmanyarteriesbutnotthecoronaryarteriesIntravascular

thrombosisB650%ofpatientsresponsivetoB6

Homocystinuriadueto

cystathionine胱硫醚synthasedeficiencyIncreasegeneexpression

fromtheWTormutantlocusIncreasingtheamountofmessengerRNAtranscribedfromtheWTlocusassociatedwithadominantdiseaseorformamutantlocus,ifthemutantproteinretainssomefunction.Hereditaryangioedema(血管神经性水肿)ADdeficiencyoftheC1inhibitorC1inhibitorisneededtocontrolthecoagulationcascadeinbloodclottingHereditaryangioedemaTypes:TypeI:Low[C1inhibitor]TypeII:Normal[C1inhibitor],butdefectedsymptoms:laryngealedema(喉头水肿)Treatment:DanazolIncreasestheabundanceoftheC1inhibitormRNASeriousattackreducedSideeffectinlong-termuseIncreasegeneexpression

fromalocusnotaffectedIncreasetheexpressionofanormalgenethatcompensatesfortheeffectofmutationatanotherlocus.Extremelypromisinginthemanagementofsicklecelldiseaseandbeta-thalassemia.Drug:decitabine(地西他滨)Inhibitsmethylationofγ-globinProducemoreHbFReducingtheexpression

dominantmutantgeneApproach:RNAinterference(RNAi)CanbeusedtodegradeaspecifictargetRNA.ShortdesignedRNA,onceintroducedinthecell,itwillbindtotargetRNAandinitiatedegradation.Stillinearlystage.IntrialwithHuntingtonDiseaseRNAinterferenceModificationofthesomaticgenomebytransplantationIndication:CellsororgansmaybetransplantedtointroduceWTcopiesofageneintoapatientwithmutationsinthatgene.Forcellreplacement,tocompensateforanorgandamagedbygeneticdisease.StemcelltransplantationFirstsuccessfultransplants—late1960s30,000-40,000transplantsperformedyearlyworldwide>20,000patientshavesurvived>5yearsStemcelltransplantationcandividethroughmitosisanddifferentiateintodiversespecializedcelltypesandcanselfrenewtoproducemorestemcellsSources:ClonedFromhumandonorsAllogeneic:fromanotherpersonSyngeneic:fromanidenticaltwinAutologous:fromthepatientNucleartransplantationTransferofadiploidnucleusfromanadultdonorsomaticcell,intoanoocytecytoplasm.NewtechnologyProblems:EthicalissuesHighlyaberrantUnabletocorrectgenomeRiskinintroducingmitochondrialdiseasesTherapeuticcloning:theuseofembryonicstemcellsgeneratedbynucleartransplantation.NoimmunerejectionStemcellfromhumandonorsType:HematopoieticSource:bonemarrow,placentalcorebloodCancerorLysosomalstoragediseaseCornealRegeneratingcornealepitheliumothers:embryoticHematopoieticstemcellscharacterisedbythepresenceofCD34SeenintheumbilicalcordandfetalliverHaveahighercloningefficiencyandgeneratesmoreprogenitorsthanadultbonemarrow.Theyhaveahugecompetitiveengraftmentadvantagerelativetotheadultbonemarrow.FetalliverisnowusedtotreatfetuseshavingX-LinkedSCID.HematopoieticstemcellsTheyareusedinProvidingafunctionalimmunesysteminapersonwithSCID.Replacingadefectivebloodsystemwithafunctionalonewhohasnonmalignantgeneticdisorderlikesicklecellanaemiaandthallasemia.Restoringthehaematopoieticsystemincancerpatientsaftertreatment.OtherfetalcellsMesenchymalstemcells(间充质细胞）-differentiatetobone,fatandcartilageliketheadultcounterpart.Neuralstemcelltheydifferentiateintoneurons,astrocytesandoligodendrocytes.TheyarethemainsourceofcellsfordegenerativeCNSinjuryforreplacement.StemcelltransplantationcantreatHistocyticdisordersInheritederythrocyteabnormalitiesInheritedimmunesystemdisorderslikeataxiatelangectesia,DiGeorgesyndrome,SCIDetcPlasmacelldisorderInheriteddisorderslikeLeschNyhansyndrome,betaThallesemiaetcInheritedplateletabnormalitiesInheritedmetabolicdisorderslikeMucopolysaccharidosis,Hurler’ssyndrome,Krabbedisease,Niemann-pickdiseaseetc处于试验中的病种有：CardiacdiseaseDiabetesMultipleSclerosisMuscularDystrophyParkinson’sdiseaseSpinalcordinjuryStroke干细胞移植的并发症-短期EarlyGraftRejectionHostversusgraftDruginjurytomarrowViralinfections:CMV,HHV-6&8InterstitialPneumonitisDiffusealveolarhemorrhageToofewdonorstemcellsARDSoftencausedbyCMV干细胞移植的并发症-长期SecondaryTumorsAcuteleukemias,solidtumors,MDSMonthstoyearsaftertransplantIncreasedincidence