基因轉(zhuǎn)移與基因治療

基因轉(zhuǎn)移與基因治療復(fù)旦大學(xué)遺傳學(xué)研究所薛京倫2003，101醫(yī)學(xué)ppt基因：染色體上的DNA片段，是遺傳信息結(jié)構(gòu)和功能的基本單位?；驔Q定生老病死控制高矮胖瘦影響喜怒哀樂基因組：某一生物的細(xì)胞中所帶有的全部遺傳信息。2醫(yī)學(xué)ppt基因與疾病基因與人類的疾病密切相關(guān)：遺傳病是由于基因先天缺陷所致;腫瘤的發(fā)生涉及多種基因改變,包括癌基因激活或抑癌基因失活;高血壓,糖尿病等多基因病也涉及到多種基因的改變;由病原體所致的傳染病也和人體基因密切相關(guān),存在易感人群和耐受人群.3醫(yī)學(xué)ppt

一、基因治療概念

基因治療(GeneTherapy)指將正?；蚧蛴兄委熥饔玫幕蛲ㄟ^一定方式導(dǎo)入靶細(xì)胞,糾正基因的缺陷或者發(fā)揮治療作用，從而達(dá)到治療疾病目的的生物醫(yī)學(xué)高技術(shù)。

4醫(yī)學(xué)pptTheapplicationofgeneticprinciplesinthetreatmentofhumandiseaseByintroductionofgenetic

materialintotargetcellsinordertocounteracttheeffectofadiseasegeneorintroduceanewfunctionSomaticandGermlineapproachespossible5醫(yī)學(xué)ppt基因治療的必要條件UnderstandingofthediseaseprocessStructure/functionofgenetobeintroducedEfficientdeliveryofgenecontrolofgeneexpressionPrevention/controlofimmuneresponsesAnimalmodelandassessmentoffunctionClinicaltrial6醫(yī)學(xué)ppt研究基因治療的三個基本步驟

尋找適當(dāng)?shù)陌屑?xì)胞

導(dǎo)入目的基因

基因表達(dá)7醫(yī)學(xué)ppt體細(xì)胞基因治療始終需要考慮的問題

基因轉(zhuǎn)移的效率

治療的特異性

基因表達(dá)的持久性及其調(diào)節(jié)

治療的毒副作用

什么疾病

什么基因

什么載體

什么靶器官和靶細(xì)胞

什么基因?qū)敕椒?醫(yī)學(xué)ppt體細(xì)胞基因治療 將基因作為一種特殊“藥物”，通過體細(xì)胞基因轉(zhuǎn)移治療疾病 慢性治療 急性治療 預(yù)防 遺傳性疾病 獲得性疾病 功能喪失 功能獲得9醫(yī)學(xué)ppt二、基因治療和傳統(tǒng)的基因工程的區(qū)別

二者都著眼于尋找可治病或有其他應(yīng)用價值的“目的基因”?；蚬こ蹋耗康幕颉d體——導(dǎo)入大腸桿菌、酵母和哺乳動物細(xì)胞——體外表達(dá)所需要的蛋白——經(jīng)過分離純化獲得能用于治療或其他用途的蛋白純品,最終是制造出一種蛋白類的藥物。10醫(yī)學(xué)ppt基因治療——目的基因——載體——導(dǎo)入人體，目的基因在人體內(nèi)的細(xì)胞中制造所需要的蛋白——達(dá)到治病的目的?；蛑委熢诩夹g(shù)上一旦成功,其優(yōu)勢：①制品為基因及其載體，非基因表達(dá)蛋白產(chǎn)物,不需復(fù)雜的蛋白產(chǎn)物分離和純化工藝②生產(chǎn)成本遠(yuǎn)遠(yuǎn)低于基因工程產(chǎn)品③從理論上講,凡能治病的基因，都有可能開發(fā)成為“藥物”④半衰期11醫(yī)學(xué)ppt但基因治療難度高，技術(shù)要求極為苛刻。例如，針對各種疾病，必須具有能夠達(dá)到治病目的的基因。在此基礎(chǔ)上，還必須具有能有效地將基因?qū)肴梭w的載體系統(tǒng)，這種系統(tǒng)要求高效，而且能定向地導(dǎo)入人體某種細(xì)胞?；?qū)肴梭w后，必須能夠控制它的表達(dá)。12醫(yī)學(xué)ppt因此，基因治療是生物高技術(shù)的高度集成，是遺傳學(xué)、分子生物學(xué)、細(xì)胞生物學(xué)、分子病毒學(xué)等多種學(xué)科知識和技術(shù)的高度綜合。

13醫(yī)學(xué)ppt三、基因治療的主要策略

GenereplacementGeneaugmentationtherapy(GAT)Genecorrection(Chimeraplasty)TargetedkillingofspecificcellsTargetedinhibitionofgeneexpression(Geneablation)14醫(yī)學(xué)pptGenereplacement:

Deficientgenecorrectedbyreplacingthemutatedallelewithanintactallele;usedforthetreatmentofautosomaldominantdisorders.Thestrategiesareasfollows:a)kockoutmutationbysinglecrossing-over(insertionalinactivation)

b)genereplacementbydouble(reciprocal)crossing-over

c)targetgeneinhibitionindominantnegativegeneticdisorders;usuallymutatedproteinswhichinteractwithnormalcellularproteinstocreatealteredproperties;mutationalinactivationoftheonemutatedcopywillalleviatethismutantphenotype;themutationcanbecarriedoutbyinsertionalinactivation,orantisensetechnology.15醫(yī)學(xué)pptGeneAugmentationTherapy(GAT)

FordiseasescausedbylossofgenefunctionmorecopiesofnormalgeneraiselevelsofgeneproductrestorenormalphenotypeApplyto:monogenicrecessivediseasescysticfibrosis,haemophilia,musculardystrophy16醫(yī)學(xué)pptGeneCorrection-Chimeraplasty17醫(yī)學(xué)ppttumourcelltkthymidinekinasegeneviralvectorganciclovirganciclovirphosphateTargetedKilling–GeneticPro-drugActivationTherapy18醫(yī)學(xué)pptTargetedinhibitionofgeneexpression

Ribozymes

-cancleave(orrepair)mRNATriplehelixoligonucleotides-blockgenetranscription

Antisense

oligos-blockmRNAtranslation19醫(yī)學(xué)pptAllgenetherapystrategiesdependongettingthegeneorgeneticmaterialintothetargetcells!!!

20醫(yī)學(xué)ppt四、基因轉(zhuǎn)移的主要方法：

若干相關(guān)術(shù)語：

Transfection轉(zhuǎn)染

：過去：將病毒DNA或RNA

轉(zhuǎn)入細(xì)胞。

現(xiàn)在：將外源DNA轉(zhuǎn)入動物細(xì)胞。

DNATransfer轉(zhuǎn)移：基因?qū)牖蜣D(zhuǎn)移廣義的概念。

Transduction轉(zhuǎn)導(dǎo)：噬菌體介導(dǎo)的細(xì)菌之間的基因

轉(zhuǎn)移。

Transformation轉(zhuǎn)化：將裸DNA或質(zhì)粒導(dǎo)入原核

和真核細(xì)胞。21醫(yī)學(xué)pptTwomainroutesofgenetransfer:Invivo: i.v.ori.m.injectable;or non-invasive(eg“sniffable”)Exvivo: hepatocytes,skinfibroblasts haematopoietic cells “bioreactors”22醫(yī)學(xué)pptaaaaaaEx-vivoIn-vivotopicaldeliveryIn-vivosystemicdeliveryVExamples:-bonemarrow-livercells-skincellsExamples:-brain-muscle-eye-joints-tumorsExamples:-intravenous-intra-arterial-intra-peritonealTHREEclassesofanatomicalgenedelivery23醫(yī)學(xué)ppt基因治療過程中的基因轉(zhuǎn)移方法

Viralvectors

Non-viralvectors

Physicalmethods

目前最有效的是病毒載體，但存在插入大小有限、免疫原性強(qiáng)和生產(chǎn)難等局限24醫(yī)學(xué)ppt1、病毒載體類型反轉(zhuǎn)錄病毒（RV）載體腺病毒（AV）載體腺相關(guān)病毒（AAV）載體單純皰疹病毒（HSV）載體慢病毒（lentivirus)載體等25醫(yī)學(xué)ppt對載體的基本要求：特異并有效的基因轉(zhuǎn)移特異、高效、持續(xù)性表達(dá)，具有可控性免疫原性低易于生產(chǎn)26醫(yī)學(xué)ppt載體特征：

reproducibility stablepropagated purifiedtohightiters mediatedtargeteddelivery27醫(yī)學(xué)pptAdvantageanddisadvantageofgene-transfervector(1)

Vector AdvantageDisadvantage

AVVeryhightransfectionexvivorepeatdosingineffectiveowingto

&invivostrongimmuneresponse

Transfectsproliferating&non-Insert-sizelimit0f7.5kb

proliferatingcells,substantialmanufacture,storage,QCare

clinicalexperienceacquiredmoderatelydifficult

efficientretargetedtransfctionshoutdurationofexpression

RVfairlyprolongedexpressionlowertransfectionefficiencyinvivo

hightransfectionefficiencyexvivoinsert-sizelimitof8kb

substantialclinicalexperienceexvivotransfectonlyproliferatingcells,safty

lowimmunogenicityconcernofinsertionalmutagenesis

Lentivirustransfectsproliferating&non-safetyconcernfromimmunodeficiency

proliferating,haematopoieticvirusorigins,manufacturing,storage,

stemcellsQCareextremelydifficult,insert-size

limitof8kb,noclinicalexperience

AAVefficientlytransfectsawidevarietyinsert-sizelimits4.5kb

0fcellsinvivo,veryprolongedmanufacture,QCareverydifficult

expressioninvivolittleclinicalexperience,safetyconcern

lowimmunogenicityofinsertionalmutagenesis,repeat

dosingaffectedlybyneutralizing

antibodyresponses28醫(yī)學(xué)ppt基因治療病毒載體比較

RVAVAAVHSVLV基因組成ssRNAdsDNAssDNAdsDNAdsRNA基因組長度10kb36kb5kb152kb10kb裝載容量<8kb8kb4.5kb30kb9kb病毒滴度107

1011

108

108108感染細(xì)胞譜窄寬寬窄寬感染能力中很強(qiáng)強(qiáng)強(qiáng)中整合能力有無有無強(qiáng)毒性作用遺傳毒細(xì)胞毒遺傳毒細(xì)胞毒遺傳毒免疫原性弱中等弱強(qiáng)弱29醫(yī)學(xué)pptAdvantageanddisadvantageofgene-transfervector(2)

Vector AdvantageDisadvantage

NakedDNAmanufacturing,storage,QCveryshortdurationofexpressionin

aresimpleandcheap,verymosttissues,veryineffecient

lowimmunogenicity,clinicaltransfectionexvivoandinvivo

limbischaemia,goodsafetyretargetingtransfectionvery

profiledifficult

Cationiclipidsrelativesimplemanufacturing,inefficienttransfectioninvivo

storage,QCefficienttransfectionveryshortdurationofexpression

exvivo,lowimmunogenicitylittleclinicalexperience

condensedrelativesimplemanufacturing,inefficienttransfectioninvivo

DNAparticlesstorage,QC.Efficienttrancfectionveryshortdurationofexpression

exvivo,lowimmunogenicitynowclinicexperience

goodsafetyprofile

retargetedtransfectiondemostraded30醫(yī)學(xué)pptaaaaaaTransfectionInfectionexposedto106particles/cell12hoursexposedto1particle/cell30minvirallymediatedgenetransferismillionsoftimesmoreefficentthannonviraltransfer(whencalculatedintermsoftransfer/particle)TransfectionversusInfection31醫(yī)學(xué)pptVectorusedingenetherapyTypeapproximateintegrationdurationofinsert-sizecapacitypersistenceNucleic-acidbased

Oligonucleotides:Decoys,antisense,Ribozomes,siRNA10-100kbnohours-daysExpressionplasmids2-10kbextremelyraredaysTransposons2-10kbefficientstablerelativerandomBacteriohageintegrase2-10kbefficient,siterestrictedstableArtificialchromosome50-300kbepisomal+/-stableVirusbasedRV2-6kbefficientstablerelativerandomLentiviral2-10kbefficientstablerelativerandomAAV2-5kbraremonths-yearsAV2-30kbNOweeks-monthsHerpesvirus2-40kbepisomalmoths-yearsEBvirus2-40kbepisomalmoths-yearsSV401-5kbmoderateunproven32醫(yī)學(xué)ppt腺病毒載體：雙鏈DNA，在基因轉(zhuǎn)移中的應(yīng)用相當(dāng)廣泛。其優(yōu)點是易制備、高滴度、宿主廣、能感染非增殖細(xì)胞，低毒，不整合入機(jī)體細(xì)胞染色體，容量達(dá)36kb，但免疫原性強(qiáng)，表達(dá)時間短。

33醫(yī)學(xué)ppt改進(jìn)的腺病毒載體1.早期AVE1+E3區(qū)缺失的有限性2.E2,E4區(qū)的置換:Wilson報道:溫度敏感E2突變AV降低免疫性,表達(dá)延長;未根本解決；制備293/E2A,293/E4細(xì)胞;相應(yīng)載體;擴(kuò)大容量;降低毒性和免疫性;效果有限;滴度下降.293T細(xì)胞:轉(zhuǎn)染SV40大T抗原34醫(yī)學(xué)ppt降低腺病毒免疫原性

腺病毒載體插入B7反義基因,阻斷共刺激途徑;腺病毒載體插入ICAM-1反義基因,阻斷粘附因子作用.腺病毒載體插入CTLA4基因,胞內(nèi)結(jié)合B7.

腺病毒載體插入MHC-I,II基因的調(diào)控基因反義片段.35醫(yī)學(xué)ppt增強(qiáng)腺病毒的感染效率腺病毒與葡聚糖,聚凝胺,多聚賴氨酸,脂質(zhì)體混合感染細(xì)胞,組織,提高感染效率10-100倍,降低炎癥反應(yīng).重組腺病毒感染前,進(jìn)行腺病毒封閉不同亞型腺病毒互換應(yīng)用.36醫(yī)學(xué)pptaaaaaaEfficiency+++PersistenceSpecificityToxicity++RecombinantAdenovirusesApproachesGenerationIGenerationIIIHybridadenos:Adeno-RVAdeno-AAVAdeno-Transposase

Advantages/Limitations8KbcapacityGenerationI

>30KbcapacityGenerationIII

Adenocanbegrownatveryhightiters,

HoweverDonotintegrateCancontainRCAsAretoxic/immunogenicExamplesOTCdeficiency(clin,---)CysticFibrosis(clin,---)Oncolyticviruses(clin,+++)37醫(yī)學(xué)ppt腺病毒載體研究進(jìn)展與展望進(jìn)展展望Ad載體用于治療性的angiogenesis降低毒性的方法將繼續(xù)獲得進(jìn)展,取得進(jìn)展最終將提供安全的Ad載體系統(tǒng).Ad引起的毒性問題得到進(jìn)一步認(rèn)識靶向方法的應(yīng)用將進(jìn)一步改進(jìn).提高Ad載體的靶向性，改善療效缺陷型的Ad載體生產(chǎn)將不斷進(jìn)展.新一代Ad載體能降低毒性，改善表達(dá)將面臨再次注射的挑戰(zhàn)38醫(yī)學(xué)ppt反轉(zhuǎn)錄病毒載體基因轉(zhuǎn)移系統(tǒng)輔助細(xì)胞:

將含有病毒結(jié)構(gòu)基因但缺失了順式包裝信號的缺陷型反轉(zhuǎn)錄病毒導(dǎo)入哺乳動物細(xì)胞（ψ—2，PA317，ψCRIP）?？僧a(chǎn)生病毒包裝蛋白但不產(chǎn)生病毒顆粒；反轉(zhuǎn)錄病毒載體:以外源目的基因替換病毒結(jié)構(gòu)基因,含病毒包裝信號輔助細(xì)胞提供載體包裝蛋白，使后者產(chǎn)生含外源目的基因的病毒顆粒39醫(yī)學(xué)ppt

缺陷型的反轉(zhuǎn)錄病毒顆粒的RNA進(jìn)入靶細(xì)胞后，變成前病毒并整合到宿主細(xì)胞的染色體上，可穩(wěn)定表達(dá)外源基因。反轉(zhuǎn)錄病毒只能感染處于增殖期的細(xì)胞。40醫(yī)學(xué)pptaaaaaaPersistenceSpecificityEfficiencyToxicityApproachesMurineRetrovirusesVSV-pseudotypedRVLentivirusesSelf-inactivatingRVCombinationvirusesAdvantages/Limitations9Kbcapacity+integrationthroughtranspositionalsoinquiescentcells(HIV),permitinprinciplelong-termtreatments,howeverdisturbedby:InsertionalmutagenesisGenesilencingHighmutationrateLowtiterofproductionExamplesSCID(IL2Rdefect,Paris)(clin,+++)AdenosineDeaminasedeficiency(clin,+++!!!)Parkinson(preclin,+++)Anticancer(clin+/-)RecombinantRetroviruses(includesHIV-based)41醫(yī)學(xué)ppt

反轉(zhuǎn)錄病毒安全性問題病毒感染的可能性病毒在靶細(xì)胞基因組整合可導(dǎo)致:

破壞細(xì)胞正常生長必需的抑癌基因

LTR激活原癌基因染色體重排激活原癌基因42醫(yī)學(xué)ppt腺相關(guān)病毒（AAV）載體：4.7kb單鏈DNA，結(jié)構(gòu)為ITR-rep-cap-ITR，病毒基因組簡單，易于消除，可降低細(xì)胞毒性和T-淋巴細(xì)胞反應(yīng)的危險性；病毒DNA可在人19號染色體上進(jìn)行位點特異性整合，需要輔助因子出現(xiàn)才復(fù)制；其Rep蛋白可能介導(dǎo)這種定向整合宿主范圍寬，易感染造血干細(xì)胞，能潛伏感染非分裂期細(xì)胞；在動物模型中表達(dá)可持續(xù)半年以上。但AAV載體接納的外源DNA小于4.5kb，載體整合效率較低，制備困難43醫(yī)學(xué)pptaaaaaaEfficiencyPersistenceSpecificityToxicityRecombinantadeno-associated-virus(AAV)ApproachesHelper-dependentproductionHelperindependentproductionCis-complementingvectorsCo-infectionAdvantages/LimitationsPersistenceinthegenomepermitslong-termexpression,hightitersareeasilyobtained,immunogenicityisverylow,HoweverSmallcapacity(<4.5kb)whichdoesnotallowtoaccommodatelargegenesorgeneclusters.ExamplesHemophiliaB(clin,animal,+++)Gaucher(clin,animal,+++)BrainIschemia(animal,+++)Cysticfibrosis(animal,+/-)44醫(yī)學(xué)pptLentivirus（慢病毒載體）優(yōu)點:感染非分裂細(xì)胞，有RV優(yōu)勢結(jié)構(gòu):LTR-gag-pol--env--LTR

TARtat,rev,tev應(yīng)用:宿主廣泛;艾滋病基因治療問題:安全性45醫(yī)學(xué)pptHSV病毒雙鏈DNA,包括:HSV-1,2,VZV,EB,CMV.

主要以HSV-1型為主.HSV載體含包裝信號,復(fù)制位點等順序結(jié)構(gòu),HSV病毒啟動子,目的基因表達(dá)框架.由輔助病毒共轉(zhuǎn)染輔助細(xì)胞M64A,制備重組病毒.

輔助病毒的改造:IE3溫度敏感突變型;

缺失突變型:IE3缺失;

應(yīng)用:感染心肌細(xì)胞,神經(jīng)元,神經(jīng)膠質(zhì)細(xì)胞.VEGF---血管形成;腦腫瘤;帕金森病

46醫(yī)學(xué)pptaaaaaaRecap:currentlimitationsofpopularvectorsAdenovirus-nopersistence-limitedpackagingtoxicity,immunogenicityRetrovirus(incl.HIV)-limitedpackaging-randominsertion-unstablegenomeGeneral-antibodyresponse-limitedpackaging-genesilencingSolutions:-syntheticviruses(“Virosomes”)Biolisticbombardmentorlocaldirectinjection-limitedareaElectroporation-limitedorganaccessLiposomes,genecorrection&Co.-veryinefficienttransferGeneral-lowtransferefficiency-noorlittlegenomicintegrationSolutions:-improvedliposomeswithviralproperties(“Virosomes”)47醫(yī)學(xué)ppt非病毒載體除了基因轉(zhuǎn)移的效率比較低以外，比病毒載體具有更多的優(yōu)越性，將來會有viral-like,butartificialvectors.48醫(yī)學(xué)pptPharmacologicalconsiderationsforDNAtransfer

ClassicaldrugMW50-500DaltonsSyntheticallypreparedRapiddiffusion/actionOraldeliverypossibleCellulardelivery:

-actatcellsurface

-permeatecellmembrane

-importedthroughchannelsCanbedeliveredassolublemolecules?ngstrom/nmsizerapidlyreversibletreatment49醫(yī)學(xué)pptMw20,000-100,000DaBiologicallypreparedSlowerdiffusion/actionOraldeliverynotpossibleCellulardelivery

-actextracellularly

CanbedeliveredassolublemoleculesnmsizerapidlyreversibletreatmentProteindrug50醫(yī)學(xué)pptNucleicacidMwNx1,000,000DaBiologicallypreparedSlowdiffusionOraldeliveryinconceivableCellulardelivery:

-nomembranetranslocation

-nonucleartranslocation

-nobiologicalimportMustbedeliveredascomplexcarrierparticles50-200nmsizeslowlyornotreversible51醫(yī)學(xué)pptTherapywithnucleicacidsrequiresparticulatedformulationismuchmorecomplexthanpreviousdrugdeliverieshasadifferentdegreeofreversibility(dosageproblem)52醫(yī)學(xué)ppt2、非病毒及物理方法脂質(zhì)體介導(dǎo)法磷酸鈣轉(zhuǎn)染法機(jī)械法（顯微注射、基因槍等）DEAE-葡聚糖和polybrene轉(zhuǎn)染法電穿孔法超聲波輔助法納米材料等53醫(yī)學(xué)ppt中性脂質(zhì)體

由脂類形成的可高效包裝DNA的人造單層膜，其結(jié)構(gòu)和性質(zhì)與細(xì)胞膜極為相似，二者易于融合，細(xì)胞的內(nèi)吞作用使其進(jìn)入細(xì)胞，操作簡單，轉(zhuǎn)染效率可高達(dá)50%，可用于體內(nèi)試驗，但目的基因表達(dá)不穩(wěn)定54醫(yī)學(xué)ppt陽離子脂質(zhì)體：陽離子脂質(zhì)體在水中可形成大小約100-400nm單層脂質(zhì)體。其帶正電，帶負(fù)電的DNA可自動結(jié)合到帶正電的脂質(zhì)體上，形成DNA-陽離子脂質(zhì)體復(fù)合物，與細(xì)胞膜作用使DNA進(jìn)入細(xì)胞。在該系統(tǒng)體內(nèi)基因?qū)胄实?，且無靶向性。在體內(nèi)應(yīng)用，除腫瘤瘤內(nèi)注射外，其前景仍存在問題。55醫(yī)學(xué)ppt56醫(yī)學(xué)ppt磷酸鈣轉(zhuǎn)染法：將氯化鈣、目的DNA和磷酸緩沖液混合，沉淀形成含有DNA的極小的不溶性磷酸鈣顆粒。磷酸鈣-DNA復(fù)合物粘附到細(xì)胞膜，通過胞飲進(jìn)入受體細(xì)胞的細(xì)胞質(zhì)，轉(zhuǎn)染過程簡單有效，適用于貼壁、非貼壁細(xì)胞，轉(zhuǎn)染效率可達(dá)20%左右，但目的基因表達(dá)不穩(wěn)定57醫(yī)學(xué)ppt機(jī)械法

如顯微注射和基因槍（biolisticparticle）。顯微注射使用一根細(xì)針頭將外源DNA直接轉(zhuǎn)入受體細(xì)胞核?；驑屖褂酶邏篋NA分子導(dǎo)入細(xì)胞。

基因槍工作原理顯微注射58醫(yī)學(xué)pptaaaaaaSpecificityEfficiencyPersistenceToxicityApproachesAntisenseRibozymes/DNAzymesTriplehelixDecoy/competitorsGene-correctingoligosAdvantages/Limitationstheseproceduresmaybesuitablefor:handlingdominantdefectstransienttreatments(genemodulation)permanenttreatments(genecorrection)ExamplesAnticancer(clin,preclin.,+/-)Restenosis(clin,+++)MuscularDistrophy(animal,+++)Oligonucleotides59醫(yī)學(xué)ppt電穿孔法通過將細(xì)胞暴露在短暫的高場強(qiáng)電脈沖中轉(zhuǎn)導(dǎo)目的DNA。細(xì)胞懸浮液置于電場中會誘導(dǎo)沿細(xì)胞膜的電壓差異，這種電壓差異會導(dǎo)致細(xì)胞膜暫時穿孔。一般，成功的電穿孔過程都伴隨高水平（50%或更高）的毒性。60醫(yī)學(xué)pptDEAE-葡聚糖和polybrene轉(zhuǎn)染法

帶正電的DEAE-葡聚糖或polybrene多聚體復(fù)合物和帶負(fù)電的外源DNA分子作用，使得DNA可以結(jié)合在細(xì)胞表面。通過使用DMSO或甘油獲得的滲透休克將外源DNA導(dǎo)入。兩種試劑都已成功用于轉(zhuǎn)染。DEAE-葡聚糖僅限于瞬時轉(zhuǎn)染。61醫(yī)學(xué)pptaaaaaaSpecificityPersistenceEfficiencyToxicityApproachesNakedDNAinjection/biolisticNakedDNA+pressureNakedDNA+electroporationLiposomalformulationsCombinationsAdvantages/LimitationsUnlimitedsizecapacity+lowerimmunogenicityandlowerbio-riskofnonviralformulationsisdisturbedbyLowefficiencyofgenetransferEvenlowerstableintegrationExamplesCriticallimbIschemia(clin,+++)CardiacIschemia(clin,+/-)Vaccination(clin,+/-)Antirestenosis(preclin.+/-)Naked/complexedDNA62醫(yī)學(xué)pptaaaaaaIdealpropertiesofasystemicallydelivered

non-viralformulationStabilityparticleshouldresistseruminactivationparticleshouldbeinerttoimmuneinactivation

Addressabilityparticleshouldpossessavascularaddressingsignatureparticleshouldbearatissue-dockingspecificityDNAconstructshouldincludetissue-specificregulatoryelements

Efficiencycargoshouldbeprotectedfromcytoplasmicinactivation(ex.lysosomes)cargoshouldcontainnuclear-translocatingsignalsDNAcargoshouldincludegenome-integrationfunctionsDNAelementmustbeguaranteedtofunctionaftergenomicintegration(nosilencing)63醫(yī)學(xué)pptOtherpropertiesParticleshouldnotincludeimmunogenic/toxicsurfacescargoshouldnotencodeimmunogenic/toxicproductsCargoshouldincludeanti-apoptoticfunctions∴

severalindependentproblemsmustbesolvedforanonviralformulationtobesuitableforclinicaltreatmentandforindustrialproductionmostviralvectorsincludemany,ifnotallthoseproperties64醫(yī)學(xué)ppt

裸DNA基因轉(zhuǎn)移和治療進(jìn)展 展望裸DNA肌肉注射，活體轉(zhuǎn)移 非病毒基因轉(zhuǎn)移越來越重要電擊促進(jìn)質(zhì)粒DNA進(jìn)入肌肉皮膚 未來幾年遺傳病臨床試驗將首選血友病血管內(nèi)導(dǎo)入質(zhì)粒DNA，肝細(xì)胞基因轉(zhuǎn)移 Duchenne肌營養(yǎng)不良，關(guān)節(jié)炎等尾靜脈導(dǎo)入pDNA，簡單有效，轉(zhuǎn)染大、嚙齒類的尾靜脈注射作為快速檢測表小鼠肝細(xì)胞達(dá)狀況和基因治療方法廣泛應(yīng)用pDNA表達(dá)載體可高水平持續(xù)表達(dá) 血管內(nèi)裸DNA導(dǎo)入為細(xì)胞有效涉入pDNA RNAi將在基因治療領(lǐng)域中成為重要手裸DNA導(dǎo)入法已經(jīng)在臨床研究（PAOD，段外周動脈閉鎖癥）SiRNA可通過血管內(nèi)基因轉(zhuǎn)移法有效導(dǎo)入Knockout小鼠65醫(yī)學(xué)pptaaaaaa∴problemsthatmustbesolvedtobesuitableforclinicaltreatmentandforindustrialproductionaredifferentbetweenviralandnon-viralvectorswhenignoringtheirlowefficiency,nonviralvectorsappearslargelysuperiorMostrelevantissuesinthetwomain'vectorology'sectors(viralversusnonviral)ViralvectorsPackagingcapacityfrom4to30kbproblemforsomelargegenes(ex.dystrophingeneorCFTRgene)importanttoxicload:ratioinfectious/non-infectiousparticlesfrom1/10to1/100strongimmunogenicity:capsidandenvelopeproteins,residualviralgenescontaminants:replication-competentviruses(ex.wildtyperevertantviruses)Viralamount(titre)obtainablewithrecombinants(ex.10exp5=poor,10exp10=excellent)Complexityofproduction(existenceornotofpackagingcellsystems)Emotionalproblemslinkedtopathogenicityofdonorvectors(ex.lentiviruses)NonviralvectorsPackagingcapacitynotanissue,evenverylargeconstructscanbeused(exampleentirelociupto150kb)minortoxicload:smallpercentageofnonrelevantadventitiousmaterialsmoderateimmunogenicity:methylationstatusofDNA(exampleCpGmotifs)contaminants:adventitiouspathogensfrompoorDNApurification(exendotoxins)AmountofDNAmoleculesisusuallynotaproblem,theothercomponentsdependsonchemicalsynthesisNoparticularcomplexity,exceptforspeciallyformulatedliposomesnoparticularemotionalproblemslinkedtothenatureofthereagents66醫(yī)學(xué)pptaaaaaaRandomintegratingvectorsr-lentivirusesr-retrovirusesr-AAVplasmids(lowfrequency)plasmids+transposase(eg'sleepingbeauty')

Transient,nonintegratingvectorsadenovirusplasmidRNAvirusbasedoligonucleotides(SiRNA,antisense,ribozymes)artificialchromosomes

Genecorrectionvectorschimeroplasts(RNA-DNAchimericoligos)singlestrandedDNA(homologousrecom)genotoxicnon-genotoxicSpecificallyintegratingvectorshybri