CN1860127A - 对Syk激酶表达的抑制 - Google Patents
对Syk激酶表达的抑制 Download PDFInfo
- Publication number
- CN1860127A CN1860127A CNA2004800191126A CN200480019112A CN1860127A CN 1860127 A CN1860127 A CN 1860127A CN A2004800191126 A CNA2004800191126 A CN A2004800191126A CN 200480019112 A CN200480019112 A CN 200480019112A CN 1860127 A CN1860127 A CN 1860127A
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- CN
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- Prior art keywords
- cell
- sirna
- sirna molecule
- syk
- nucleotide
- Prior art date
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Abstract
本发明大体上涉及Syk激酶，特别涉及使用小干扰RNA(siRNA)来抑制Syk激酶表达的方法。
Description
本申请要求申请日为2003年7月3日的临时申请60/484,299的优先权，该临时申请的全部内容以参考的方式引入本文。
技术领域
本发明大体上涉及Syk激酶，特别涉及一种使用小干扰RNA(siRNA)抑制Syk激酶表达的方法。
背景技术
已显示在包括美丽线虫(C.elegans)和果蝇(Drosophila)以及植物在内的许多生物体中，双链RNA为一种用于干扰基因表达的有效物质(Bernstein等，RNA 7：1509～2151(2001)；McManus等，Nat.Rev.Genet.3：737～747(2992)；Hutvagner等，Curr.Opin.Genet.Dev.12：225～232(2002)；Zamore，Nat.Struct.Biol.8：746～750(2001)；Tuschl等，GenesDev.13：3191～3197(1999))。用双链RNA使哺乳动物的基因沉默引发了早期问题，因为哺乳动物的免疫系统通过例如干扰素应答等机制破坏了含有双链RNA的细胞，所述干扰素应答等机制逐渐成为抵制入侵性RNA病毒的防御措施(Clarke等，RNA 1：7～20(1995))。然而，已经证明被称为小干扰RNA(siRNA)的很短的RNA片段(例如，长度为20nt～23nt)，能避开免疫应答。因此导入的siRNA能在哺乳动物细胞中很好地发挥基因沉默剂的功能(Elbashir等，Nature 411：494～498(2001)；Elbashir等，Genes Dev.15：188～200(2001)；Paddison等，Genes Dev.16：948～958(2002)；Wianny等，Nat.Cell Biol.2：70～75(2000))。
据目前已知，RNAi(RNA干扰)包括一个多步骤的过程。双链RNA由内切核酸酶Dicer(切酶)剪切产生21～23个核苷酸的片段(siRNA)。siRNA双链体解离成2个单链RNA，一条链被加入至含有蛋白的复合体，在这种情况下，该链发挥作为向导RNA的功能而指导靶RNA的剪切(Schwarz等，Mol.Cell.10：537～548(2002)；Zamore等，Cell 101：25～33(2000))，从而使特定的遗传信息沉默(也见Zeng等，Proc.Natl.Acad.Sci.100：9779(2003))。
反义DNA也被广泛地用于抑制基因表达(Roth等，Annu.Rev.Biomed.Eng.1：265～297(1999))。一旦在细胞内部，反义寡核苷酸(ASO)识别并紧紧地结合于互补的mRNA，从而阻止mRNA与细胞的蛋白质翻译组件的相互作用。
已经证明，通过用Syk激酶mRNA的ASO抑制Syk激酶的表达，显著地降低了Fcγ受体信号传导(Matsuda等，Molec.Biol.of the Cell 7：1095～1106(1996))，以及通过气溶胶导入大鼠肺部的Syk激酶mRNA的ASO防止了Fcγ受体诱导的肺部炎症(Stenton等，J.Immunol.169：1028～1036(2002))。
至少在某些系统中，siRNA比ASO作为基因表达的抑制剂更加有效和可靠。本发明产生自设计测试siRNA作为Syk激酶表达的抑制剂的功效的研究。
发明内容
本发明大体上涉及Syk激酶。在一个优选实施方案中，本发明涉及一种使用小干扰RNA(siRNA)抑制Syk激酶表达的方法和基于该方法的治疗性策略。
从以下描述中，会清楚本发明的目的和优点。
附图说明
图1.除了起始模板的腺嘌呤二聚体和末尾突出端的胸苷二聚体以外，各Syk激酶的siRNA的有义链是与靶序列相同的序列。siRNA的反义链是靶序列的反向互补体。
图2.用靶向于Syk激酶mRNA的siRNA转染的RBL-2H3细胞中Syk激酶的表达。RBL-2H3细胞是用siRNA-1(第2泳道)、siRNA-2(第3泳道)或脂转染胺(lipofectamine)转染对照(第1泳道)转染的。细胞溶解物中的蛋白质通过SDS-PAGE(十二烷基磺酸钠-聚丙烯酰胺凝胶电泳)分离并转移至硝酸纤维素上。上面为Syk激酶的免疫印迹；下面为肌动蛋白的免疫印迹。
图3.用靶向于Syk激酶mRNA的siRNA转染的人单核细胞中Syk激酶的表达。单核细胞是用siRNA(第2泳道)或脂转染胺转染对照(第1泳道)转染的。细胞溶解物中的蛋白质通过SDS-PAGE分离，转移至硝酸纤维素上，并用抗Syk激酶抗体进行免疫印迹。
图4.蛋白质印迹分析：HS-24细胞中Syk蛋白的表达。
图5A和5B.(图5A)经siRNA处理后将HS-24细胞溶解，等量的HS-24细胞溶解物中的总蛋白质用10％SDS凝胶电泳分离，使用Syk或肌动蛋白的单克隆抗体通过蛋白质印迹进行分析。第1泳道：未处理；第2泳道：siRNA-1(对照)处理24小时；第3泳道：siRNA-1处理48小时；第4泳道：siRNA-2处理24小时；第5泳道：siRNA-2处理48小时。(图5B)分离RNA并对Syk和β-肌动蛋白进行RT-PCR(反转录-聚合酶链式反应)。第1泳道：未处理；第2泳道：siRNA-1(对照)处理48小时；第3泳道：siRNA-2处理48小时。
图6A和6B.将在包被有聚赖氨酸的平板上涂布的HS-24细胞(非刺激的、静息的)或包被有纤连蛋白的平板上涂布的HS-24细胞(刺激的)用siRNA-2或siRNA-1(对照)或四羟反式芪(piceatannol)处理。过夜培养期间用10ng/ml的TNF(肿瘤坏死因子)处理细胞。(图6A)用siRNA(48h)或四羟反式芪(16h)处理后，除去细胞，用抗CD54(ICAM-1)免疫染色，通过流式细胞计数器进行分析。(图6B)用IL-6ELISA(酶联免疫吸附测定)试剂盒分析细胞培养上清液的IL-6释放。与用TNF刺激的未处理细胞(例如未用siRNA处理)相比较，*P＜0.05、**P＜0.005。结果代表了3至5个独立的实验。该数据显示通过siRNA-2抑制了Syk的表达，向下调节了TNF诱导的ICAM-1(细胞间黏着分子-1)的表达和IL-6的释放，这在炎症反应中是重要的。
图7A和7B.三次处理后，靶向于Syk激酶的siRNA(经气溶胶传递)对卵清蛋白(OA)致敏并攻击的Brown Norway大鼠的支气管肺泡灌洗(BAL)液中全部细胞数目的影响。(图7A提供了柱状图数据，图7B显示了单个体数据点(单个动物)。
图8A～8D.三次处理后，靶向于Syk激酶的siRNA(经气溶胶传递)对OA致敏并攻击的Brown Norway大鼠的BAL液中的巨噬细胞、嗜中性粒细胞、淋巴细胞和嗜曙红细胞的数目的影响。(图8A提供了柱状图数据，图8B显示了巨噬细胞数目的单个体数据点(单个动物)，图8C显示了嗜中性粒细胞数目的单个体数据点，图8D显示了嗜曙红细胞数目的单个体数据点)。
具体实施方式
本发明涉及靶向于Syk激酶mRNA的RNA分子。例如，本发明涉及长度为大约19、20或21至大约23个核苷酸的RNA分子，其指导Syk激酶mRNA的剪切和/或降解。
在一个优选的实施方案中，本发明涉及siRNA分子的用途，该siRNA分子为典型地包含2条20个核苷酸～23个核苷酸(nt)的链的双链RNA分子。适用于本发明的siRNA能使用各种方法中的任一方法产生。可在体外制备siRNA，然后将其直接导入细胞中(例如通过转染)。可选择地，通过转染到在细胞内表达siRNA的细胞构建体(例如基于DNA的载体或表达盒)可实现细胞内的表达。
更具体地，例如通过化学合成、体外转录、使用核酸酶III型酶如切酶或核酸酶III酶解较长的dsRNA、由siRNA表达质粒或病毒载体在细胞中表达，或者由源自PCR的siRNA表达盒在细胞中表达，可制备适用于本发明的siRNA。这些各种方法的详细描述可容易地获得，并且可例如在
http：//www.ambion.com/techlib/tn/103/2.html、
www.bdbiosciences. com、
www.oligoengine.com、
www.genetherapysystems.com、
www. dharmacon.com、http：//www.mpibpc.gwdg.de/abteilungen/100/105/sirna.html和/或在此引用的参考文献中(这些参考文献也以参考的方式引入本文)找到。(也见Sui等，Proc Natl Acad Sci USA 99：5515-20(2002)；Brummelkamp等，Science 296：550-3(2002)；Paul等，Nature Biotechnology20：505-8(2002)；Lee等，Nature Biotechnology 20：500-5(2002)；Castanotto等，RNA 8：1454-60(2002)和美国申请20030108923)
可利用各种方法提高本发明的RNA的稳定性(见例如美国申请20020086356、20020177570和20020055162和美国专利6,197,944、6,590,093、6,399,307、6,057,134、5,939,262和5,256,555和它们引用的参考文献)。
如上所述，适用于本发明的siRNA可以用化学方法制备。优选在合成过程中利用例如，酸不稳定的原酸酯保护基团对2′羟基进行保护以防止降解(见Scaringe等，J.Am.Chem.Soc.120：11820(1998)和www.dharmacon.com(例如其中所描述的ACE技术))。RNA寡聚体在使用前可同时进行2′去保护和退火。
在化学合成的siRNA中，双链分子的至少1条链可具有长度为大约1至大约6个核苷酸(例如嘧啶和/或嘌呤核苷酸)的3′突出端。优选长度为大约1至大约5个核苷酸(例如胸苷或尿苷)，更优选大约1至大约4个核苷酸，最优选2或3个核苷酸的3′突出端。有利地，每一条链均具有突出端。各条链突出端的长度可以相同的或不同。典型地，两条链具有相同长度的突出端。在一个特别的实施方案中，本发明的RNA包含21或22个核苷酸的链，其是成对的且在两条RNA链的3′末端具有大约1至3，特别是大约2个核苷酸的突出端。
如上所述，适用于本发明的siRNA可通过使用核酸酶III型酶(例如切酶)酶解较长的dsRNA而制备。(见上述引用的参考文献和网址。)例如，可以使用商购可获得的切酶siRNA产生试剂盒，其允许由全长靶基因产生大量的siRNA(Gene Therapy Systems，Inc，MV062603)。使用基于PCR的克隆，可由靶DNA和T7RNA聚合酶启动子序列产生siRNA。在靶序列的RNA转录后，重组切酶可以将转录的RNAi(干扰RNA)剪切成22bp的siRNA。
也如上所述，也可使用本领域已知的方法重组产生适用于本发明的siRNA分子。(见上述引用的参考文献和网址。)重组技术允许在哺乳动物细胞中体内转录siRNA。根据该方法，可以使用包含例如RNA聚合酶III或U6启动子序列的载体。这样的载体(包括病毒载体和质粒载体(如pSIREN))可用作与病毒系统(例如腺病毒或逆转录病毒系统)联合的表达载体或穿梭载体，以将siRNA导入哺乳动物细胞中。载体可被工程化以表达siRNA的有义链和反义链，它们在体内退火以产生功能性siRNA。可选择地，可通过将靶的有义链(例如大约20nt)、接着将短的间隔(例如大约4至大约10nt)、然后将靶的反义链(例如大约20nt)，和例如作为转录终止子的大约5～6个T插入到载体中，来表达发夹RNA。所得RNA转录物回折，以形成包含例如大约20bp的茎和大约10nt的环以及3′末端的2～3个U的茎环结构。(也见Paddison等(Proc.Natl.Acad.Sci.99：1443-1448(2002)。)本领域的技术人员能容易地设计适用于实现体内生产的构建体(包括载体和启动子的选择)，其将根据例如细胞/组织靶和所需功效而改变。
倘若dsRNA与靶向的Syk激酶的mRNA具有足够的同源性，则dsRNA可以用于本发明的方法中。可以例如通过查找Syk激酶cDNA中的靶基序“AA(N)19”来设计siRNA双链体，其中N是任意核苷酸，优选G/C含量大约为30％至70％的基序，更优选G/C含量大约为50％的基序。siRNA双链体的有义链可对应于所选择的AA(N)19基序的核苷酸3至核苷酸21。siRNA双链体的反义链可具有所选择的AA(N)19基序的核苷酸1至核苷酸21的互补序列。进一步的设计细节提供于http：//www.mpibpc.gwdg.de/abteilungen/100/105/sirna.html。
优选的靶序列包括Syk激酶mRNA特有的序列。例如，靶序列可以选自介于Syk激酶的2个SH2结构域之间的序列或介于第二个SH2结构域和激酶结构域之间的序列。某些特殊的DNA靶序列描述在以下非限制性的实施例中。另外的靶包括但不限于如下的靶：
*序列 ％GC 经鉴定的16～18/19个核苷酸的同源性
AATATGTGAAGCAGACATGGA 42 线粒体核糖体prot15
AATCAAATCATACTCCTTCCC 42
AAGAGAGTACTGTGTCATTCA 42
AAGGAAAACCTCATCAGGGAA 47 肌醇六磷酸激酶，Chr1上的β球蛋白
AATCATACTCCTTCCCAAAGC 47
AATTTTGGAGGCCGTCCACAA 53 催产素酶(oxytokinase)
AAGACTGGGCCCTTTGAGGAT 58
AAGCAGACATGGAACCTGCAG 58 组胺受体H3，GTP结合蛋白
AACTTCCAGGTTCCCATCCTG 58
AAGCCTGGCCACAGAAAGTCC 63
AAGCCCTACCCATGGACACAG 63
AACCTGCAGGGTCAGGCTCTG 68
AAGGGGTGCAGCCCAAGACTG 68 γ谷氨酰转移酶，rb prot L27a
AACTTGCACCCTGGGCTGCAG 68 钙通道α1E亚单位
AAGTCCTCCCCTGCCCAAGGG 74 NADH；泛醌氧化还原酶MLRQ亚单位
AAGGCCCCCAGAGAGAAGCCC 74
AATCTCAAGAATCAAATCATA 26
AATGTTAATTTTGGAGGCCGT 42
AATCCGTATGAGCCAGAACTT 47
AATCGGCACACAGGGAAATGT 53
AACCGGCAAGAGAGTACTGTG 58
AAGGAGGTTTACCTGGACCGA 58
可以采用各种方式使用本文描述的siRNA。例如，siRNA分子在细胞或生物体中可用于靶向Syk激酶mRNA。在一个具体的实施方案中，可将siRNA导入人细胞或人体中以介导所述细胞或所述个体的细胞中的RNAi，从而预防或治疗疾病或与Syk激酶表达有关的不期望的症状(例如肺部、关节、眼睛或膀胱的炎症)。siRNA也可用于治疗血细胞的免疫损坏，例如自身免疫性溶血性贫血症中的红细胞和免疫性紫色血小板减少症(ITP)中的血小板(例如通过在巨噬细胞、脾和肝细胞中靶向Syk激酶mRNA)。根据该方法，靶向Syk激酶基因并通过RNAi降解相应的mRNA(靶向的Syk激酶基因的转录产物)。当肺细胞为靶时，可将包含siRNA的组合物雾化给药，例如经过吸入给药。对于关节的给药可以通过注射包含siRNA的溶液而实现。对于眼睛的给药可以通过例如注射或施用容器中装有包含siRNA的滴液而实现。对于膀胱等的给药，可以通过例如用包含siRNA的组合物清洗或冲洗靶组织而实现。对于皮肤的给药可通过局部给药(例如作为液体、乳剂或凝胶剂)。
根据本发明，个体的细胞(例如血单核细胞、嗜碱性细胞或肥大细胞)可进行离体(ex vivo)处理以实现Syk激酶mRNA的降解。待处理的细胞可使用已知方法获自所述个体，可将介导相应Syk激酶mRNA降解的siRNA导入细胞中，然后所述细胞再导入该个体中。
在一个具体的实施方案中，本发明涉及使用上述siRNA来抑制中介体(例如组胺)从具有Fcε受体的细胞如肥大细胞中释放。在哮喘的治疗方面，例如抑制组胺(一种肥大细胞中介体)的释放具有重要的疗效。
本发明的siRNA(或适用于实现细胞内生产siRNA的构建体)可以全身给药(例如通过IV(静脉注射))或直接施用于靶组织(例如通过气溶胶给药至肺)。使用这里描述的技术(包括脂质体制剂)可实现传递。除了脂质体制剂外，可使用聚合物制剂。聚乙烯亚胺(PEI)是合适的阳离子聚合物的例子。可使用不同大小的PEI，包括线性的22kDa和分枝的25kDa的PEI(也可使用其它大小的、修饰和未修饰的，以及生物可降解形式的PEI)。使用例如无毒性的病毒传递系统(例如腺相关病毒传递系统)也可以实现传递。最佳的剂量要根据患者、siRNA、给药方式和所需功效而定。本领域的技术人员不需要进行过度的试验就可确定最佳的条件。
本发明的某些方面可在以下非限制性的实施例中进行更加详细的描述。(也见美国申请20030084471、20030108923和20020086356)。
实施例1
实验详述
试剂
脂转染胺2000和Opti-Mem购自Invitrogen(San Diego，CA)。Eagle′sMEM(EMEM)、FCS、青霉素和链霉素购自Life Technologies(Grand Island，NY)。兔抗鼠Syk激酶多克隆抗体(Ab)和抗肌动蛋白Ab购自Santa CruzBiotechnology(Santa Cruz，CA)，F(ab′)2山羊抗兔Ab由Jackson实验室(Bar Harbor，ME)提供。化学发光试剂购自DuPont NEN(Boston，MA)。
细胞和细胞系
获自宾夕法尼亚大学健康志愿者的外周血单核细胞按照以前所述进行分离(Matsuda等，Molec.Biol.of the Cell 7：1095～1106(1996))。简言之，将肝素化的血液在Ficoll-Hypaque(淋巴细胞分离介质；OrganonTeknika，Durham，NC)上离心，将界面细胞重悬浮于含有RPMI 1640(GIBCO BRL，Grand Island，NY)、10％热灭活的胎牛血清(FCS)(Intergen，Purchase，NY)和2mM L-谷氨酰胺的完全培养基中。使细胞在37℃粘附于预先用FCS包被的组织培养瓶30min。45min～90min之后，通过Hanks平衡盐溶液大量清洗而除去未粘附细胞。通过剧烈搅动收获细胞。根据锥虫蓝排除法判断单核细胞一般＞98％存活。在37℃的5％CO2中，将所分离的单核细胞保持在补充有L-谷氨酰胺(2mM)和10％热灭活的FCS的RPMI 1640中。
在37℃的5％CO2中，将大鼠嗜碱性细胞(RBL-2H3)在补充有17％FBS、100U青霉素、100μg/ml链霉素和4mM谷氨酰胺的EMEM中培养。
siRNA双链构建体
Syk激酶的siRNA由Dharmacon Research Inc.(Lafayette，Co)制备。在根据生产商提供的指南来设计siRNA时，首次鉴定了人Syk激酶RNA中潜在的siRNA靶(位于AA碱基对直接下游的19个核苷酸)。然后将在大鼠和小鼠的Syk激酶RNA序列中扫描这些位点以鉴定这些物种中共有的Syk激酶RNA靶序列。鉴定了2个合适的位点，构建了2条21个核苷酸单体(21-mer)的RNA，每一条RNA由19个互补核苷酸和3′末端非互补胸苷二聚体组成(Elbashir等，Nature 411：494～498(2001))。除了末端的胸苷二聚体外，siRNA的有义链是与靶mRNA序列相同的序列。siRNA的反义链是靶序列的反向互补体。
1)siRNA-1：人，bp 296至bp 316；小鼠和大鼠，bp 307至bp 327。
有义5′-gaagcccuucaaccggccc dTdT 3′
反义3′-dTdT cuucgggaaguuggccggg 5′
2)siRNA-2：人，bp 364至bp 382；小鼠和大鼠，bp 375至bp 393
有义5′-ccucaucagggaauaucug dTdT 3′
反义3′-dTdT ggaguagucccuuauagac 5′
转染
通过转染将siRNA导入到RBL-2H3细胞和单核细胞中。对于转染，24孔板的每一孔中接种5×104RBL-2H3细胞或1×105单核细胞。24小时后，用400μl缺少血清和抗生素的新鲜培养基替换完全培养基，将siRNA/脂转染胺2000复合物加入到每孔中。对于RBL细胞，根据生产商规定，将3μl siRNA双链体(20μM)和3μl脂转染胺2000加入到100μl无血清或抗生素的Opti-mem中形成siRNA/脂转染胺2000复合物。对于单核细胞，将3μl siRNA双链体(20μM)和1μl脂转染胺2000加入到100μl无血清或抗生素的Opti-mem中形成siRNA/脂转染胺2000复合物。在用蛋白质印迹检测激酶蛋白的表达前，将细胞在37℃孵育48小时。
Syk激酶蛋白的蛋白质印迹分析
通过在Laemmli样品缓冲液(2％SDS，10％甘油，100mM DTT(二硫苏糖醇)和60mM Tris(pH 6.8))中将细胞煮沸5分钟来制备溶解物。用SDS-PAGE(10％聚丙烯酰胺)分离所述溶解物中的蛋白质并转移至样品缓冲液(25mM Tris，190mM甘氨酸和20％甲醇)中的硝酸纤维素膜上。在用山羊抗兔HRP(辣根过氧化物酶)孵育(室温1.5h)之前，将硝酸纤维素膜在4℃用1μl/ml的兔抗鼠Syk激酶多克隆抗体(Ab)孵育过夜。根据生产商规定，用化学发光试剂使膜上的蛋白质条带显现。检测Syk激酶蛋白之后，将膜在含有100mM 2-ME(2-巯基乙醇)、2％SDS和62.5mM Tris-HCl(pH 6.7)的剥离缓冲液中于50℃孵育30分钟并偶尔搅动，以除去抗Syk激酶抗体。然后用抗肌动蛋白抗体再次探查该膜并用化学发光试剂使膜上的条带显现。通过密度测定法定量Syk激酶的蛋白水平(Personal Densitometer，Molecular Dynamics)。
结果
siRNA对Syk激酶的表达的作用
用定向于大鼠、小鼠和人Syk激酶RNA的共有序列的siRNA转染大鼠嗜碱性细胞(RBL-2H3细胞)和人单核细胞。用抗Syk激酶抗体通过蛋白质印迹来分析Syk激酶蛋白的表达(图2和3)。用siRNA处理的RBL细胞中Syk激酶表达的抑制示于图2，表1中提供了Syk激酶蛋白的水平，该水平用肌动蛋白的水平进行标准化。肌动蛋白是用作检测蛋白表达的标准的通用蛋白。相对于未处理的RBL细胞(第1泳道)，siRNA处理的RBL细胞中Syk激酶蛋白的表达被抑制了45％～51％(图2，第2和3泳道)。在培养细胞中用siRNA抑制Syk激酶基因表达的量是鼓舞人心的，因为先前采用Syk激酶mRNA ASO的实验显示在体外培养的非增殖性细胞如单核细胞与在增殖性细胞相比可达到更高的Syk激酶抑制水平。还观察到保持培养状态的单核细胞中siRNA对Syk激酶表达的抑制(图3)。siRNA处理的单核细胞中Syk激酶的表达被抑制(图3，第2泳道)。此外，siRNA处理的U937细胞和siRNA处理的THP-1细胞中Syk激酶的表达被抑制(U937和THP-1是巨噬细胞样细胞系)。由此这些数据证明了针对Syk激酶RNA的siRNA有效抑制该基因的表达，并显示针对Syk激酶RNA的siRNA可用作有效的抗炎症治疗工具。
表1.RBL细胞中Syk激酶表达的密度测定法定量
| 转染对照 | Syk激酶siRNA1 | Syk激酶siRNA2 | |
| Syk*Syk修正**％Syk抑制 | 2040-0 | 953111445 | 62799751 |
*密度测定单位
**每泳道中经％肌动蛋白表达量修正的Syk密度测定单位
讨论
这些研究中所使用的siRNA是化学合成的(Dharmicon Research Inc.，Lafayette，CO)，但也可通过重组技术制备siRNA。siRNA双链体可包含21个核苷酸的有义链和21个核苷酸的反义链，以具有2个核苷酸(dT)的3′突出端的方式配对。
SiRNA的靶向的区域可以是选自所设计的开始于起始密码子下游50至100核苷酸的cDNA序列中的序列AA(N19)(N为任意核苷酸)。优选G/C含量大约为50％。由于只在第一个转录的核苷酸是嘌呤时，从pol III启动子来表达RNA才有效，因此优选有义和反义siRNA起始于嘌呤核苷酸，从而不改变靶向位点它们就能从pol III表达载体进行表达。
在上述研究中，选择人Syk激酶mRNA中潜在的siRNA靶，然后扫描大鼠和小鼠Syk激酶mRNA中的序列，以鉴定这些物种中共有的Syk激酶mRNA靶序列。鉴定了2个合适的靶序列：
靶向的区域(1)(cDNA)：5′aagaagcccttcaaccggccc；
靶向的区域(2)(cDNA)：5′aacctcatcagggaatatctg。
靶序列和Syk激酶siRNA示于图1。双链体的RNA非常不容易受到核酸酶的降解，使用脱氧核苷酸(胸苷(T)而不是尿苷(U))可影响3′突出端的稳定性。
实施例2
用3μl siRNA-1(对照)
DNA靶：5′AAGAAGCCCTTCAACCGGCCC
有义siRNA 5′-gaagcccuucaaccggccc dTdT 3′
反义siRNA 3′-dTdT cuucgggaaguuggccggg 5′
或siRNA-2
DNA靶：5′AACCTCATCAGGGAATATCTG
有义5′-ccucaucagggaauaucug dTdT 3′
反义3′-dTdT ggaguagucccuuauagac 5′或Syk反义链，和脂转染胺2000在12孔板中预处理HS-24细胞(2×105)24或48hr，用10ng/ml的TNF刺激过夜。读取：培养上清液中的IL-6(ELISA)和ICAM-1的细胞表面表达(流式细胞技术)。如蛋白质印迹分析所示(见图4)，48hr处理后，siRNA-2引起Syk蛋白表达的减少。
更具体地，用siRNA-2或siRNA-1(对照)瞬时转染HS-24细胞。然后检测ICAM-1的细胞表面表达，以及IL-6的释放。用siRNA-2但不用siRNA-1处理HS-24细胞48小时，同时显著地抑制了Syk蛋白(图5A)和mRNA(图5B)的表达。
HS-24细胞组成性表达的低水平ICAM-1(图6A)不受siRNA-2处理的影响(未显示)。过夜培养期间用10ng/ml的TNF刺激HS-24细胞，同时引起了在静息细胞(置于聚L-赖氨酸上)和粘附于纤连蛋白的细胞中ICAM-1表达的显著增加(图6A)。在用TNF刺激后，粘附于纤连蛋白的细胞相对于粘附于聚L-赖氨酸的细胞显示出更高的ICAM-1的表达(P＜0.05)。但是在铺有纤连蛋白的HS-24细胞中用siRNA-2进行的转染向下调节TNF诱导的ICAM-1的表达(P＜0.005)，但是对于铺有聚L-赖氨酸的细胞中的ICAM-1没有显著的影响。用药理学上的Syk抑制剂四羟反式芪(10μM)过夜处理，则在聚L-赖氨酸和纤连蛋白粘附条件下，TNF刺激的细胞中都引起了显著的ICAM-1的向下调节(图6A)。正如锥虫蓝染料排除测定，用siRNA或四羟反式芪处理细胞对于存活率没有显著影响(在所有实验中，存活率为＞96％)。
虽然无TNF刺激的HS-24细胞释放的IL-6是最小的，但在粘附于纤连蛋白的细胞的培养上清液中存在较高的IL-6水平的趋势(图6B)。如所预计的，在两种培养条件下，TNF刺激后观察到IL-6水平的巨大上升，而在纤连蛋白粘附的细胞中具有更高的水平(P＜0.05)。siRNA-2处理引起IL-6释放的向下调节(55％～58％)，其在纤连蛋白粘附的培养物中达到了统计学上的显著性(P＜0.05)。四羟反式芪几乎完全抑制TNF诱导的IL-6释放(图6B)。
因此，对Syk激酶的抑制向下调节了TNF诱导的ICAM-1表达和IL-6释放，它们为气道上皮中炎症反应的显著特点。该作用在粘附于纤连蛋白的细胞中是显著的，显示了Syk在参与这些有关的促炎事件中至少部分依赖于β1整联蛋白。
实施例3
在用卵清蛋白(OA)诱导哮喘的Brown Norway大鼠模型中体内研究靶向于Syk激酶的siRNA的作用。
如(Laberge等，Am.J.Respir.Crit.Care Med.151：822(1995))所述，用OA进行I.P.(腹膜内注射)使Brown Norway大鼠致敏并在致敏后第21天使用。
该实施例中所使用的siRNA如下所示：
DNA靶：5′AACCTCATCAGGGAATATCTG
有义5′-ccucaucagggaauaucug uu 3′
反义3′-uu ggaguagucccuuauagac 5′
关于图7和8中所使用的siRNA 2M的描述，参照上面，用Dharmacon修饰以提供额外的稳定性。关于图7和8中所使用的siRNA-2的设计，参照上面未修饰形式的序列。
使用无脂质体的单独siRNA或在形成siRNA/脂质体复合物后使用。如先前所述(见Stenton等，J.Immunol.169：1028(2002)及其所引用的参考文献)制备1，2二油酰基-3-三甲基铵-丙烷(DOTAP)/二油酰基-磷酯酰基-乙醇胺(DOPE)脂质体(Lipo)。将阳离子型DOTAP∶DOPE脂质体以脂质体比siRNA为2.5∶1的比率进行孵育，通过雾化后的气溶胶给予125微克siRNA(有或无脂质体)。
如Stenton等，J.Immunol.164：3790(2000)所述，进行靶向于Syk激酶的siRNA的雾化给药。如Stenton等，J.Immunol.169：1028(2002)所述，使用侧流雾化器雾化给予每只大鼠9毫升盐水、siRNA或siRNA/脂质体45min。24小时后，重复该过程，接着在第48小时进行第3次处理。第3次处理后立即将大鼠用雾化的盐水或含5％OA的盐水攻击5min。攻击24小时后，处死动物。
如Stenton等，J.Immunol.169：1028(2002)所述，进行支气管肺泡灌洗(BAL)。
对所分离的BAL细胞进行计数并用Cytospin制备细胞涂片。用HEMA-3试剂染色后以盲检的方式对细胞差异进行计数(BiochemicalSciences，Swedesboro，NJ)。
总之，在上述研究中使用OA诱导的过敏性哮喘和肺部炎症的BrownNorway大鼠模型。根据将细胞募集到BAL液来确定的肺部炎症，显著地受到的靶向于Syk激酶的siRNA(存在或不存在脂质体)的抑制。
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将上面引用的所有文献和其它信息来源的全部内容以参考的方式引入本文。
Claims (21)
1.一种在细胞中抑制Syk激酶表达的方法，该方法包括：将小干扰RNA分子导入所述细胞中，所述小干扰RNA分子指导所述细胞中存在的靶Syk激酶mRNA序列的剪切，从而实现所述的抑制。
2.如权利要求1所述的方法，其中所述的小干扰RNA分子的长度为大约20～23个核苷酸。
3.如权利要求1所述的方法，其中所述的小干扰RNA分子被直接导入所述细胞中。
4.如权利要求1所述的方法，其中所述的小干扰RNA分子是在编码该小干扰RNA分子的核苷酸序列导入所述细胞后在细胞内产生的。
5.如权利要求1所述的方法，其中所述的小干扰RNA分子包含2条链，且所述小干扰RNA分子的至少一条链具有长度为大约1～大约6个核苷酸的3′突出端。
6.如权利要求5所述的方法，其中所述小干扰RNA分子的两条链都具有长度为大约2～3个核苷酸的3′突出端。
7.如权利要求6所述的方法，其中所述的3′突出端包含尿苷或胸苷。
8.如权利要求1所述的方法，其中所述的小干扰RNA分子具有茎环结构。
9.如权利要求8所述的方法，其中所述茎环结构的3′末端具有长度为大约1～大约6个核苷酸的3′突出端。
10.如权利要求9所述的方法，其中所述3′突出端的长度为大约2～大约3个核苷酸。
11.如权利要求1所述的方法，其中所述的靶序列是Syk激酶mRNA特有的序列。
12.如权利要求11所述的方法，其中用cDNA表示的所述靶序列包含选自以下组的核苷酸序列：
AATATGTGAAGCAGACATGGA，
AATCAAATCATACTCCTTCCC，
AAGAGAGTACTGTGTCATTCA，
AAGGAAAACCTCATCAGGGAA，
AATCATACTCCTTCCCAAAGC，
AATTTTGGAGGCCGTCCACAA，
AAGACTGGGCCCTTTGAGGAT，
AAGCAGACATGGAACCTGCAG，
AACTTCCAGGTTCCCATCCTG，
AAGCCTGGCCACAGAAAGTCC，
AAGCCCTACCCATGGACACAG，
AACCTGCAGGGTCAGGCTCTG，
AAGGGGTGCAGCCCAAGACTG，
AACTTGCACCCTGGGCTGCAG，
AAGTCCTCCCCTGCCCAAGGG，
AAGGCCCCCAGAGAGAAGCCC，
AATCTCAAGAATCAAATCATA，
AATGTTAATTTTGGAGGCCGT，
AATCCGTATGAGCCAGAACTT，
AATCGGCACACAGGGAAATGT，
AACCGGCAAGAGAGTACTGTG，
AAGGAGGTTTACCTGGACCGA，和
AACCTCATCAGGGAATATCTG。
13.如权利要求1所述的方法，其中所述的细胞是哺乳动物细胞。
14.如权利要求13所述的方法，其中所述的细胞是人类细胞。
15.如权利要求14所述的方法，其中所述的细胞存在于人体中。
16.如权利要求15所述的方法，其中所述的人体是患有炎性症状的患者，并且所述小干扰RNA分子被施用足够的量以实现对所述症状的治疗。
17.如权利要求16所述的方法，其中所述的炎性症状是所述患者的支气管、肺部、眼睛、膀胱或皮肤的症状。
18.如权利要求17所述的方法，其中所述的炎性症状是支气管或肺部的症状，并且所述小干扰RNA分子通过吸入被导入支气管或肺部细胞中。
19.一种小干扰RNA分子，该小干扰RNA分子包含与选自以下组的序列互补的序列：
AATATGTGAAGCAGACATGGA，
AATCAAATCATACTCCTTCCC，
AAGAGAGTACTGTGTCATTCA，
AAGGAAAACCTCATCAGGGAA，
AATCATACTCCTTCCCAAAGC，
AATTTTGGAGGCCGTCCACAA，
AAGACTGGGCCCTTTGAGGAT，
AAGCAGACATGGAACCTGCAG，
AACTTCCAGGTTCCCATCCTG，
AAGCCTGGCCACAGAAAGTCC，
AAGCCCTACCCATGGACACAG，
AACCTGCAGGGTCAGGCTCTG，
AAGGGGTGCAGCCCAAGACTG，
AACTTGCACCCTGGGCTGCAG，
AAGTCCTCCCCTGCCCAAGGG，
AAGGCCCCCAGAGAGAAGCCC，
AATCTCAAGAATCAAATCATA，
AATGTTAATTTTGGAGGCCGT，
AATCCGTATGAGCCAGAACTT，
AATCGGCACACAGGGAAATGT，
AACCGGCAAGAGAGTACTGTG，
AAGGAGGTTTACCTGGACCGA，和
AACCTCATCAGGGAATATCTG。
20.一种包含如权利要求19所述的小干扰RNA分子和载体的组合物。
21.一种包含如权利要求19所述的小干扰RNA分子和脂质体或聚合物的组合物。
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| CA2447360A1 (en) * | 2001-05-09 | 2002-11-14 | Alk-Abello A/S | Pharmaceutical compositions for preventing or treating th1 and th2 cell related diseases by modulating the th1/th2 ratio. |
| DE60233574D1 (de) * | 2001-07-10 | 2009-10-15 | Univ R | Verfahren und zusammensetzungen zum nachweis des aktivierungszustands multipler proteine in einzelzellen |
| US7381535B2 (en) * | 2002-07-10 | 2008-06-03 | The Board Of Trustees Of The Leland Stanford Junior | Methods and compositions for detecting receptor-ligand interactions in single cells |
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| WO2004052364A1 (en) * | 2002-12-06 | 2004-06-24 | The Trustees Of Boston University | METHODS FOR SUSTAINING eNOS ACTIVITY |
| US20040219575A1 (en) * | 2002-12-26 | 2004-11-04 | Toomas Neuman | Methods and compositions for the diagnosis, prognosis, and treatment of cancer |
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| JP2007527240A (ja) * | 2004-03-01 | 2007-09-27 | マサチューセッツ インスティテュート オブ テクノロジー | アレルギー性鼻炎および喘息のためのＲＮＡｉベースの治療 |
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| US20070219152A1 (en) | 2007-09-20 |
| US7173015B2 (en) | 2007-02-06 |
| AU2004257167A1 (en) | 2005-01-27 |
| EP2371835A1 (en) | 2011-10-05 |
| WO2005007623A8 (en) | 2006-03-23 |
| JP2011200238A (ja) | 2011-10-13 |
| US20120093913A1 (en) | 2012-04-19 |
| CN100577680C (zh) | 2010-01-06 |
| CN101961497A (zh) | 2011-02-02 |
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