修回日期: 2005-04-05
接受日期: 2005-04-09
在线出版日期: 2005-06-15
RNA干扰现象自被发现后, 已被广泛应用于基因功能、抗病毒治疗和信号转导系统方面的研究.本文对这一现象从研究历史、作用机制、抗病毒应用以及存在的问题等方面进行初步的探讨.
引文著录: 潘金水, 任建林, 董菁, 王小众. RNA干扰的机制及其抗肝炎病毒的应用. 世界华人消化杂志 2005; 13(11): 1322-1326
Revised: April 5, 2005
Accepted: April 9, 2005
Published online: June 15, 2005
N/A
- Citation: N/A. N/A. Shijie Huaren Xiaohua Zazhi 2005; 13(11): 1322-1326
- URL: https://www.wjgnet.com/1009-3079/full/v13/i11/1322.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v13.i11.1322
RNA干扰(RNA interference, RNAi)是指由特定双链RNA(double stranded RNA, dsRNA)引发同源mRNA降解的转录后基因静默机制.这类特定dsRNA被裂解成小干扰RNA(small interfering RNA, siRNA), 并导致同源mRNA在RNA诱导的静默复合体(RNA-induced silencing complex, RISC)作用下降解.DNA甲基化也可触发这一过程.RNAi是真核生物中普遍存在的与抵抗病毒入侵、抑制转座子活动、调控基因表达、染色体修饰有关的监控机制, 并可能有维持干细胞数量的功能及稳定有丝分裂、减数分裂期染色体结构的作用[1-3].目前已成功应用于基因功能、抗病毒治疗和信号转导系统上下游分子相互关系的研究.总的来讲, RNAi可以用于治疗任何与某一特定基因表达增强有关的疾病, 如病毒性疾病(例如病毒性肝炎)、肿瘤、炎症性疾病、神经元病变、糖尿病等, 有可能为上述疾病治疗提供新策略.
1990年Napoli et al[4]在向牵牛花转导入色素合成基因后, 发现不仅转入的基因未表达, 牵牛花自身的色素合成也减弱了.他们把这种现象称为"共抑制"(co-suppression).类似的现象也发生在真菌中, 1994年Cogni et al[5]把胡萝卜素基因转入到野生型粗糙链孢霉菌中, 发现与转基因同源的内源性基因表达水平明显减弱, 他们称之为"压制"(quelling).1995年康奈尔大学的Guo和Kemphues[6]研究秀丽隐杆线虫(Caenorhabditis elegans, C.elegans)par-1基因表达时发现一个令人不解的现象:注射反义RNA(antisense RNA)及正义RNA(sense RNA)均能阻断par-1基因的表达.Fire et al[7]对这一现象的机制进行深入的研究, 比较了反义RNA、正义RNA和dsRNA在C.elegans中的抑制效应, 发现dsRNA可以产生至少10倍以上的抑制靶基因表达的效果, 并将dsRNA抑制同源基因表达的现象称为RNA干扰.后来发现在许多类型的病原微生物中都存在同样的RNA干扰现象, 例如果蝇、锥虫、涡虫、线虫以及高等的哺乳动物细胞, 甚至人的细胞中也都发现类似的RNA干扰现象[8-9].最初认为在人体中不存在RNAi现象, 因为dsRNA进入哺乳动物细胞内可以激发机体的以干扰素产生为标志的强有力抗病毒反应, 并导致基因表达抑制、细胞凋亡, 从而限制了病毒扩散及其在机体内的播散.但是后来发现小于30 nt的dsRNA并不激发干扰素效应.因此, siRNA进入体内不会发生机体基因表达关闭, 能以一种与植物、蠕虫、果蝇非常相似的方式来诱导同源mRNA的降解[10-13].
随着研究的深入, RNAi的机制被逐步阐明.
RNAi是一种古老的保护机体免受病毒入侵的机制.许多病毒在其生命周期中以RNA而不是DNA作为其遗传信息的载体, 并且在生命史至少有一个阶段存在dsRNA.所有多细胞生物均可产生一种相对保守的可以识别dsRNA的蛋白, 即Dicer.Dicer属于RNaseIII超家族的一员, 是一种核酸酶二齿螯合物.Dicer与RNaseIII超家族另一成员Drosha共同参与siRNA的成熟, 如发生突变则RNAi过程受抑制[14-15].Dicer2与另一蛋白R2D2(C.elegans中RNAi蛋白RDE-4的同源物)形成的复合体与siRNA结合, siRNA转运至RISC上可促进RISC介导特定序列mRNA降解[16].在果蝇属(Drosophila)中, RISC是一至少由5个分子组成的超分子结构[AGO2, VIG-1, ARMI, AUB 及FXR相关分子][17-18].在果蝇属、C.elegans及哺乳动物中RISC尚包括一种可能具有催化作用的组分TUDOR-SN[18].当RISC结合至mRNA时, mRNA被降解而不能被翻译.其他参与RNAi的成分还有RNA依赖的RNA聚合酶、解螺旋酶、dsRNA核酸内切酶[19]以及Exportin-5等, Exportin-5介导siRNA前体的出核转运[20].
RNAi主要在转录后水平发挥作用, 与同源mRNA的降解有关[21].转录后RNAi包括高度保守的两个步骤[22]:(1)dsRNA被转运入细胞内(在C.elegans可能是由一种RNA转运蛋白SID-1转运的[23]), 并被Dicer切割成长约21-23 nt的siRNA[14,24], 有2nt的3'突出端;(2)iRNA双链以一种ATP依赖的方式展开[25], 并自行黏附至RISC上, 然后结合至具有完全互补序列的靶mRNA上, 在RISC的作用下, 同源mRNA被降解[26-27].
RNAi的特点:(1)RNAi发生在转录后水平, 不能产生任何代偿性转录;(2)RNAi有高度特异性, 即使仅有一个碱基错配亦可使干扰效应明显下降;(3)RNAi有剂量依赖性, 但在C.elegans中这种现象不明显, 因为siRNA可以通过RNA-依赖的RNA聚合酶活性而发生扩增;(4)RNAi具有快速、费用低等优点, 但却可以产生几乎与基因缺失相媲美的效果.例如, 应用抗lush、white及dGq 的dsRNA发挥的RNAi效应与基因敲除相似[28-29], 而且特别容易被导入到C.elegans中[30-31];(5)RNAi可以有效地与其他较成熟的基因功能研究方法结合使用[32].
在培养的组织模型中, 应用RNAi抗原癌基因及抗获得性免疫缺陷病病毒(HIV)、流感病毒、登革热病毒及脊髓灰质炎病毒已经取得令人振奋的效果[33-37].丙型肝炎病毒(HCV)是导致慢性肝病和肝细胞癌的一个主要致病因子, 感染HCV后20年内约有20%可发展至进展期肝病.虽然其危害大, 但目前治疗费用昂贵且效果并不理想, 标准干扰素结合利巴韦林(ribavirin)治疗48 wk后血清学持续阴转率仅约为43%[38].由于其基因组为单链正股RNA, 长约9.5 kb, 同时充当mRNA及复制模板, 特别适合于RNAi研究[39-44].因体外细胞培养不能稳定繁殖HCV, 限制了RNAi研究的进行.近年来有学者将含有HCV基因组全序列或部分序列的质粒转染传代细胞株(Huh-7)构建了能稳定表达HCV RNA的细胞模型解决了这一问题[45].Randall et al[40]在含有HCV复制系统的Huh-7肝癌细胞系中引入siRNA, 发现4 d后HCV RNA水平下降80多倍, 并且98%以上的细胞不再表达HCV抗原及HCV RNA, 他们还发现RNAi具有剂量依赖性并需要严格的碱基配对等特点.McCaffrey et al[46]设计合成了针对HCV非结构蛋白5B(NS5B)基因的dsRNA, 当与NS5B表达载体进行共转染时, 在鼠肝细胞中观察到dsRNA对于NS5B基因表达水平的抑制率达到75%;如果dsRNA与NS5B的表达载体进行共转染抑制率可以达到98%.由于HCV变异率高, Zhang et al[44]针对HCV感染的辅因子如La、polypyrimidine tract-binding protein(PTB)、真核起始因子eIF2B的γ亚基设计了siRNA, 以腺病毒为载体转染Huh-7细胞, 发现HCV复制水平明显下降.
Kronke et al[41]则采用其他方法进行RNA干扰.简述如下:以Huh-7 20-1、9B、9-13株及新近建立的A-3/3细胞株作为宿主细胞, 其中A-3/3细胞株能表达嵌合状态的HCV 1a/1b复制子I389/NS3-3'/H77/DR, 培养在经Dulbecco改良的Eagle培养基(Life Techno-logies, Gaithersburg, Md.)中.培养基中含100 mL/L胎牛血清, 每毫升含200 IU青霉素G及200 μg链霉素, 并含不同数量的G418(Life Technologies).并加入10 μg/mL的zeocin(Invitrogen, Carlsbad, Calif.)作阳性选择.以质粒pCV-H77为载体构建复制子I389/NS3-3'/H77/DR导入Huh-7细胞.以Moloney鼠白血病病毒(Moloney murine leukemia virus, Mo-MuLV)为基础的载体pBABE/puro经改造成为载体pBABE/H1/SV40/EGZ/ΔU3, 并将短发夹RNA(short hairpin RNA, shRNA)编码序列插入其XhoI及BglII位点间导入Huh-7细胞.利用HCV数据库(http://hepatitis.ibcp.fr)或VECTOR NTI ADVANCE程序(INFORMAX, Bethesda, Md.)分析HCV序列.分别设计11条针对位于5'端非翻译区或前C区高度保守序列的shRNA及1条针对变异度相对较高的NS4B编码区的shRNA进行RNA干扰.他们发现针对结构域IV的shRNA具有显著的抗病毒效应, 针对结构域II、III的shRNA亦有较好的抗病毒效应.
以上试验表明RNAi可能是一种极有潜力的抗HCV方法, 并且不仅可以针对HCV基因组也可以针对HCV感染相关的辅因子设计siRNA片段, 均有显著效果.
乙型肝炎是治疗较为困难的病毒性疾病之一, 患者数量众多而抗病毒治疗效果又欠佳.Wong et al[47]荟萃分析了15项随机对照的标准干扰素治疗HBeAg阳性的乙肝患者显示HBV-DNA、HBeAg及HBsAg阴转率约为37%、33%及7.8%.拉米夫啶(lamivudine)单一治疗1年 HBeAg阴转率约为16-18%[48-50].作为新发展起来的一项技术, RNAi在抗乙型肝炎病毒(HBV)方面的研究也日益广泛, 并且显示出明显的优势[51-56].Konishi et al[52]在稳定表达HBV的HepG2 2.2.15细胞系中导入抗多聚腺苷酸尾、前C区、S区的siRNA, 发现HBsAg表达率分别下降78%、67%及42%.
McCaffrey et al[53]的试验表明不仅体外RNAi效果显著, 而且在哺乳动物体内进行RNAi亦有良好的抗病毒效应:分别合成shRNA No.1-4(针对编码S抗原的mRNA), No.5(X区及其转录子), No.6-7(C抗原、多聚酶).在每个试验中, 均有三种质粒共转染入Huh-7细胞中, 分别是:4 μg含有HBV基因组的质粒pTHBV2;5 μg的No.6 shRNA表达载体或阴性对照载体;5 μg表达分泌型蛋白人抗胰蛋白酶-1的质粒(pThAAT).在第8 d测定培养基中HBsAg水平, 发现除shRNA No.1外, 治疗组HBsAg水平均有下降, 其中shRNA No.2(94.2% vs 0.59%)及No.6(91.5% vs 1.4%)效果最为显著.体内试验方法为:以12 μg pTHBV2、5 μg 的No.2及6 shRNA表达载体或阴性对照载体、5 μg的pThAAT用注射法共转染具有免疫力及免疫缺陷的两组小鼠, 第7 d以Northern杂交法测定肝内RNA含量.结果发现, 与未进行RNAi的小鼠相比, 转染No.2 shRNA的两组具有免疫力、免疫缺陷小鼠HBV RNA分别下降77%及92%, 转染No.6 shRNA则分别下降31%及58%.
此外, 还发现在鼠暴发性肝炎模型中, 引入抗Fas蛋白的siRNA可以拮抗Fas特异性抗体引起的损害[57].由于RNAi抗HBV机制的特殊性, 不但体外、体内试验效果均令人满意, 而且不受拉米夫啶耐药突变的影响, 因而有望成为抗HBV的一个有力武器[58].但是与HCV一样, HBV有些位点也存在高度变异现象, 可能影响RNAi的效率, 因此设计siRNA时必须考虑到这一问题.
发展至今, 阻断mRNA表达、翻译的方法有转基因法、反义核酸法、RNAi法.从1970年初至1980年末, 基础科学推动着转基因治疗于1988年第一次进入临床应用.几位患有严重联合免疫缺陷病年轻患者的治愈标志着转基因治疗在临床上取得的第一个巨大进步[59-60].然而, 至今临床试验并不是很成功, 这使得许多医学家及科学家怀疑这一方法的可行性.近来一些事件的发生更加动摇了人们对基因治疗的信心, 如参与宾西法尼亚大学临床试验的一位患者的死亡, 激起了公众对基因治疗安全性的反思, 人们采用更加审慎的态度来对待基因治疗[61-62].另一个方法是反义核酸技术, 即将单链DNA与具有互补序列的mRNA形成RNA-DNA杂合体, 从而阻断mRNA的翻译并进一步导致其降解.然而, 反义核酸技术在临床上也没有取得很大成功, 部分原因是因为其基因抑制效应较弱, 另一个重要原因是无法确保反义核酸在到达靶细胞前不被降解.
RNAi技术的出现无疑为人们带来新的希望.但是, RNAi也存在许多亟待解决的问题:(1)病毒载体的安全性 在哺乳动物模型开展RNAi研究存在许多困难, 特别是如何将体外合成的siRNA导入细胞内, 因为哺乳动物细胞比较不容易摄取裸露的核酸.导入siRNA的方法有物理法(如阳离子脂质体法)及生物载体法.在哺乳动物体细胞内引入长dsRNA后通常可以激活一种导致非特异性细胞mRNAs缩短的防御机制.然而, Elbashir et al[10]认为直接引入双股siRNA可以绕过这一防御反应并导致有效的基因静默.后来发展了几种可以在哺乳动物细胞内表达siRNA的质粒表达系统, 其siRNA是由shRNA前体演变而来的[63-65], 后来更进一步发展了数种用于shRNA介导的RNAi的病毒载体[66-68].该类载体可以在细胞内表达短RNA, 并依靠其互补序列形成发夹.在细胞内shRNA被除去环状结构并进一步形成siRNA二聚体.病毒载体如逆转录病毒、腺病毒、慢病毒属可以作为载体, 并可以获得持续的RNAi效应.但是病毒载体的安全性是个不容小觑的问题.近来的报道显示在部分应用病毒载体进行转基因治疗的患者中出现白血病样综合征[69].而且, 白血病被认为是治疗干预的直接后果, 因为当病毒载体整合入患者基因时可能有插入诱变效应[70];(2)另一问题是如何安全有效地将RNAi物质转运至特定的靶器官, 因为siRNA在转运过程中常被核酸酶非特异性水解;而且如何定向运输也是个需要解决的问题, RNAi物质只有转运至特定器官才能发挥作用, 或许将来生物导向技术的发展可以解决这一问题;(3)病毒为了逃避机体的免疫监视, 常发生变异, 表现为核酸序列某些位点的高变性.而RNAi则需要高度精确的核酸配对才能有效地进行, 针对高度保守的关键性序列设计RNAi物质可以解决这一问题;(4)有些mRNA具有二、三级结构, 或者与蛋白质结合, 可能会影响RNAi物质的配对.因此, 进行RNAi前有必要了解靶mRNA的结构.
总之, 虽然目前RNAi技术还存在不少需要解决的问题, 但相信随着技术的发展, 这些问题可以得到妥善解决.可以预言, RNAi将为病毒性、肿瘤性等疾病的基因治疗提供一强有力的全新工具, 为攻克病毒性肝炎这一顽疾注入新的希望.
编辑:徐协群 审读:张海宁
| 1. | Bernstein E, Kim SY, Carmell MA, Murchison EP, Alcorn H, Li MZ, Mills AA, Elledge SJ, Anderson KV, Hannon GJ. Dicer is essential for mouse development. Nat Genet. 2003;35:215-217. [PubMed] [DOI] |
| 2. | Hall IM, Noma K, Grewal SI. RNA interference machinery regulates chromosome dynamics during mitosis and meiosis in fission yeast. Proc Natl Acad Sci USA. 2003;100:193-198. [PubMed] [DOI] |
| 3. | Carrington JC, Ambros V. Role of microRNAs in plant and animal development. Science. 2003;301:336-338. [PubMed] [DOI] |
| 4. | Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell. 1990;2:279-289. [PubMed] [DOI] |
| 5. | Cogoni C, Romano N, Macino G. Suppression of gene expression by homologous transgenes. Antonie Van Leeuwenhoek. 1994;65:205-209. [PubMed] [DOI] |
| 6. | Guo S, Kemphues KJ. par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell. 1995;81:611-620. [PubMed] [DOI] |
| 7. | Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998;391:806-811. [PubMed] [DOI] |
| 10. | Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001;411:494-498. [PubMed] [DOI] |
| 11. | Caplen NJ, Fleenor J, Fire A, Morgan RA. dsRNA-mediated gene silencing in cultured Drosophila cells: a tissue culture model for the analysis of RNA interference. Gene. 2000;252:95-105. [PubMed] [DOI] |
| 12. | Silverstein AM, Mumby MC. Analysis of protein phosphatase function in Drosophila cells using RNA interference. Methods Enzymol. 2003;366:361-372. [PubMed] [DOI] |
| 13. | Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001;15:188-200. [PubMed] [DOI] |
| 14. | Ketting RF, Fischer SE, Bernstein E, Sijen T, Hannon GJ, Plasterk RH. Dicer functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans. Genes Dev. 2001;15:2654-2659. [PubMed] [DOI] |
| 15. | Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, Lee J, Provost P, Radmark O, Kim S. The nuclear RNase III Drosha initiates microRNA processing. Nature. 2003;425:415-419. [PubMed] [DOI] |
| 16. | Liu Q, Rand TA, Kalidas S, Du F, Kim HE, Smith DP, Wang X. R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway. Science. 2003;301:1921-1925. [PubMed] [DOI] |
| 17. | Williams RW, Rubin GM. ARGONAUTE1 is required for efficient RNA interference in Drosophila embryos. Proc Natl Acad Sci USA. 2002;99:6889-6894. [PubMed] [DOI] |
| 18. | Caudy AA, Ketting RF, Hammond SM, Denli AM, Bathoorn AM, Tops BB, Silva JM, Myers MM, Hannon GJ, Plasterk RH. A micrococcal nuclease homologue in RNAi effector complexes. Nature. 2003;425:411-414. [PubMed] [DOI] |
| 19. | Agrawal N, Dasaradhi PV, Mohmmed A, Malhotra P, Bhatnagar RK, Mukherjee SK. RNA interference: biology, mechanism, and applications. Microbiol Mol Biol Rev. 2003;67:657-685. [PubMed] [DOI] |
| 20. | Lund E, Guttinger S, Calado A, Dahlberg JE, Kutay U. Nuclear export of microRNA precursors. Science. 2004;303:95-98. [PubMed] [DOI] |
| 21. | Wadhwa R, Kaul SC, Miyagishi M, Taira K. Know-how of RNA interference and its applications in research and therapy. Mutat Res. 2004;567:71-84. [PubMed] [DOI] |
| 22. | Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell. 2000;101:25-33. [PubMed] [DOI] |
| 23. | Feinberg EH, Hunter CP. Transport of dsRNA into cells by the transmembrane protein SID-1. Science. 2003;301:1545-1547. [PubMed] [DOI] |
| 24. | Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature. 2001;409:363-366. [PubMed] [DOI] |
| 25. | Nykanen A, Haley B, Zamore PD. ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell. 2001;107:309-321. [PubMed] [DOI] |
| 26. | Hammond SM, Bernstein E, Beach D, Hannon GJ. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature. 2000;404:293-296. [PubMed] [DOI] |
| 27. | Martinez J, Patkaniowska A, Urlaub H, Luhrmann R, Tuschl T. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell. 2002;110:563-574. [PubMed] [DOI] |
| 28. | Helwani FM, Kovacs EM, Paterson AD, Verma S, Ali RG, Fanning AS, Weed SA, Yap AS. Cortactin is necessary for E-cadherin-mediated contact formation and actin reorganization. J Cell Biol. 2004;164:899-910. [PubMed] [DOI] |
| 29. | Kalidas S, Smith DP. Novel genomic cDNA hybrids produce effective RNA interference in adult Drosophila. Neuron. 2002;33:177-184. [PubMed] [DOI] |
| 30. | Kamath RS, Martinez-Campos M, Zipperlen P, Fraser AG, Ahringer J. Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol. 2001;2:RESEARCH0002. [PubMed] |
| 31. | Tabara H, Grishok A, Mello CC. RNAi in C. elegans: soaking in the genome sequence. Science. 1998;282:430-431. [PubMed] [DOI] |
| 32. | Mariol MC, Segalat L. Muscular degeneration in the absence of dystrophin is a calcium-dependent process. Curr Biol. 2001;11:1691-1694. [PubMed] [DOI] |
| 33. | Damm-Welk C, Fuchs U, Wossmann W, Borkhardt A. Targeting oncogenic fusion genes in leukemias and lymphomas by RNA interference. Semin Cancer Biol. 2003;13:283-292. [PubMed] [DOI] |
| 34. | Ge Q, McManus MT, Nguyen T, Shen CH, Sharp PA, Eisen HN, Chen J. RNA interference of influenza virus production by directly targeting mRNA for degradation and indirectly inhibiting all viral RNA transcription. Proc Natl Acad Sci USA. 2003;100:2718-2723. [PubMed] [DOI] |
| 35. | Gitlin L, Karelsky S, Andino R. Short interfering RNA confers intracellular antiviral immunity in human cells. Nature. 2002;418:430-434. [PubMed] [DOI] |
| 36. | Coburn GA, Cullen BR. Potent and specific inhibition of human immunodeficiency virus type 1 replication by RNA interference. J Virol. 2002;76:9225-9231. [PubMed] [DOI] |
| 37. | Adelman ZN, Sanchez-Vargas I, Travanty EA, Carlson JO, Beaty BJ, Blair CD, Olson KE. RNA silencing of dengue virus type 2 replication in transformed C6/36 mosquito cells transcribing an inverted-repeat RNA derived from the virus genome. J Virol. 2002;76:12925-12933. [PubMed] [DOI] |
| 38. | Poynard T, Marcellin P, Lee SS, Niederau C, Minuk GS, Ideo G, Bain V, Heathcote J, Zeuzem S, Trepo C. Randomised trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon alpha2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. International Hepatitis Interventional Therapy Group (IHIT). Lancet. 1998;352:1426-1432. [PubMed] [DOI] |
| 39. | Kapadia SB, Brideau-Andersen A, Chisari FV. Interference of hepatitis C virus RNA replication by short interfering RNAs. Proc Natl Acad Sci USA. 2003;100:2014-2018. [PubMed] [DOI] |
| 40. | Randall G, Grakoui A, Rice CM. Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs. Proc Natl Acad Sci USA. 2003;100:235-240. [PubMed] [DOI] |
| 41. | Kronke J, Kittler R, Buchholz F, Windisch MP, Pietschmann T, Bartenschlager R, Frese M. Alternative approaches for efficient inhibition of hepatitis C virus RNA replication by small interfering RNAs. J Virol. 2004;78:3436-3446. [PubMed] [DOI] |
| 42. | Randall G, Rice CM. Interfering with hepatitis C virus RNA replication. Virus Res. 2004;102:19-25. [PubMed] [DOI] |
| 43. | Takigawa Y, Nagano-Fujii M, Deng L, Hidajat R, Tanaka M, Mizuta H, Hotta H. Suppression of hepatitis C virus replicon by RNA interference directed against the NS3 and NS5B regions of the viral genome. Microbiol Immunol. 2004;48:591-598. [PubMed] [DOI] |
| 44. | Zhang J, Yamada O, Sakamoto T, Yoshida H, Iwai T, Matsushita Y, Shimamura H, Araki H, Shimotohno K. Down-regulation of viral replication by adenoviral-mediated expression of siRNA against cellular cofactors for hepatitis C virus. Virology. 2004;320:135-143. [PubMed] [DOI] |
| 45. | Lohmann V, Korner F, Koch J, Herian U, Theilmann L, Bartenschlager R. Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science. 1999;285:110-113. [PubMed] [DOI] |
| 46. | McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, Kay MA. RNA interference in adult mice. Nature. 2002;418:38-39. [PubMed] [DOI] |
| 47. | Wong DK, Cheung AM, O'Rourke K, Naylor CD, Detsky AS, Heathcote J. Effect of alpha-interferon treatment in patients with hepatitis B e antigen-positive chronic hepatitis B. A meta-analysis. Ann Intern Med. 1993;119:312-323. [PubMed] [DOI] |
| 48. | Dienstag JL, Schiff ER, Wright TL, Perrillo RP, Hann HW, Goodman Z, Crowther L, Condreay LD, Woessner M, Rubin M. Lamivudine as initial treatment for chronic hepatitis B in the United States. N Engl J Med. 1999;341:1256-1263. [PubMed] [DOI] |
| 49. | Lai CL, Chien RN, Leung NW, Chang TT, Guan R, Tai DI, Ng KY, Wu PC, Dent JC, Barber J. A one-year trial of lamivudine for chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. N Engl J Med. 1998;339:61-68. [PubMed] [DOI] |
| 50. | Schalm SW, Heathcote J, Cianciara J, Farrell G, Sherman M, Willems B, Dhillon A, Moorat A, Barber J, Gray DF. Lamivudine and alpha interferon combination treatment of patients with chronic hepatitis B infection: a randomised trial. Gut. 2000;46:562-568. [PubMed] [DOI] |
| 51. | Klein C, Bock CT, Wedemeyer H, Wustefeld T, Locarnini S, Dienes HP, Kubicka S, Manns MP, Trautwein C. Inhibition of hepatitis B virus replication in vivo by nucleoside analogues and siRNA. Gastroenterology. 2003;125:9-18. [PubMed] [DOI] |
| 52. | Konishi M, Wu CH, Wu GY. Inhibition of HBV replication by siRNA in a stable HBV-producing cell line. Hepatology. 2003;38:842-850. [PubMed] [DOI] |
| 53. | McCaffrey AP, Nakai H, Pandey K, Huang Z, Salazar FH, Xu H, Wieland SF, Marion PL, Kay MA. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol. 2003;21:639-644. [PubMed] [DOI] |
| 54. | Shlomai A, Shaul Y. Inhibition of hepatitis B virus expression and replication by RNA interference. Hepatology. 2003;37:764-770. [PubMed] [DOI] |
| 55. | Wu J, Nandamuri KM. Inhibition of hepatitis viral replication by siRNA. Expert Opin Biol Ther. 2004;4:1649-1659. [PubMed] [DOI] |
| 56. | Zhang XN, Xiong W, Wang JD, Hu YW, Xiang L, Yuan ZH. siRNA-mediated inhibition of HBV replication and expression. World J Gastroenterol. 2004;10:2967-2971. [PubMed] [DOI] |
| 57. | Song E, Lee SK, Wang J, Ince N, Ouyang N, Min J, Chen J, Shankar P, Lieberman J. RNA interference targeting Fas protects mice from fulminant hepatitis. Nat Med. 2003;9:347-351. [PubMed] [DOI] |
| 58. | Ying C, De Clercq E, Neyts J. Selective inhibition of hepatitis B virus replication by RNA interference. Biochem Biophys Res Commun. 2003;309:482-484. [PubMed] [DOI] |
| 59. | Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, Selz F, Hue C, Certain S, Casanova JL. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science. 2000;288:669-672. [PubMed] [DOI] |
| 60. | Hacein-Bey-Abina S, Fischer A, Cavazzana-Calvo M. Gene therapy of X-linked severe combined immunodeficiency. Int J Hematol. 2002;76:295-298. [PubMed] [DOI] |
| 61. | Marshall E. Gene therapy death prompts review of adenovirus vector. Science. 1999;286:2244-2245. [PubMed] [DOI] |
| 62. | Vogel G. Gene therapy. FDA moves against Penn scientist. Science. 2000;290:2049-2051. [PubMed] [DOI] |
| 63. | Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science. 2002;296:550-553. [PubMed] [DOI] |
| 64. | Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS. Short hairpin RNAs (shRNAs)induce sequence-specific silencing in mammalian cells. Genes Dev. 2002;16:948-958. [PubMed] [DOI] |
| 65. | Miyagishi M, Taira K. U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol. 2002;20:497-500. [PubMed] [DOI] |
| 66. | Barton GM, Medzhitov R. Retroviral delivery of small interfering RNA into primary cells. Proc Natl Acad Sci U S A. 2002;99:14943-14945. [PubMed] [DOI] |
| 67. | Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, Kopinja J, Rooney DL, Ihrig MM, McManus MT, Gertler FB. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet. 2003;33:401-406. [PubMed] [DOI] |
| 68. | Xia H, Mao Q, Paulson HL, Davidson BL. siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol. 2002;20:1006-1010. [PubMed] [DOI] |
| 69. | Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, McIntyre E, Radford I, Villeval JL, Fraser CC, Cavazzana-Calvo M. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med. 2003;348:255-256. [PubMed] [DOI] |