bb基因診斷治療

基因診斷與基因治療

GeneDiagnosisandGeneTherapyLaboratoryofMolecularVirology,InstituteofVirology,WuhanUniversitySchoolofMedicine1/31/20241第一節(jié)基因診斷〔genediagnosis〕一、定義：Definitionofgenediagnosis基因診斷：用分子生物學(xué)技術(shù)對導(dǎo)致疾病的緣由進(jìn)展病原學(xué)和細(xì)胞遺傳基因的檢測分析,從而對相應(yīng)疾病進(jìn)展診斷。Geneticdiagnosisisatestusingmolecularbiologytechnologiestoseekthecausesofdiseasesthroughanalysisofpathogensandgenes.1/31/20242基因診斷的靶分子是基因。Thetargetsofgenediagnosisaregenes1/31/20243傳統(tǒng)的疾病診斷：Conventionaldiagnosticapproaches

細(xì)胞形狀、數(shù)量celluarmorphology&celluarquantity生化反響、酶活性biochemicalreaction,activitiesofenzymes免疫學(xué)檢驗(yàn)Immunologictests

1/31/20244檢測表型或形狀學(xué)改動detectionofphenotypicormorphologicfeatures

檢測對象是基因的產(chǎn)物--蛋白質(zhì)、多肽。Detectionobjectsareproductsofgenes,suchasprotein,polypeptide

1/31/20245二、基因診斷常用的技術(shù)：Technologiesincommonuse

分子雜交技術(shù)(Nucleicacidmolecularhybridization)PCR技術(shù)(Polymerase-Chain-Reaction)3.以上二者的衍生技術(shù)〔包括DNA芯片〕derivativetechnologiesfromabovetwotechnologies,DNA-Chips4.DNA序列測定DNAsequencing1/31/20246基因診斷獨(dú)特的優(yōu)點(diǎn)：Advantagesofgenetherapy

從分子程度上找到特異的致病基因Tofindpathogenicgenesatthemolecularlevels

具有很高的靈敏度Highsensitivity1/31/20247獲得穩(wěn)定的結(jié)果StableResults核酸比蛋白質(zhì)穩(wěn)定Nucleicacidismorestablethanthatofprotein.快速、早期診斷技術(shù)Speedy&early甚至在未出現(xiàn)病癥的情況下早期診斷運(yùn)用廣、適用性強(qiáng)Wideapplicability1/31/20248三、基因診斷戰(zhàn)略Strategiesforgenetherapy〔一〕經(jīng)過致病基因的檢測診斷疾病Diagnosisofdiseasesviapathogenicgenes

1/31/20249基因的構(gòu)造清楚、突變點(diǎn)明確、致病機(jī)制根本清楚。Structure,mutationandpathogenicmechanismofpathogenicgenesareclearlyunderstood.1/31/2024101/31/2024111)單基因遺傳病eg.monogeneticorsingle-genedisease鐮狀細(xì)胞貧血Sicklecellanemia→珠蛋白基因globingeneCodon6GAG→GTGA-T突變(mutation)谷氨酸(glutamic)→纈氨酸(valine)1/31/2024122)、Gene缺失遺傳病的診斷1)α地貧的Gene診斷α基因不同程度的缺失引起不同類型的α地貧，α基因缺失1～4個(gè)。正常Gene組用BamHI切割，可得到14kb的片段，而缺失一個(gè)αGene時(shí)可得到10kb片段。BamHIBamHIα2α1α210kb14kbprobe1/31/202413以αGene為探針，Southernblot方法檢測αα/αααα/α-αα/--α-/----/--正常缺1缺2缺3缺414kb10kb1/31/2024143)DMD/BMD的缺失診斷

〔進(jìn)展性肌營養(yǎng)不良或假肥大型〕DMD是一種嚴(yán)重致死性疾病〔XR〕，臨床病癥表現(xiàn)肌肉進(jìn)展性萎縮和乏力。BMD臨床病癥與DMD類似，但病癥較輕。Gene定位Xp21，Gene2300kb,79個(gè)Exon，cDNA全長74bp編碼3685Aa，分子量427kD。DMD/BMD70%左右為缺失型，基因很大，缺失能夠發(fā)生在不同的部位。運(yùn)用多重PCR擴(kuò)增檢測，判別缺失形狀。1/31/202415〔二〕經(jīng)過連鎖遺傳標(biāo)志檢測診斷疾病Diagnosisvialinkagegeneticmarker

基因構(gòu)造、突變情況不清楚，但知定位在染色體的特定位置上。Chromosomelocationofpathologicgenesisclear,butthestructureandmutationofthegenesareunclear.

1/31/202416同一染色體上位置非常接近的基因或其他遺傳標(biāo)志,經(jīng)常一同遺傳,構(gòu)成連鎖〔linkage〕.Thegenesorothergeneticmarkerslocatednearatthesamechromosomecanbeinheritedtogether,andtoformlinkage.1/31/202417例如：詳細(xì)基因不明的遺傳病Hereditarydiseasecausedbyunidentifiedgenes用內(nèi)切酶切正常母親DNA，雜交后出現(xiàn)7.6kb帶；同酶同方法檢查有遺傳病的父親，發(fā)現(xiàn)6.0和7.6kb兩種片斷，闡明6.0kb與基因病有連鎖關(guān)系。這樣當(dāng)胎兒羊水或胚胎絨毛細(xì)胞同樣處置后，僅出現(xiàn)7.6kb片段，闡明胎兒正常，假好像時(shí)有6.0和7.6時(shí)，闡明胎兒攜帶有父親的遺傳病基因。

1/31/2024181、Gene異常不明確的遺傳病的診斷例：成年型多囊腎病——APKD，AD，臨床表現(xiàn)為腰痛，蛋白尿，血尿，高血壓，腎盂性腎炎，腎結(jié)石。最終導(dǎo)致腎功能衰竭和尿毒癥。APKD——Gene定位16p13，但致病基因尚未克隆，基因產(chǎn)物的生化性質(zhì)和疾病發(fā)病機(jī)理也尚未闡明，但已證明APKDGene與α珠蛋白基因3`端附近的一段小衛(wèi)星DNA序列〔3`HVR〕嚴(yán)密連鎖，因此，可以經(jīng)過RFLP連鎖分析進(jìn)展診斷。1/31/202419以3`HVR為probe與PvuII酶切后的家系有關(guān)成員的Gene組DNA雜交——SouthernBlot雜交Gene組DNAprobe〔3`HVR〕PvuII酶切

DNA片段

雜交結(jié)果1/31/202420英國女王維多利亞。她帶有血友病的基因，并將其傳給了她的兒女。血友病是一種遺傳性凝血妨礙疾病，患者能夠因很小的傷口而出血不止導(dǎo)致死亡。血友病在女性普通表現(xiàn)為隱性遺傳，較少發(fā)病，但會傳給后代；在男性那么表現(xiàn)為顯性遺傳，顯示出病癥。維多利亞女王在科堡主持的一次歐洲王室成員集會，與會者有17人是她的后裔，其中有她的孫女亞歷山德拉〔21〕，她同俄國沙皇尼古拉二世結(jié)婚，導(dǎo)致他們的兒子患有血友病。1/31/202421PCR—RFLP診斷甲型血友病的基因診斷，知甲型血友病XR，獲得家系成員基因組DNA。PCR142bpBcLI

142bp99bp43bp電泳結(jié)果分析？p1p2142bp99bp43bpBclIBclI-BclI+1/31/202422問:1/Ⅱ2、Ⅲ1診斷；2/Ⅲ1是男或是女發(fā)??？

142bp99bp12123ⅠⅡIII11/31/202423結(jié)果分析1〕先癥者II2具有99bp片段而發(fā)病，該片段來自母親。2〕II2有142bp/99bp雜合子。142bp來自父親，為正常片段，99bp片段是來自母親而成為攜帶者。3〕Ⅲ1為99bp片段假設(shè)是男性為患者〔99bp片段來自母親〕；女性為攜帶者〔來自父母雙方〕1/31/202424連鎖分析(geneticlinkageanalysis)就是在家系成員中尋覓患病成員共同具有的比正常成員出現(xiàn)頻率更高的染色體片段.Thesearchforcertainchromosomefragmentsthatarefoundinpeoplewithadisorderaremorefrequentlythanyouwouldexpectinpeoplewithoutthedisorder.1/31/202425在這個(gè)區(qū)域內(nèi)具有疾病傾向的基因Thegenewithsusceptibilitytodiseaseislocatedintheregion.1/31/202426遺傳標(biāo)志選擇Geneticmarkerselection：多標(biāo)志原那么Mult-markerprinciple接近原那么Convergenceprinciple1/31/202427不是尋覓DNA缺陷(DNAdeficiency),而是經(jīng)過分析DNA遺傳標(biāo)志多態(tài)性來判別被檢者能否具有帶致病基因的染色體,判別被檢者患病的能夠性Itisananalysisofpolymorphismofgeneticmarkertodecidewhetherthechromosomeiscarryingthepathogenicgene,butnotasearchforDNAdeficiency.1/31/202428〔三〕經(jīng)過基因表達(dá)的定量分析診斷疾病Diagnosisofdisordersviaquantitativeanalysisofgeneexpression基因表達(dá)程度的變化,可引起相應(yīng)蛋白質(zhì)的含量變化。variationingeneexpressionlevelsisrelatedtovariationinitsproteinlevels

1/31/202429定量PCR技術(shù)可用于基因診斷，也可用于治療效果監(jiān)控。Applicationofreal-timepolymerasechainreactiontogenediagnosisandmonitoringoutcomeoftherapy1/31/2024301/31/202431四、基因診斷存在的問題及前景Problemsandprospectsinthegenediagnosis1、實(shí)際問題theoryproblems.1/31/202432已找到的致病基因不多，弄清機(jī)制的就更少。基因診斷失去了根底。Littleisknownaboutpathogenicgenes,andlessisknownaboutthemechanism.Genediagnosislosesitsfoundation1/31/2024332、技術(shù)問題technicalproblems對設(shè)備及條件要求高、所需試劑價(jià)錢昂貴。highrequirementsforequipmentsandconditions&highexpenseforessentialregents1/31/202434技術(shù)復(fù)雜、精細(xì)，對操作人員的要求高。Thevarietyandcomplexityoftechnologyhasalsogrownandweneedaprofessionthatdealswiththissituation1/31/202435技術(shù)靈敏度高，很容易遭到污染而出現(xiàn)假陽性。Falsepositiveresultsmaybeobtainedbycontaminationduetothehighsensitivityoftechniques.1/31/2024363、倫理問題ethicalconcerns能夠引起基因歧視〔Itmaycauseageneticdiscriminationbygenediagnosis〕

1/31/2024371/31/202438基因診斷的開展前景Futureprospectsforgenediagnosis

呈現(xiàn)非常寬廣的運(yùn)用前景，開展?jié)摿甏?。genediagnosishasawideapplicationandagreatpotentialinfuture.1/31/202439五、已進(jìn)展基因診斷的遺傳病Inheriteddisordersdetectedbygenediagnosis〔一〕血紅蛋白病〔hypochromia〕鐮狀細(xì)胞貧血〔sicklecellanemia〕→珠蛋白基因globingeneCodon6GAG→GTGA-T突變谷氨酸(Ghe)→纈氨酸〔vol〕1/31/202440α〔β〕地中海貧血α〔β〕MediterraneanSeaanemiaα〔β〕珠蛋白基因缺失，珠蛋白mRNA含量減少，珠蛋白α〔β〕鏈合成不平衡。Defectinα〔β〕globin,andreductionofglobinmRNAproduction,whichleadstoanimbalanceinα:βchainratio.1/31/202441診斷Diagnosis：PCR-ASO〔allelespecificoligonucleotide,ASO)1/31/202442定性分析(qualitativeanalysis)：PCR定量分析(quantitativeanalysis)：實(shí)時(shí)定量RT-PCRReal-timequantitativePCRassay1/31/202443杜氏肌營養(yǎng)不良〔Duchennemusculardystrophy,DMD)---性連鎖隱性遺傳病〔Sex-LinkedRecessiveInheritance〕，抗肌萎縮蛋白〔dystrophin〕基因突變所致DuchenneMuscularDystrophyiscausedbythegenemutationofdystrophin.1/31/202444苯丙酮尿癥〔phenylketonuria，ＰＫＵ〕－－常染色體隱性遺傳氨基酸代謝病autosomalrecessivegeneticdisordersofaromaticaminoacidmetabolism苯丙氨酸羥化酶基因突變phenylalaninehydroxylasegenemutation丙氨酸羥化酶缺乏phenylalaninehydroxylasedeficiency1/31/202445脆性Ｘ綜合癥fragileXsyndromeFMR-1基因的微衛(wèi)星序列〔CGG〕n改動，導(dǎo)致功能變異。ThemutationinCGGrepeatofFMR1geneleadstoalteredfunction.1/31/202446可經(jīng)過檢測串聯(lián)反復(fù)序列的長度來進(jìn)展。Wecanexaminethediseasebydetectingthelengthoftandemrepeatsequences.1/31/202447基因診斷技術(shù)檢測腫瘤Therolesofgenediagnosistechnologyindetectionofcancer１〕腫瘤的早期診斷和預(yù)警診斷Earlydiagnosis&earlywarningoftumours1/31/202448２〕高?！惨赘小橙巳汉Y查及危險(xiǎn)性評價(jià)Screenforhighriskorsusceptiblegroup&RiskAssessment1/31/202449３〕腫瘤鑒別診斷、分期、預(yù)后判別Distinctivelydiagnosis,stagingandprognosis４〕微小轉(zhuǎn)移灶識別Detectionofmicrometastases1/31/202450５〕治療效果監(jiān)測及評價(jià)Assessment&surveillanceoftherapyeffects1/31/202451對不同腫瘤、不同目的，采取不同的基因診斷戰(zhàn)略

Differentstrategiesfordifferenttumorcells,differentpurposes1/31/202452腫瘤基因診斷主要戰(zhàn)略有Mainstrategiesforidentifyinggenesinvolvedintumours檢查腫瘤染色體易位及交融基因Detectionofchromosometranslocation&fusiongenes1/31/202453檢查癌基因與抗癌基因Detectionofoncogenes&tumorsuppressorgenes1/31/202454檢測腫瘤相關(guān)基因Detectionoftumor-relatedgene檢測腫瘤標(biāo)志物或mＲＮＡ等DetectionofmarkersandmRNAoftumor1/31/202455基因診斷技術(shù)檢測感染性疾病Genetherapytechnologiesinthediagnosisofinfectiousdiseases細(xì)菌、病毒、寄生蟲感染(infectionofbacteria,virusandparasite〕1/31/2024561/31/202457第二節(jié)基因治療〔genetherapy〕是現(xiàn)代分子生物學(xué)技術(shù)與醫(yī)學(xué)學(xué)科交叉浸透而構(gòu)成的一個(gè)全新治療領(lǐng)域。Genetherapyisanewcross-discipline,whichcoverstechnologiesinmodernmolecularbiologyandmedicalscience.1/31/2024581/31/202459基因治療早期是將目的基因?qū)氚屑?xì)胞內(nèi)，成為宿主細(xì)胞遺傳物質(zhì)的一部分，Earlypurposeofgenetherapyistomaketargetgenesbecomeapermanentpartofthehostcell'sgeneticmaterialbyinsertingtheirgeneticmaterialintothechromosomesofhostcells.1/31/202460目的基因的表達(dá)產(chǎn)物對疾病起治療作用。Therapeuticeffectofexpressionproductsofgenesofinterestondiseases.1/31/202461如今:將正?；?qū)氩∽兗?xì)胞中，替代或與缺陷基因共存，產(chǎn)生正常表達(dá)產(chǎn)物補(bǔ)充缺陷蛋白質(zhì)Delivernormalcopyofageneintopathologicalcellstosupplementtheproteinthatarenotexpressingenough.1/31/202462抑制細(xì)胞內(nèi)過度表達(dá)的基因；Delivernucleicacidthatwillinactivateanover-expresseddeleteriousprotein.1/31/202463將特定基因?qū)敕遣∽兗?xì)胞，在體內(nèi)表達(dá)特定產(chǎn)物；andtransfergivengeneintonormalcellstoletitexpressgivengeneproducts.1/31/202464向功能異常細(xì)胞中導(dǎo)入該細(xì)胞中本來不存在的基因，利用表達(dá)產(chǎn)物到達(dá)治療疾病的目的。addafunctionalcopyofthetherapeuticgenetocellswithdysfunctionthatoriginallydonotexpressit.

利用基因產(chǎn)生治療效應(yīng)。andmakeuseofgenestoproducethetreatmenteffect1/31/202465第一節(jié)基因治療戰(zhàn)略

Strategiesforgenetherapy基因置換〔genereplacement〕或基因矯正〔genecorrection〕－－將特定目的基因?qū)胩囟?xì)胞置換原有的缺陷基因。Anormalcopyofagenemaybeintroducedintocellstoreplaceadefectivecopy.1/31/202466必要條件是：Essentialconditions對導(dǎo)入的基因及其產(chǎn)物有詳盡的了解.Fullunderstandingaboutthetransferredgeneanditsgeneproductisessential.能有效的導(dǎo)入靶細(xì)胞Theabilityofbeingtransferredefficientlyintotargetcells在靶細(xì)胞中長期穩(wěn)定存在Long-termandstablegeneexpressionintargetcellsisdesired.1/31/202467導(dǎo)入基因有適度程度的表達(dá)Keepingtransferredgeneexpressingatamoderatelevel.導(dǎo)入方法及載體對細(xì)胞無害Themethodsofgenetransferandvectorsmustbeharmlesstothetargetcells.1/31/202468普通采用同源重組技術(shù)〔homologousrecombination〕又稱基因打靶〔genetargeting〕Generallyadoptthistechniqueofhomologousrecombination,genetargeting.1/31/2024691/31/2024701/31/202471２.基因替代(genereplacement)、基因添加(geneaddition)、基因補(bǔ)充(genesupplement)、

.1/31/2024721)針對特定的缺陷基因,導(dǎo)入相應(yīng)的正?；?缺陷基因并未除去)Transferafunctionalcopyofthegeneintotargetcellsbyaimingataparticulardefectgene(andthedefectedgenecanbekept).2)導(dǎo)入靶細(xì)胞本來不表達(dá)的基因(將細(xì)胞因子導(dǎo)入腫瘤細(xì)胞)Transfertheinterestgeneintotargetcellsthatareoriginallynotexpressingthegeneproduct(bygettingcytokineintotumorcells)1/31/2024733.基因干涉(geneinterference)采用某些方式抑制某個(gè)基因的表達(dá)或經(jīng)過破壞某個(gè)基因的構(gòu)造使之不能表達(dá),到達(dá)治療目的.Toattainthetreatmentpurpose,geneinterferenceiseithertoextinguishexpressionfromthemutationalleleorbreakthestructureofacertaingene.1/31/202474三種戰(zhàn)略是以恢復(fù)細(xì)胞正常功能或干涉細(xì)胞功能為目的.Thepurposeofabovethreestrategiesistoresumethenormalfunctionorintervenethefunctionofcells.1/31/2024754.細(xì)胞自殺效應(yīng)Suicidegenetherapy

經(jīng)過導(dǎo)入自殺基因,誘發(fā)細(xì)胞自殺效應(yīng)也是重要的治療戰(zhàn)略.Inductionofsuicideeffectviadeliveryof“suicide〞genesintotargetcells,suchastumorcellsisalsoanimportanttreatmentstrategy

1/31/202476自殺基因是病毒、細(xì)菌等原核生物中具有特殊功能的酶類基因，此類基因轉(zhuǎn)入哺乳動物細(xì)胞后產(chǎn)生的酶能將無毒或毒性極低的藥物前體轉(zhuǎn)化成細(xì)胞毒性代謝產(chǎn)物，導(dǎo)致腫瘤細(xì)胞自殺，因此又稱為病毒導(dǎo)向酶解藥物前體療法(VDEPT).1/31/2024771/31/2024785、基因修飾(genemodification)也屬于基因添加，但目的不是修復(fù)、改善細(xì)胞功能，而是改動細(xì)胞功能。1/31/2024791〕基因修飾腫瘤細(xì)胞的“疫苗〞療法：將IL-2、TNF-導(dǎo)入腫瘤細(xì)胞，誘發(fā)抗腫瘤細(xì)胞毒性淋巴細(xì)胞反響；另分泌的細(xì)胞因子加強(qiáng)CTL和效應(yīng)細(xì)胞反響。1/31/2024802〕基因修飾TIL的過繼療法：將細(xì)胞因子導(dǎo)入腫瘤侵潤淋巴細(xì)胞中，使TIL具有更強(qiáng)的抗腫瘤作用1/31/2024813〕免疫加強(qiáng)基因療法：將MHC-I類基因?qū)肽[瘤細(xì)胞，添加其免疫原性。1/31/202482第二節(jié)基因轉(zhuǎn)移技術(shù)Genetransfertechnologies基因治療方式有兩種：Mannersofgenetherapy1〕體內(nèi)法〔invivo〕2〕回體法〔exvivo〕1/31/202483CellsremovedfrombodyTransgenedeliveredCellsculturedCellsreturnedtothebodyExVivoInVivoTransgenedelivereddirectlyintohostStrategiesforTransgeneDelivery1/31/202484invivo即將外源性功能基因以逆轉(zhuǎn)錄病毒、質(zhì)粒載體或脂質(zhì)體類包囊，或以裸露DNA的方式直接注入體內(nèi)，讓基因在體內(nèi)表達(dá)。Amethodoflettingaforeignfunctionalgeneexpressinvivobytransferofretrovirusvector,plasmidvector,liposomeorthedirectinjectionof'nude'DNA.

1/31/202485exviro即將受體細(xì)胞〔例如淋巴細(xì)胞、骨髓干細(xì)胞、皮膚或纖維細(xì)胞等〕在體外培育導(dǎo)入外源性功能基因，然后把這種經(jīng)遺傳修飾的受體細(xì)胞輸回或移植入患者體內(nèi)，讓外源基因表達(dá)。Amethodoflettingaforeignfunctionalgeneexpressbytransferringthegeneintotargetcells(eg.Lymphocytes,hematopoieticstemcells,skinorfibercells,etc.)culturedexviro,andthentransfusingortransplantingthegeneticmodifiedcellsintothepatientbody.

1/31/2024861/31/2024871/31/202488基因治療必需經(jīng)過適當(dāng)?shù)幕蜣D(zhuǎn)移(genetransfer)技術(shù),將外源基因轉(zhuǎn)移至特定細(xì)胞內(nèi).Genetherapyreliesintheconceptthat“foreign〞genesmaybetransfertotargetcellsusingappropriategenetransfertechnologies1/31/202489基因轉(zhuǎn)移技術(shù)有兩大類Methodsofgenedelivery:生物學(xué)方法Biologicalmethods(病毒介導(dǎo)的基因轉(zhuǎn)移系統(tǒng)Viralmethods)非生物學(xué)方法Non-Biologicalmethods(物理或化學(xué)方法PhysicalorChemicalmethods)1/31/202490逆轉(zhuǎn)錄病毒載體(retrovirusvector)是基因治療中最常用的載體,轉(zhuǎn)移效率較高.Themostcommonvirusvectorsthatareusedingenetherapyareretrovirusvectors一、基因轉(zhuǎn)移生物學(xué)方法Biologicmethodsofgenetransfer1/31/2024911/31/202492基因組是二倍體ss+RNAThegenomeisdiploid.類似真核mRNA，similarinstructuretoeucaryoticmRNA5`端帽狀構(gòu)造，5`cappingstructure3`端多聚A尾,3`poly-Atail1/31/202493前病毒基因組構(gòu)造特點(diǎn)：Charactersofprovirusgenome1)兩端各有一長末端反復(fù)序列LTR〔由U3、R、和U5組成〕，LTR中含有啟動子、加強(qiáng)子、加尾信號。Alongterminalrepeat(LTR)ofendogenousretrovirusislocatedateitherendofgenome(composeofU3,RandU5).LTRcontainstheelementsincludingpromoter,enhancerandtailingsignal.1/31/202494

是病毒整合進(jìn)細(xì)胞基因組的過程的關(guān)鍵構(gòu)造。Itisthekeystructurefortheintegrationofviralgenomeintothechromosomeofhostcells,formingtheprovirus.

1/31/2024952〕有三個(gè)構(gòu)造基因:gag、plo和envItcontains3structuregenes,gag,ploandenv

分別編碼中心蛋白、逆轉(zhuǎn)錄酶和外殼〔包膜〕糖蛋白,encodingcoreprotein,reversetranscriptaseandenvelopeglycoprotein,respectively.1/31/202496

1/31/2024973〕5‘端LTR下游有病毒包裝所必需的序列、引物結(jié)合位點(diǎn)等。Thereareasequencerequiredforefficientpackagingofvirusandaregionofprimerbindingsiteatthedownstreamof5’LTR.1/31/202498retrovirusvector1/31/202499用于基因治療的逆轉(zhuǎn)錄病毒載體系統(tǒng)由兩部分組成：Retroviralvectorsystemthatcanbeusedforgenetherapyisconstitutedbytwoparts.將前病毒DNA中編碼蛋白的基因切除，Cutoffthegenegenesencodingviralproteins,保管順式元件、包裝信號,andkeepcis-actingelementsandpackagingsignals插入質(zhì)粒toinsertaplasmid----逆轉(zhuǎn)錄病毒載體formingaretroviralvector.1/31/2024100包裝細(xì)胞〔packagingcell〕是將缺失了包裝信號及相關(guān)序列的缺陷型逆轉(zhuǎn)錄病毒〔輔助病毒〕導(dǎo)入哺乳動物細(xì)胞而制備成的特殊細(xì)胞。Packagingcellsareakindofspecialcelllinesthatcanenabledefectiveretrovirus(helpervirus)thatlackpackagingsignalsandcorrelationsequencetotransferintomammalcells.1/31/2024101將上兩部分結(jié)合運(yùn)用即可產(chǎn)生攜帶治療基因，而只需一次感染才干的病毒顆粒------假病毒顆粒〔pseudoviralparticles〕Combineabovetwoparttoformpseudoviralparticlesthatcarrytherapygeneandcaninfect(ortransduce)targetcellsonlyonce.1/31/2024102假病毒感染效率非常高Highefficiencyoftransfection.用于體內(nèi)基因治療時(shí)，只能感染增殖期細(xì)胞，有一定局限性。Thelimitedabilityofonlyinfectingdividingcellswhenusedingenetherapy.1/31/2024103逆轉(zhuǎn)錄病毒載體(retrovirusvectors)腺病毒載體〔adenovirus,Advectors〕腺相關(guān)病毒〔adenoassociatedvirus，AAvvectors〕載體單純皰疹病毒〔herpessimpexvirusvectors〕載體TypesOfViruses1/31/2024104NakedDNATargetCellTherapeuticProteinAAVRetrovirus/LentivirusAdenovirusNucleusGeneTherapyPrinciples1/31/20241051/31/20241061/31/2024107二、基因轉(zhuǎn)移非生物學(xué)方法脂質(zhì)體〔liposome〕介導(dǎo)基因轉(zhuǎn)移利用陽離子脂質(zhì)單體與DNA混合，可自動構(gòu)成包埋外源DNA的脂質(zhì)體，再經(jīng)過細(xì)胞的內(nèi)吞作用〔endocytosis〕將外源DNA轉(zhuǎn)移至細(xì)胞內(nèi)。Liposome,positivelychargedlipiddroplets,caninteractwithnegativelychargedforeignDNAtowrapitupanddelivertocellsviaendocytosis.1/31/20241082.細(xì)胞外表受體介導(dǎo)的基因轉(zhuǎn)移Genetransferviacellsurfacereceptors

受體介導(dǎo)的細(xì)胞內(nèi)吞作用是轉(zhuǎn)移特異性外源物質(zhì)進(jìn)入細(xì)胞的一種方式。Receptor-mediatedendocytosisisoneofmannersoftransferringforeignmatterintocells.1/31/2024109細(xì)胞膜上存在一些專注性受體，與相應(yīng)的配體結(jié)合后，經(jīng)過細(xì)胞的內(nèi)吞作用，實(shí)現(xiàn)配體向細(xì)胞內(nèi)轉(zhuǎn)移。Thespecificreceptorsofcellmembranecancombinewithcorrespondingligands,andtransfertheligandsintocellsviaendocytosis.1/31/20241101/31/2024111將外源DNA與已證明有細(xì)胞或組織親和性的配體偶聯(lián)，可使DNA具有靶向性。Thetarget-specificactionofgenedeliverycanbeincreasedbycouplingligandswithhighcellortissueaffinitywithforeignDNA..1/31/2024112偶聯(lián)通常用多聚陽離子(condensedcation)，如多聚賴氨酸(polylysine)來實(shí)現(xiàn)。Couplingcanberealizedbycondensedcation,suchaspolylysine1/31/2024113如：僅在肝細(xì)胞外表的去唾液酸糖蛋白〔ASGP〕受體，它的天然配體是ASGP。帶有這種配體的DNA復(fù)和物可被定向送入肝細(xì)胞。Asialoglycoproteinreceptor(ASGP—R)isonlyexpressonthesurfaceofhepatocytes,anditsnaturalligandisasialoglycoprotein.DNAcomplexcarryingtheligandcanbetransferreddirectionallyintohepatocytes.1/31/20241143.直接注射進(jìn)展基因轉(zhuǎn)移(DNAinjection)–直接將裸DNA注入動物或器官組織內(nèi)?！甆ude’DNAisdirectlyinjectedintoanimalsortissues.

1/31/2024115外源基因在肌肉中的表達(dá)量與注入的DNA含量成正比；TheamountofforeigngeneexpressionsinmuscleisindirectratiotothatofDNAdeliveredbyintramuscularinjection.反復(fù)注射比一次注射效果好。Theeffectofrepeatedinjectionismoreeffectivethanthatofsingleinjection.1/31/20241161/31/2024117優(yōu)點(diǎn)：Advantages制備簡單easytohandle排除病毒致癌的潛在危險(xiǎn)withoutcarcinogenicriskofvirus1/31/2024118不需整合即可表達(dá)expressionwithoutneedingintegration可反復(fù)運(yùn)用andcanbeadministeredagainandagain.1/31/2024119三、基因轉(zhuǎn)移的靶細(xì)胞

Targetcellsusedingenetransfer目前條件下，基因治療僅限于體細(xì)胞〔嚴(yán)禁進(jìn)展生殖細(xì)胞的基因治療操作〕Allcurrenthumangenetherapyapproachesaretargetedtosomaticcellsnotgerm-linecells.1/31/20241201/31/2024121Somaticgenetherapy1/31/2024122選擇轉(zhuǎn)移基因的靶細(xì)胞，要思索幾點(diǎn)：Howtoselecttargetcellsforgenetransfer?1〕最好是組織特異性細(xì)胞Tissuespecificcellsarebetter.2〕細(xì)胞較易獲得，Itiseasytoobtain.3)生命周期較長Lifecycleislong.

1/31/20241234〕易受外源物質(zhì)轉(zhuǎn)化Itiseasytobetransformbyforeignfactors5〕轉(zhuǎn)染后細(xì)胞仍易成活I(lǐng)tiseasytosurviveaftertransfection.

1/31/2024124目前已開展的研討的靶細(xì)胞有：Targetcellsinpresentstudies造血干細(xì)胞(hematopoieticstemcells)、淋巴細(xì)胞(lymphocytes)、皮膚成纖維細(xì)胞(skinfibroblasts)、肝細(xì)胞(hepatocytes)、血管內(nèi)皮細(xì)胞(vascularendothelialcells,)、肌細(xì)胞(muscularcells,)、神經(jīng)元和神經(jīng)膠質(zhì)細(xì)胞(neurons&glialcells)、腫瘤細(xì)胞(tumourcells)等andsoon.1/31/2024125exvivoretroviralvectors(oncoretroviralorlentiviral)invivoadenoviralvectorsAAVvectorslentiviralvectors1/31/2024126第三節(jié)基因治療的運(yùn)用

applicationsofgenetherapy1990年基因治療的第一個(gè)病例是先天性腺苷脫氨酶(ADA)缺乏癥Thefirstdiseaseapprovedfortreatmentwithgenetherapywasadenosinedeaminase(ADA)deficiencyin1990.

1/31/2024127

美國醫(yī)學(xué)家W·F·安德森等人對腺甘脫氨酶缺乏癥〔ADA缺乏癥〕的基因治療，是世界上第一個(gè)基因治療勝利的范例。1990年9月14日，安德森對一例患ADA缺乏癥的4歲女孩謝德爾進(jìn)展基因治療。這個(gè)4歲女孩由于遺傳基因有缺陷，本身不能消費(fèi)ADA，先天性免疫功能不全，只能生活在無菌的隔離帳里。他們將含有這個(gè)女孩本人的白血球的溶液輸入她左臂的一條靜脈血管中，這種白血球都曾經(jīng)過改造，有缺陷的基因曾經(jīng)被安康的基因所替代。在以后的10個(gè)月內(nèi)她又接受了7次這樣的治療，同時(shí)也接受酶治療。經(jīng)治療后，免疫功能日趨健全，可以走出隔離帳，過上了正常人的生活。

謝德爾，19991/31/2024128一、對遺傳病進(jìn)展基因治療Genetherapyofhumangeneticdisease研討范圍非常有限，須符合以下條件：Therangeofresearchisverylimited,whichmustmatchthefollowingconditions.1〕在DNA程度上明確發(fā)病緣由及機(jī)制TodefinethecausesandmechanismsofdiseasesattheDNAlevel.1/31/20241292〕必需是單基因遺傳病Itmustbeamonogeneticorsingle－genedisease.1/31/20241303〕表達(dá)不需求準(zhǔn)確調(diào)控Accurateregulationofgeneexpressionisnotessential.4〕該遺傳病不經(jīng)治療有嚴(yán)重的后果Severeresultsmaybegotwithouttreatment.1/31/2024131二、對惡性腫瘤的基因治療CancerGeneTherapy較少涉及倫理問題，Lessethicsproblems臨床迫切需求，Itisneededurgentlyintheclinic.腫瘤的基因治療已走在前列。Thegenetreatmentoftumorshasalreadywalkedinadvance.1/31/2024132基因療法成為不少癌癥病人挽救生命的獨(dú)一選擇。Theonlychoiceistosavecancerpatients.1/31/2024133治療戰(zhàn)略StrategiesofTherapy：導(dǎo)入抑癌基因以抑制癌的開展、轉(zhuǎn)移Transferoftumorsuppressorgenestoinhibittumorgrowthandmetastasis.1/31/2024134干擾癌基因的轉(zhuǎn)錄和翻譯，抑制癌基因活性Interferewithtranscriptionortranslationofoncogeneinordertoinhibitoncogeneactivity1/31/2024135對瘤組織進(jìn)展細(xì)胞因子修飾，加強(qiáng)腫瘤細(xì)胞的免疫原性Modifycytokinegeneexpressionintumortissuebytransferringgenestoenhanceimmuneresponse.導(dǎo)入自殺基因，殺傷癌細(xì)胞Transferofsuicidegenestocausedeathofcancercells.1/31/2024136三、病毒病的基因治療GeneTherapyofvirusdisease抗病毒基因治療非常引人注目。包括調(diào)理機(jī)體免疫功能和針對病毒本身兩個(gè)方面。Theanti-virusgenetherapyisveryarrestive.Regulationofimmunologicalfunctionanddirecttreatmentofvirusaretwowaysofanti-virusgenetherapy.1/31/2024137前者主要是將細(xì)胞因子〔干擾素、白介素〕的基因?qū)朊庖呒?xì)胞，誘導(dǎo)機(jī)體對病毒的細(xì)胞和體液免疫應(yīng)對。Transductionofcytokinegeneseg.interferonandinterleukintoinduceshumoralandcellularimmuneresponsesagainstvirus.1/31/2024138后者Thelatter：1〕誘導(dǎo)對病毒RNA的降解inducedegradationofviralRNA2〕抑制病毒與宿主細(xì)胞結(jié)合inhibitvirustocontactwithhostcells3〕干擾病毒基因組的轉(zhuǎn)錄起始interferethetranscriptioninitiationofviralgenome.1/31/2024139抑制病毒復(fù)制和蛋白合成inhibitviralreplicationandprotein

synthesisRNA干擾(RNAinterference,RNAi)1/31/2024140.干擾RNA，RNAi哺乳動物中的研討1999年才開場被以為是最有出路的核酸藥物<Science>雜志將這一新發(fā)現(xiàn)評為2002年度世界十大科學(xué)成就之首.1/31/2024141In1990,RNAsilencingwasfirstobservedinplants.Thisphenomenawascalled“cosuppression〞or“PTGS〞(posttranscriptionalgenesilencing).PlantCell,vanderKroletal.1990,2(4):291-299ThehistoryofsiRNA1/31/2024142a起始階段：由于RNA病毒感染、轉(zhuǎn)座子轉(zhuǎn)錄和外源導(dǎo)入基因表達(dá)等緣由,生物體細(xì)胞內(nèi)出現(xiàn)雙鏈RNA(dsRNA),dsRNA在一種具有RNaseⅢ樣活性的dsRNA特異性核酸內(nèi)切酶(dsRNAspecificendonuclease,Dicer)作用下,被裂解成由正義和反義鏈組成的21～23nt的干擾性小RNA(smallinterferingRNA,或shortinterferingRNAs,siRNA).1/31/2024143b效應(yīng)階段：目前以為該階段存在兩種假說方式,一種假說方式是以為siRNA作為導(dǎo)游序列,與RNA誘導(dǎo)的沉默復(fù)合物(RNAinducedsilencingcomplex,RISC)的無活性前體結(jié)合,在ATP參與下siRNA雙鏈構(gòu)造解旋誘導(dǎo)構(gòu)成有活性的RISC,活性RISC隨即與同源性靶向mRNA結(jié)合,在與siRNA反義鏈配對區(qū)域的中部切斷同源性靶向mRNA,使其降解,從而干擾基因表達(dá),這稱為內(nèi)源性核苷酸裂解(endonucleolyticcleavage)1/31/2024144另一種假說方式為隨機(jī)降解性多聚酶鏈反響(randomdegradativePCR),即siRNA作為針對靶向mRNA特殊引物,在RNA依賴性RNA多聚酶(RNAdependentRNApolymerase,RdRP)的作用下以靶向mRNA為模板合成靶向mRNA的互補(bǔ)鏈,使單鏈mRNA成為dsRNA,然后在Dicer酶的作用下被降解構(gòu)成新的siRNA,新的siRNA又進(jìn)入上述過程,反復(fù)循環(huán)，從而構(gòu)成信號擴(kuò)增系統(tǒng).1/31/2024145MechanismofShortinterferingRNAs—post-transcriptionalgenesilencing.1/31/2024146四、其他疾病的基因治療Genetherapyofotherdisorders人神經(jīng)退化疾病humanneurodegenerativediseases〔如帕金森氏病Parkinson'sdisease)1/31/2024147Morethan1.5millionAmericanssufferfromParkinson'sdisease.Thereisnocure.Butnowthereisanewtreatmentthatmayimprovepatients'qualityoflife.1/31/20241