第六章高分子納米藥物-siRNA藥物病變組織的基因活性正常表達過度表達低表達小分子抑制劑?蛋白療法?抑制劑:RNA干擾療法,反義RNA,…下調(diào)上調(diào)增強表達:

質(zhì)粒DNAViralVectorsEmulateDrug?

抗體:Bindprotein,butonlyextracellularepitopesLowthroughput,highcost反義RNA/核酶:Stopgeneexpression(production),mostproteinsChemicalstabilizationessentialBarriers:sequenceselection,potency,toxicities,…

RNA干擾:Stopgeneexpression(production),mostproteinsGoodactivitywithoutchemicalstabilizationRapidlyreplacingantisense:reallybetter?特異性基因下調(diào)手段RNA干擾（RNAinterference,RNAi）技術(shù)功能：特異性地沉默內(nèi)源或外源性靶基因影響：2001年《Science》雜志十大科學(xué)成就之二 2002年《Science》雜志十大科學(xué)成就之首醫(yī)學(xué)領(lǐng)域的應(yīng)用：基因功能研究；基因敲除；基因治

療；基因表達調(diào)控等;

抗病毒以及糖尿病、肥胖癥和癌癥、 黃斑變性的眼科疾病治療等。RNAi作用機制Nature，2004，430：161Log[Oligo](M)NormalizedGeneExpressionLevelAntisensesiRNAControlsFromH.Winkler,IBCConference“RNAInterference”,SanDiego,CA,Feb2003siRNAdsRNAsiRNARNAseshRNAInVitroInVivoInVivomiRNAAdaptedFromDykxhoornetal.,NatureReviews4,457,2003具有RNAi機制的藥物TargetIDsiRNATargetValidationGrowingClinicalLeadDiscovery&OptimizationPreclinical藥物遞送是關(guān)鍵挑戰(zhàn)siRNA藥物?siRNA藥物:革命性突破

Company

siRNA

Disease

Routeofadministration

Stage

ALN-TTR01ATTRintravenousALN-RSVRSVinfectionIntranasalALN-VSPLiverCancerIntravenousQPI-1007NAIONIntravenousI5NPARFIntravenousPF-4523655AMDIntravitrealBevasiranibAMDIntravitrealDMEIntravitrealTKM-080301CancerIntravenousPRO-040201HypercholesterolemiaIntravenous

ATU027AdvancedSolidTumorsIntravenoussiG12DLODERPancreaticCancer

intratumoralTD101PCIntradermalsir027AMDIntravitreal

RTP801i-14AMDIntravitreal

CALAA-01SolidtumoursIntravenousPotentialofsiRNA-BasedTherapyPhase1Phase2Phase3RNA干擾療法的潛力siRNA藥物迅速進入臨床研究難以被細胞攝?。捍蠓肿?，陰離子脫靶效應(yīng)：非特異性沉默，引起免疫刺激等難以富集在腫瘤部位：體內(nèi)不穩(wěn)定，半衰期短

1

2

3PotentialofsiRNA-BasedTherapyRNA干擾療法的潛力siRNA藥物臨床引用的關(guān)鍵Nature2010,468,p487,Nov.25,2010PotentialofsiRNA-BasedTherapyRNA干擾療法的潛力siRNA給藥技術(shù)成為關(guān)鍵挑戰(zhàn)表達siRNA的質(zhì)粒DNA的傳遞病毒（NatureBiotechnology,2002,20:1006-10）非病毒類載體化學(xué)合成的siRNA的傳遞 非病毒類載體：脂質(zhì)體和高分子 物理手段:electroporation,highpressure/highvolumeinjectionPotentialofsiRNA-BasedTherapyRNA干擾療法的潛力siRNA的傳遞：體內(nèi)RNAi技術(shù)應(yīng)用的關(guān)鍵脂質(zhì)類Figure2.ChemicalmodificationsofsiRNAs.Shownarestructuresofsugar,backboneandbasemodificationsandofthecholesterolconjugate.NatueChemicalBiology,2(2006),711-719.Naturereviewsdrugdiscovery,

6(2007),443-453高分子納米載體抗體類化學(xué)修飾siRNAsiRNA綴合物PotentialofsiRNA-BasedTherapyRNA干擾療法的潛力siRNA的非病毒載體種類內(nèi)涵體逃避靶向結(jié)合吞噬核轉(zhuǎn)運DNA釋放到細胞質(zhì)形成RISC內(nèi)涵體逃避靶向結(jié)合吞噬siRNA釋放到細胞質(zhì)siRNA的傳遞DNA的傳遞組織轉(zhuǎn)運DAN和siRNA傳遞的比較蛋白質(zhì)表達轉(zhuǎn)錄mRNA化學(xué)修飾的siRNAFigure2.ChemicalmodificationsofsiRNAs.Shownarestructuresofsugar,backboneandbasemodificationsandofthecholesterolconjugate.NatueChemicalBiology,2(2006),711-719.Naturereviewsdrugdiscovery,

6(2007),443-453Nature，2004，432：173-178

化學(xué)修飾的siRNAChallenge1:穩(wěn)定Challenge2:長循環(huán)Challenge3:靶向Challenge4:滲透Challenge5:細胞攝取Challenge6:制劑化基于納米載體的siRNA給藥系統(tǒng)EngineeringNanoparticlesforsiRNADeliveryExtendedCirculationTissueTargetingPenetrationCellinternalization脂質(zhì)體類siRNA納米載體脂質(zhì)體類siRNA納米載體StableNucleicAcidLipidParticle(SNALP)StableNucleicAcidLipidParticle(SNALP)Meanparticlesize:140±12nmMeanencapsulationefficiency:93%±3%Nucleicacid/lipidratio:42±4

g/

molsiRNA-SNALPwithalongerhalf-lifeinplasmaandliverreducedserumHBVDNAtitersinmousemodelfollowingintravenousinjectionNatureBiotechnology

23,1002-1007(2005)ApoB-siRNA-SNALPresultedinsilencingofApoBmRNAexpressioninliverandsignificantreductionsinApoBprotein,serumcholesterolandlow-densitylipoproteinlevelsNature441,111-114(4May2006)高分子類siRNA給藥系統(tǒng)StericCoat(Inert)ActiveCoreExposedTargetingLigandsCleavableLink(Shedding)LigandsStericpolymerCarrierCleavablelinkage+++++++siRNAPayload水溶性陽離子高分子納米載體:NUCLEICACIDSRESEARCH32(19):Art.No.e1492004PNAS2007,104,5715-5721靶向修飾的水溶性陽離子高分子納米載體:CancerResearch2005,65,8984-8992LigandPEGDegradablelinkagesiRNAJACS,127,1624-1625靶向修飾的水溶性高分子-siRNA鍵合物:JACS,126(42),13612-13613LoadingsiRNApostnanoparticleformationPEG:prolongedbloodcirculationBiocompatibleDegradable,potentiallyintracellularsiRNAreleaseCo-deliveryofchemotherapeuticdrugforsynergisticeffect++++++++++++++++++++++++++++++siRNA親水嵌段疏水嵌段帶正電荷嵌段陽離子膠束/siRNA復(fù)合納米顆粒:abc30minFAM-siRNA12h24hRho-PTX30min12h24hRho-PTXMicelleplexFAM-siRNAFAM-siRNAPBSabcdeabcdeabcdeRho-PTXMicelleplexNCsiRNA+MicelleplexFAM-siRNAbcdeaSunTM,etal.,ACSNano,2011,5,1483-1494Dayspost-xenografting121620242832364044TumorVolume(mm3)100200300400500600PaclitaxelMicelleplexNCsiRNAPaclitaxelMicelleplexsiPlk1PaclitaxelMicelleplexNCsiRNA+MicelleplexsiPlk1MicelleplexsiPlk1PBSBlankMicellesMicelleplexNCsiRNASunTM,etal.,ACSNano,2011,5,1483-1494β-actin020406080100120Plk1mRNA(%ofControl)MicelleplexNCsiRNAPBSBlankmicellesMicelleplexsiPlk1PaxlitaxelMicelleplexPaxlitaxelMicelleplexsiPlk1PaxlitaxelMicelleplexNCsiRNA+MicelleplexsiPlk1Plk1KimA.Woodrow,W.MarkSaltzmanetal.,NatureMaterials,2009,8,526–533ChristopherJ.Cheng,

W.MarkSaltzman,Biomaterials,2011,32,6194–6203siRNA包載效率低下，僅為21%！1gsiRNA目前的價格為200萬！基于聚乙二醇