现代医药生物技术概论3/04/2012核酸技术及药物Email:zhang_张必良ProteinRNADNAtranscriptiontranslationCCTGAGCCAACTATTGATGAAPEPTIDECCUGAGCCAACUAUUGAUGAA传统中心法则传统中心法则:DNA RNA Protein中心法则之例外中心法则之例外DNAretroviruses use reverse transcriptaseto replicate their genome(David Baltimore and Howard Temin)RNA virusesmRNA introns(splicing)(Philip Sharp and Richard Roberts)RNA editing(deamination of cytosineto yield uracil in mRNA)RNA interference(RNAi)a mechanismof post-transcriptional gene silencing utilizing double-stranded RNA(Andrew Fire and Craig Mello)RNAs(ribozymes)can catalyze anenzymatic reaction(Thomas Cech and Sidney Altman)RNAProteinPrions are heritable proteins responsiblefor neurological infectious diseases(e.g.scrapie and mad cow)(Stanley Pruisner)Epigenetic marks,such as patterns ofDNA methylation,can be inherited andprovide information other than the DNAsequencemiRNA and piRNA etc.Nobel Prizes非编码RNA（ncRNAs）可以通过多种遗传机理调控DNA的结构、RNA的表达及蛋白质的翻译和功能，进而在细胞、组织或个体水平上影响生物体的正常生长发育。
RNA越来越为人们所重视，一个崭新的RNA时代已来临1957年Hoagland、Zamecnik 及Stephenson 等分离出tRNA?1965年Holley等测定了酵母丙氨酰tRNA的序列，获得1968年诺贝尔医学奖?1977年Roberts and Sharp 发现RNA splicing,荣获1993年诺贝尔医学奖?1981年中科院等完成了酵母tRNA-Ala 的人工全合成这是世界上首次人工合成的一个具有完整生物活力的RNA?1981年Cech等发现四膜虫rRNA的自我剪接，从而发现核酶（ribozyme）与Altman 荣获1989年诺贝尔化学奖?1993年Ambros 发现第一个miRNA(lin-4）?1998年Fire和Mello发现RNAi,获得2006年度诺贝尔医学奖?1999年Steitz等发现核糖体是核酶，获得2009年度诺贝尔化学奖?2000年Reinhart 等发现第二个miRNA(let-7）?2001年Tuschl 等发现siRNA(small interfering RNA)?2006年Tuschl 和Hannon等发现piRNA?2009年Rinn等发现LincRNARNARNA重大历史事件重大历史事件Overview of DNA to RNA to Protein?A gene is expressed in two steps1)Transcription:RNA synthesis2)Translation:Protein synthesis核糖核酸（RNA）?任何序列?多种多样 2结构和功能?与蛋白，代谢物和其它核酸结合?功能RNAs:Riboregulators：piRNAsmiRNAssiRNAsLincRNARiboswitchesRibozymes?调控水平:?转录?翻译LincRNA调控作用调控作用LincRNA是基因间的长链非编码RNA（large intergenic noncoding RNA，简称lincRNA）。
Part I:核酶（Ribozyme）Part II:适配体(Aptamer)和SELEX技术Part III:反义核酸（Antisense）Part IV:RNA干扰（RNA interference,RNAi)核酸技术及药物核酸技术及药物核酸技术及药物核酸技术及药物1.什么是核酶（RIBOZYME）?2.核酶有哪几类?3.什么是适配体（Aptamer）?4.SELEX的原理是什么?5.核酸药物现状?6.反义核酸治疗原理?7.Fire和Mello发现RNAi的原理是什么?8.RNAi的应用有哪些?9.RNA干扰（RNAi）技术作为治疗药物最大障碍是什么?Part I:Ribozymes(核酶)A brief HistoryHow many ribozyme?Why?Catalytic efficiency,condition3D structure of ribozyme:And a mechanism of catalysisApplication?History of enzymatic catalysis1877 W.Kuhne introdues the term“enzyme”1893 W.Ostwald shows that enzymes are catalysts1926 J.Summer crystallizes the enzyme urease and demonstrates that it is a protein.1977 P.Sharp discovers the splicing of introns.1982 T.Cech shows that the intron of one Tetrahymena rRNA is self splicing.He proposes the term“ribozyme”to refer to catalytic RNA1983 S.Altman demonstrates that the RNA moiety of RNAse P is sufficient for part of its activity.核酶核酶(Ribozyme)?由于这类酶具有类似核糖核酸酶功由于这类酶具有类似核糖核酸酶功能，而化学本质为核酸，因此被Cech称之为核酶(Ribozyme)。
核酶是一种具有核酸内切酶活性的RNA 分子，可特异性地切割靶RNA序列，具有解离后重复切割相同靶序列，具有解离后重复切割相同靶分子的能力Thomas R.CechT.R.CechT.R.Cech（1947-）,因发现RNA 的生物催化作用而与奥尔特曼共同获得 1989年诺贝尔化学奖.他独立地发现核糖核酸(RNA)不仅像过去所设想的那样仅被动地传递遗传信息,还起酶的作用,能催化细胞内的为生命所必需的化学反应.在他们的发现之前,人们认为只有蛋白质才能起酶的作用.他最先证明RNA 分子能催化化学反应,并于1982年公布其研究结果1983年证实RNA的这种酶活动1982:Self-splicing in Tetrahymenapre-rRNA(group I intron)Kruger et al,and Cech,Cell 31,147-157(1982)Sidney Altman奥尔特曼(S.Altman)（1939-）因发现RNA 的生物催化作用而获奖.Altman与Cech分别发现了核糖核酸(RNA)自身具有的生物催化作用,这项研究不仅为探索RNA 的复制能力提供了线索，而且说明了最早的生命物质是同时具有生物催化功能和遗传功能的RNA，打破了蛋白质是生物起源的定论。
1983:RNAse P is a ribozymeGuerrier-Takada et al,and Altman,Cell,35,849-857(1983)核酶发现的意义：核酶发现的意义：?1.它突破了“酶是蛋白质”的传统概念自从1926年J.Summer首先结晶了脲素酶（Urease），并证明这个结晶蛋白能催化脲素水解以来，很多酶被纯化，而且都是蛋白质从而酶被定义为“生物催化剂是一种称作酶的特殊蛋白”，“使代谢进程加速的生物催化剂”核酸性酶的发现无疑是对传统的酶和催化理论巨大的冲击核酶发现的意义：核酶发现的意义：?2.2.核酸性酶的发现对科学家们普遍感兴趣的生核酸性酶的发现对科学家们普遍感兴趣的生命的起源这一问题有了新的认识，对生物前化命的起源这一问题有了新的认识，对生物前化学（学（prebiotic chemistryprebiotic chemistry）有重要贡献有重要贡献长期以来对生命起源中是先有蛋白质还是先有核酸争论不休核酸分子含有合成蛋白质的氨基酸序列的信息，而蛋白质（酶）又是在核酸修复、转录、剪接、翻译等等所有环节中必须存在的因此谁先谁后的问题就像“鸡与鸡蛋”的问题一样令人困惑。
长期以来，人们认为所有的生命形式在冗长的相互依赖的循环中，信息分子和功能分子是分离的核酸是信息分子，而蛋白质是功能分子核酸性酶的发现给人们以启示，即RNA分子不但有复制的功能，含有复制的信息，而且还有催化的功能因此既是信息分子，又是功能分子这些新的概念无疑将对生物前化学的研究开辟新的思路How many ribozyme?Why?-the hammerhead ribozyme(plant virus)-the hairpin ribozyme(plant virus)-hepatitis delta ribozyme(human virus)-neurospora VS ribozyme(mitochondrial RNA)-group I and group II intron ribozyme(rRNA and mt RNA)-RNAse P(tRNA maturation)-Ribosome(translation)-Spliceosome?(splicing)核酶的酶活性反应种类核酶的酶活性反应种类:?核苷酸转移作用?磷酸二酯键水解作用?磷酸转移反应催化作用?脱磷酸作用?限制性内切酶作用?肽基转移作用One main reaction:Nucleolytic cleavageTransesterification(SN2)From Lilley TIBS(2003)HammerheadHaipinHepatitis deltaVS ribozymeThe hammerhead ribozyme(plant virus)-discovered in small RNA satellites of small viruses(1986)-replication by rolling circle mechanismSecondary structureThe hammerhead ribozyme(plant virus)-tertiary structureScott et al and Klug,Science 1996enzyme,substrate,and cleavage site(A-1.1 and C-17)Hammerhead ribozymesVoet.31-53?Small ribozymes?in RNAs of certain plant viruses?Reaction:transesterification.A 3-5 phosphodiester bond between two adjacent nucleotides(red)is cleaved.A 2-3 cyclic phosphodiester and a 5OH result from the clevage:Hairpin ribozymeNature 418,Doudna and Cech,2002The hairpin ribozyme is a catalytic RNA derived from the self-cleaving and ligating domain of the satellite RNA of tobacco ringspot virus.Reversible transesterification catalyzed by the ribozymeThe hepatitis delta ribozyme(human virus)From Lilley TIBS(2003)General structure of group II intronsRNA World 11.1adenine that drivesthe first nucleophilic attack(step 1).arrows point towards the exon-intron junctionsexons as dashed lines.Group I&II intron ribozyme(rRNA and mt RNA)Doudna and CechNature,2002Group I intron ribozyme(rRNA and mt RNA)Golden et al,and cechScience(1998)Catalytic efficiency,condition-ribozyme follows a Michaelis-Menten kineticsE+S ES E+P k1k2k-1-all ribozyme need cations for activity(M2+,M+)Km=k-1+k2 k1=10-5-10-7Mkcat=0.5-2 min-1kcat/Km=103-106 M-1.min-1Good catalytic efficiency!核糖体是一个核酶核糖体是一个核酶Ban,et al.,Science,289,905(2000)Peptidyl transferase centerRibozyme technologyA specifically-designed ribozyme cleaves a specific pathogenic RNA molecule to make it inactive.For example,the viral RNA causing hepatitis C.Very promising results using cell cultures.The ribozyme is synthesized in vitro and administrated to the patient.Problems:half-life too short and low potency once in the body.Reference:Reviews:Lilley TIBS(2003)De Rose Chem&Biol(2002)Ferre dAmare Biopolymer(2003)Article:Kruger et al,and Cech,Cell(1982)Guerrier-Takada et al,and Altman,Cell(1983)Scott et al Nature(1995)Science(1996)Rupert et al Nature(2001),Science(2002)Part II:SELEXA brief HistoryThe method?A few examples.Biological application?适配分子(aptamer)SELEX(指数级富集的配体系统进化技术):Systematic Evolution of Ligands by EXponential enrichmentA brief HistoryEllington and Szostak,Nature(1990)Tuerk and Gold,Science(1990)In vitroselection of RNA molecules that bind specific ligandsAndrew D.Ellington&Jack W.SzostakSubpopulations of RNA molecules that bind specifically to a variety of organic dyes have been isolated from a population of random sequence RNA molecules.Roughly one in 1010random sequence RNA molecules folds in such a way as to create a specific binding site for small ligands.Systematic evolution of ligands by exponential enrichment:RNA ligands to bacteriophage T4 DNA polymerase.Tuerk C,Gold L.High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species.Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized.In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized.Two different sequences were selected by this procedure from the calculated pool of 65,536 species.One is the wild-type sequence found in the bacteriophage mRNA;one is varied from wild type at four positions.The binding constants of these two RNAs to T4 DNA polymerase are equivalent.These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function;high-affinity ligands could conceivably be developed for any target molecule.In-Vitro Selection and In-Vitro Evolution(SELEX)AMPLICATIONSELECTIONMUTATIONInitialpoolWilson and Szostak,Ann.Rev.Bioc.(1999)-Selection against small molecules-Selection against proteins-Selection of new ribozymes(RNA world)The ATP aptamer structureIn vitro selection of an enzymeOHOOAPCON HSH3C5BioSSCONH253OHHOOAPCON HSH3CBioSCONH5RibozymeCUUUCCCA C CC CA sitemRNA3RibosomeC CCON HCONH2SH3COOOHOOACON HCONHSH3CCHOCHOOHHOOAP siteA C CCUUUCCCAGAGAGGG5SA siteP siteAGAGAGGGRibozyme catalyzing peptide bond formation like ribosomeB.Zhang&T.Cech,Nature 390,96-100(1997)B.Zhang&T.Cech,Chemistry&Biology,5,539(1998)（封面）AAACGGGUUG A C C U A U A A G A C C UGUUGC U G G A A C G C U G G ACAGCAAA10 A G G A C U G G AA G U U C U U G A C C U3-GUGCCUC U U C G U C G A G A G G G G A G G C A G C U U U C C C C G G U U A C G A A A G G C U G C CAC CCAAAAAACCCCCCCG G 5?AAAAGGGGGCUU120150170180130C C G A U G C U U U C C G A C G U140110Phe-SS-5?70 C40 50 AUAC60 ACCUUUAAAAAAGUUUUAGGC30 90 10080 20 160Aptamer优点优点l、Aptamer的确定是通过体外过程完成的，它不依赖于细胞或动物，也不在体内，其特性可根据需要改变。
2、由于不需要免疫动物和细胞，毒素和没有免疫源性的分子也可产生高亲和力的Aptamer3、Aptamer能用化学合成的方法制备，具有很高的精确性和重复性在变性条件下能纯化得到很高的纯度在制备Aptamer的过程中几乎没有批与批之间的差异4、标记分子如荧光素和生物素能标记到Aptamer的精确位置，在合成过程中亦可以衍生上去5、Aptamer能可逆变性变性后的Aptamer能在数分钟之内复原，并能长期储存和在常温下运输SELEX技术和Aptamer应用SELEX技术在基础医学研究中的应用、在药物筛选中的应用、在临床诊断中的应用等Aptamer在流式细胞分析中的应用、Aptamer在传感器中的应用、Aptamer在荧光偏振分析中的应用、Aptamer在双位点分析中的应用、Aptamer与分子灯塔的结合、Aptamer在毛细管电泳中的应用、Aptamer作为分子开关、Aptamer检测固定在膜上的蛋白质、Aptamer阵列研究蛋白组等SELEX技术的改进与发展：导向SELEX技术、复合靶分子SELEX技术、基因组SELEX技术等Part III.反义核酸技术反义核酸技术?反义核酸（Antisense Technology）Oligonucleotide(P-S)2-Alkyl oligonucleotidePNAsMorpholinosLNAAntisenseAntisense technologyA single-stranded RNA or DNA molecule that is complementary to a target mRNA pairswith the mRNA and prevents translation.This strategy works well in the laboratory on cultured cells and on model organisms.Clinical example:treatment against cancers.The tumor sizes decreased but this was mainly due tothe production of interferons in response to high doses of foreign RNA.If the dose waslowered to prevent the interferon response,the clinical benefits largely disappeared as well.Antisense MechanismsRNase-HRISCStericdependantdependantblockingSummerton J.Morpholino Antisense Oligomers:The Case for an RNase-H Independent Structural Type.Biochimica et Biophysica Acta 1999 1489:141-158Representative Antisense StructuresRNARISCdependantMorpholinoStericblockingPhosphorothioate RNase-H dependantMorpholino-RNA Heteroduplex?Watson-Crick bonds?Bases positioned for?strong binding?Usually 25 base?Morpholino oligos?are usedBlocking TranslationA steric-blocking oligo stops the initiation complex as it moves toward the start codon.Without reaching the start codon,the complete ribosome cannot form.Without the complete ribosome,the protein will not be made.Blocking TranslationBlocking SplicingWhen a Morpholino binds to boundary of an exon and an intron,the Morpholino can change pre-mRNA splicing.This results in a changed mRNA and a changed protein.Blocking SplicingFDA批准的反义核酸药物批准的反义核酸药物1998年Vitravene(Isis Pharmaceuticals):主治艾滋病患者中十分常见的巨细胞病毒视网膜炎。
2003年又有一个“重磅炸弹”级的反义药物Fuzeon在美国上市此药是新一代抗艾滋病病毒（HIV）新药，属于病毒的融合阻止剂类药物，能“锁定”HIV基因，使其无法发挥“融合”功能并在人类T淋巴细胞内复制，并最终消亡2003年11月24日EN101正式批准英、以两国共同研制的EN101（Monasen）获得美国“罕见病用药新药”证书，准予在美国销售EN101是一种直接针对产生乙酰胆碱酶的mRNA的反义类药物，而乙酰胆碱酶的过量分泌是引起重症肌无力的直接原因2006年6月9日，美国FDA又批准了一种新型反义药物在美国上市ISIS301012（RASONS）它是一种专门治疗家族性（遗传性）哮喘的新药，可从源头上遏制哮喘的发生，降低急性哮喘的死亡率FDA批准的反义核酸药物批准的反义核酸药物Mipomersen：Genzyme(GENZ)与Isis Pharmaceuticals(ISIS)发表第三阶段实验数据，以mipomersen治疗同合子家族性高胆固醇血症（Homozygous Familial Hypercholesterolemia）Mipomersen属于首个载脂蛋白B（apo-B)合成抑制剂，目前处于后期开发阶段，它可通过防止患者体内导致动脉粥样化的脂蛋白形成而降低患者体内LDL-C水平。
目前，Genzyme和Isis两家公司正进行该药的一系列临床实验，包括：一项治疗heFH的3期临床实验；一项治疗重度血胆脂醇过多的3期临床实验；一项治疗冠心病高危人群高胆固醇血症患者的3期临床实验；一项治疗对他汀类药物耐受性差，高危高胆固醇患者的2期临床实验Part IV:Introduction to RNAiA brief HistorySiRNA and miRNARNAi MechanismA few very recent structures Biological applicationA practical example of siRNA20062006年的诺贝尔生理学奖获得者年的诺贝尔生理学奖获得者Andrew Z.FireCraig C.Mello预备知识?dsRNA：双链RNA，包含正义链和反义链?Dicer：属于RNase 家族，是dsRNA的特异性核酸内切酶?siRNA：small interfering RNA，RNAi的关键效应分子，21-23个nt大小的双链RNA?RISC：RNA-inducing silencing complex，具有核酸内切、外切以及解旋酶活性?RdRP：RNA-dependent RNA polymerases，是RNAi的调节因子，使RNAi可以在生物体内传递Cloning of par-1,a gene required for embryonic polarity-Mapped par-1 to small region on genetic map-DNA rearrangements associated with par-1 alleles identify a candidate gene-Rescue mutant with wild-type gene?Problem:cant get large DNA clones comprising candidate geneSolution:Make antisense RNA from candidate gene and see if can phenocopy Par-1 phenotype.Caenorhabditis elegansRNA-mediated interferenceAntisense RNA par-1Sue Guos“antisense”experimentSue Guos“antisense”experimentSue Guo,1995Graduate Student in Ken Kemphues lab,Sue Guos“antisense”experimentCell 81,1-20 1995Molecule Injected%embryonic lethalityZC22(par-1)antisense52ZC22(par-1)sense54TS antisense0Z1 antisense0H200“Surprisingly,injection of sense ZC22 RNA also induced par-1 phenotypes.It is not clear what accounts for this effect The basis for the sense effect is under investigation and will not be discussed further”Andy Fire and Craig Mellos ExperimentNature 391,806 1998Molecules Injected%twitchingunc-22“pure”antisense0unc-22“pure”sense0unc-22sense+antisense100“The mixture was at least two orders of magnitude more effective than either single strand alone in producing genetic interference.The lowest dose tested,60,000 molecules of each strand per adult,led to twitching phenotypes in an average of 100 progeny.At this point,the original injected material would be diluted to at most a few molecules per cell.”mex-3BdsRNANo injectionNo probeNo injectionProbe with mex3AInject mex3B antisenseProbe with mex-3AInject mex3B dsRNAProbe with mex-3AEffects of mex-3 RNA interference on levels of the endogenous mRNA(胚胎的原位杂交)RNAi:double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals.Zamore PD,Tuschl T,Sharp PA,Bartel DP.Double-stranded RNA(dsRNA)directs the sequence-specific degradation of mRNA through a process known as RNA interference(RNAi).Using a recently developed Drosophila in vitro system,we examined the molecular mechanism underlying RNAi.We find that RNAi is ATP dependent yet uncoupled from mRNA translation.During the RNAi reaction,both strands of the dsRNA are processed to RNA segments 21-23 nucleotides in length.Processing of the dsRNA to the small RNA fragments does not require the targeted mRNA.The mRNA is cleaved only within the region of identity with the dsRNA.Cleavage occurs at sites 21-23 nucleotides apart,the same interval observed for the dsRNA itself,suggesting that the 21-23 nucleotide fragments from the dsRNA are guiding mRNA cleavageCell,v101 pp25-33(2000)dsRNA is cut in 21-23 nt fragmentsZamore PD,Tuschl T,Sharp PA,Bartel DP,Cell,v101 pp25-33(2000)The mRNA is cut in 21-23 nt fragments by the siRNAZamore et al,Cell,v101 pp25-33(2000)A first model for the mechanism RNAiZamore et al,Cell,v101 pp25-33(2000)Role for a bidentate ribonuclease in the initiation step of RNA interference.Bernstein E,Caudy AA,Hammond SM,Hannon GJ.RNA interference(RNAi)is the mechanism through which double-stranded RNAs silence cognate genes.In plants,this can occur at both the transcriptional and the post-transcriptional levels;however,in animals,only post-transcriptional RNAi has been reported to date.In both plants and animals,RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppressed.These 22-nucleotide sequences serve as guide sequences that instruct a multicomponent nuclease,RISC,to destroy specific messenger RNAs.Here we identify an enzyme,Dicer,which can produce putative guide RNAs.Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs,and is evolutionarily conserved in worms,flies,plants,fungi and mammals.The enzyme has a distinctive structure,which includes a helicase domain and dual RNase III motifs.Dicer also contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi.Nature v 409,pp 363-366(2001)22 ntRNAIdentification of DICERBernstein et al,Nature,409,363(2001)MacRac,et al.Science,311,195(2006)Elbashir et al,G&D,v18 pp188-200(2001)The RISC complexSiRNA and miRNAMicroRNAs.Genomics,Biogenesis,Mechanism,and Function.Bartel DP.MicroRNAs(miRNAs)are endogenous approximately 22 nt RNAs that can play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression.Although they escaped notice until relatively recently,miRNAs comprise one of the more abundant classes of gene regulatory molecules in multicellular organisms and likely influence the output of many protein-coding genes.Cell,v116,pp 281-297(2004)(review)First miRNAin C.elegansmiRNAin C.eleganswith homologsIn flies and humanmiRNA in PlantsmiRNAsiRNAPost-transcriptionalCleavage of mRNATranslational repressionof the mRNATranscriptional silencingReference:Reviews:Bartel Cell.(2004)Hannon Nature(2002)Article:Fire at al,Nature V391 pp 806-811(1998)Elbashir et al,G&D,v18 pp188-200(2001)Bernstein et al,Nature v 409,pp 363-366(2001)内源和外源内源和外源siRNAsiRNA内源的dsRNA 21-23对碱基(siRNA)处理和输出靶标可以是病毒或细胞RNA靶标RNA切断和降解RISC外源siRNA21对碱基细胞核内表达的shRNA细胞质细胞核RNAi药物离我们还有多远？siRNA药物开发现状药物开发现状Company Targets Disease StatusAcuity(Bevasiranib),PAVEGFAge-related macular degeneration Diabetic macular oedemPhase IIISirna(Sirna-027)Alnylam(ALN RSV01),MAVEGFR1Respiratory syncitial virusAge-related macular degenerationRSV infectionSpinal cord injuriesParkinsons diseasePhase IIPhase IpreclinicalpreclinicalPTC Therapeutics(PTC-299)Acugen,Berlin VEGFSolid tumoursMetabolic diseasesOcular diseasesPhase IResearch phaseResearch phaseBenitec,Mountain View,CA,CytRxHIVHBV,HCVPTP-1BHIV/AIDA lymphomaHepatitisLou Gehrigs diseaseObesityCytomegalovirusSolid tumorsPhase IPhase IResearch phase Research phaseResearch phaseIND plannedIntradigm Corp.Rockville,MDNucleonics,Horsham,PA Phytovation,NetherlandsHBV,HCVHCVHIV-1HepatitisHepatitisHIVinfluenzaPhase IResearch phaseResearch phaseResearch phaseSirna Dermatology Boulder,COHairless TX factorTopical hair removalHuntingtons diseaseIND plannedIND plannedSirna Pharma,san Francisco,CAIL-4,IL-4R,IL-13,IL-13RAsthmatype II diabetesIND plannedIND plannedRNAiRNAi药物开发可极大地节省药物开发可极大地节省时间和金钱时间和金钱药靶鉴定确认高通量筛选先导化合物鉴定先导化合物确认动物试验临床试验传统小分子药RNAi药物开发药靶鉴定确认动物试验临床试验12-15 年$3.5-5.0 亿新药-年新药首次RNAi临床试验获成功Bevasiranib1.靶基因：去年6月1日公布了以RNAi为基础的Bevasiranib能够关闭血管内皮生长因子（VEGF）基因，这种基因会刺激老年性黄斑变性患者视网膜上血管的生长。
2.可能副作用：这种siRNA分子不会与DNA反应，因此不必担心该药物会改变患者的遗传组成而在其他类型的基因治疗中，这是不可忽略的一个风险已经证明，到目前为止最成功的XSCID基因疗法（治疗X连锁重度复合性免疫缺陷病的基因疗法）的临床试验中，接受这种基因治疗的其中三名儿童在治疗后患上了白血病3.药效：Bevasiranib能够与其他药物联合使用，例如能抑制血管内皮生长因子的抗癌药物AvastinAvastin的效果只能持续几周时间，而Bevasiranib的效果则能持续更长的时间4.临床II试验结果：对129名患者的试验发现，Bevasiranib能够减缓眼睛中血管的生长并改善视力：在最低剂量时，这种效果持续了数月；最高剂量时，这种好的效果一直到研究结束还存在而且，试验中，除了药物注射位置的红肿外，没有观察到其他任何副作用首个申请临床病毒感染疾病RNAi治疗药物?去年11月1日，Alnylam制药公司宣布向美国FDA提交一项研发新药，申请开始对药物ALN-RSV01进行人类临床实验，这种药物是利用RNAi疗法治疗感染呼吸道合胞体病毒(respiratory syncytial virus,RSV)的疾病。
ALN-RSV01的作用原理是利用RNAi对病毒衣壳N基因进行沉默这个基因是病毒中最高度保守的基因之一，并且对病毒复制有重要意义在体内动物实验和体外实验数据都显示ALN-RSV01的高效性通过ALN-RS01滴鼻治疗后，在动物体内可以特异一直RSV的复制，有效防止、治疗RSV的感染另外Alnylam完成了对于ALN-RSV01的毒性实验(65位健康人），并没有发现药物有明显毒性英国基因研究小组接近突破癌症的边缘英国基因研究小组接近突破癌症的边缘今年年初，位于伦敦的SR Pharma公司获得了一个专门控制胰腺癌基因RNAi药物的专利动物试验显示，治疗能阻止肿瘤生长，并防止其向身体其它地方扩散首例人体试验将在今后几个月里进行，此种药物可能在三年内面市英国癌症研究所的黑兹尔.鲁恩说，“从实验室研究来看，核糖核酸干扰技术可能从理论上来说具有治疗一系列疾病的潜力但是在该技术能够用于治疗像胰腺癌之类的疾病之前，我们还需要在研发核糖核酸输送入人体细胞的方法上取得重大进步才行”RNAiRNAi药物面临的问题药物面临的问题1.脱靶效应(off-target effects)2.免疫反应3.干扰素反应(TLR)4.体内稳定性5.给药方式1.实践证明RNA干扰技术是一强有力的基因功能研究的工具；2.RNAi在药物靶标的确认中有着不可取代的地位；3.siRNA将是充满希望和前景的新型基因药物。
siRNA的给药问题药物开发的瓶颈的给药问题药物开发的瓶颈Delivery of siRNAs for TherapyRNAi是。