CN117137936A - 以NOX1为靶点的小干扰siRNA对心肌缺血再灌注损伤的应用 - Google Patents
以NOX1为靶点的小干扰siRNA对心肌缺血再灌注损伤的应用 Download PDFInfo
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Abstract
本发明涉及一种小干扰核酸的制药应用,属于生物医药领域。一种针对NOX1靶基因表达的小干扰RNA在制备预防和治疗心肌缺血再灌注损伤药物中的用途。本发明小干扰siRNA通过舌下静脉注射方式给药,结果显示注射siRNA药物的大鼠心肌梗死面积明显降低,并能显著降低模型大鼠血清中LDH水平。实验结果表明,针对NOX1靶基因表达的siRNA有望能为治疗心肌缺血再灌注损伤的药物。
Description
技术领域
本发明涉及一种小干扰核酸的制药用途,属于生物医药领域,具体涉及一种针对NOX1靶基因表达的小干扰RNA在制备预防和治疗心肌缺血再灌注损伤药物中的用途。
背景技术
RNA干扰(RNA interference,缩写为RNAi)是一种由双链RNA诱发的基因表达沉默的现象,其机制是通过阻碍特定基因的转录或翻译来抑制基因表达。当向细胞内导入与内源性mRNA编码区同源的双链RNA时,该mRNA被降解而导致相应的基因表达沉默。人工合成的siRNA(Small interfering RNA,小干扰核酸)序列长度一般为19-23nt,可通过化学合成,具备高效性、易合成及易操作的特点,所以该技术已被广泛用于探索基因功能和传染性疾病及恶性肿瘤的基因治疗领域。
心血管疾病是全球范围引起死亡最高的疾病,是人类健康的头号杀手。缺血性心脏病是造成心血管疾病死亡最主要的原因。冠状动脉粥样硬化性心脏病为目前导致发达国家人口死亡的主要原因之一。随着经济的发展及人们饮食习惯的改变,冠状动脉粥样硬化性心脏病的发病率及死亡率均有持续上升趋势。而心肌梗死(myocardial infarction,MI)作为冠心病主要的发病形式之一,具有发病急、进展快及预后差等特点。在急性心肌梗死发作后的几分钟内,缺血中央区的心肌细胞便会由于缺血而死亡,是严重威胁人类健康和生命的常见病之一,近年来冠心病发病率和死亡率逐步上升。《中国心血管病报告2005》是我国发布的第一部中国心血管病权威报告,揭示了我国高血压、血脂异常、肥胖、糖尿病人群的患病率逐年增加,而上述疾病均为冠心病危险因素,冠心病已经成为威胁我国人民健康的主要疾病。
急性冠状动脉闭塞引发的急性心肌梗死是冠心病的主要类型之一,临床经过溶栓或冠状动脉介入术治疗能够及时有效的恢复心肌血液的灌注,减轻缺血导致的心肌损伤及坏死。但是,心肌缺血后血液再灌注也会造成心肌损伤,影响心功能甚至危及生命,这一现象被称为心肌缺血再灌注损伤。心肌缺血再灌注损伤已经成为影响冠心病尤其是急性心肌梗死血运重建治疗效果的重要因素之一,主要表现为再灌注诱发的心律失常、心肌顿抑、微血管阻塞、致死性心肌再灌注损伤。研究发现心肌缺血再灌注损伤与细胞凋亡、氧自由基释放、细胞内钙超载、心肌能量代谢障碍、炎性反应和血管内皮细胞损伤等有关。缺血再灌注损伤不仅涉及缺血器官本身,还可能导致远处器官的全身性损伤,可能导致多***器官衰竭。
心肌缺血再灌注的过程中,心肌组织中ATP的生成将会减少,细胞内钙离子将会增多,从而激活了钙依赖性蛋白酶,使黄嘌呤脱氢酶(xanthinedehydrogenase,XD)向有害的黄嘌呤氧化酶(xanthineoxidase,XO)转变增多,同时在ATP分解过程中会产生次黄嘌呤。在黄嘌呤氧化酶两次催化过程中均会产生大量氧自由基;此外钙超载能够激活磷脂酶A2,进而发生过氧化反应降解细胞膜磷脂,也会产生大量的氧自由基。氧自由基能够氧化细胞内的多种有机物质,例如蛋白质、酶和核酸等,从而引起线粒体功能障碍,增加细胞膜的通透性,进而使细胞内的信号传导发生障碍,促使心肌细胞大量凋亡。在心肌缺血再灌损伤过程中,ROS起到主要作用。在这个过程中,NOX1的过度激活起到了关键作用。
发明内容
本发明要解决的技术问题是:一种小干扰核酸的制药用途。
为实现上述目的,本发明采用以下技术方案:
一种针对NOX1靶基因表达的小干扰RNA在制备预防和治疗心肌缺血再灌注损伤药物中的用途。
所述NOX1靶基因具有序列表SEQ ID NO:1所示的碱基序列、或其同源度不低于80%的序列。
所述针对NOX1靶基因表达的小干扰RNA还含有药学上可接受的载体,所述药学上可接受的载体为阳离子聚合物、脂质纳米粒、无机纳米粒、抗体、多肽及具备靶向递送功能的小分子化合物,其中所述抗体能靶向目标脏器中高表达的特异性抗原或受体,包括抗体和抗体片段(Fab,Fv,单链抗体,纳米抗体),所述小分子化合物为半乳糖、葡萄糖、甘露糖、叶酸、透明质酸、脂肪酸链、DUPA(2-[3-(1,3-二羧基丙基)-脲基]戊二酸)谷氨酸脲。
所述针对NOX1靶基因表达的小干扰RNA由正义链和与其反向互补的反义链组成,所述正义链具有5’-CUGAGUCUUGGAAGTGGAUCCUU-3’(序列表SEQ ID NO.2)的核苷酸序列或其同源序列,所述反义链具有5’-AAGGAUCCACUUCCAAGACUCAG-3’(序列表SEQ ID NO.3)的核苷酸序列或其同源序列,所述反义链与所述靶基因上的区域反向互补。为生成符合WIPOST.26的序列表,在本申请的序列表文件中,上述SEQ ID NO.1,SEQ ID NO.2和SEQ IDNO.3中的尿嘧啶“U”替换为英文字符“T”,本申请文件的说明书中,仍以生物学符号U来表示尿嘧啶。
所述正义链和反义链上分别自5’端起的前21个核苷酸进行2’-O-核糖修饰,和/或至少一个磷酸酯基为具有修饰基团的磷酸酯基;优选地,所述含有修饰基团的磷酸酯基为磷酸酯基中的磷酸二酯键中的至少一个氧原子被硫原子取代而形成的硫代磷酸酯基。这些修饰能够帮助核酸序列抵抗体内核糖核酸酶的降解作用,有助于提高核酸的体内稳定性。
所述2’-O-核糖修饰选自2’-O-核糖甲基修饰、2’-O-核糖氟代修饰和2’-O-MOE核糖修饰中的一种或几种。这些修饰的组合,能够提高核酸的敲低效率,并减少脱靶的反应。
优选的,所述正义链的修饰为5’-CsUsGAGUCUUGGAAGTmGmGmAmUmCmCmUmU-3’,所述反义链的修饰为mA-s-mA-s-mGmGmAmUmCCACUUCCAAGACUCAG,其中所述小写字母m表示该字母右侧的一个核苷酸的核糖基团为2’-甲氧基核糖基;小写字母s表示字母两侧的脱氧核糖核苷酸之间磷酸酯基为硫代磷酸酯基。硫代修饰能够提高核酸的体内稳定性,延长半衰期。
优选的,所述正义链和/或反义链的3’末端连接有1至4个额外的核苷酸,在所述正义链和反义链互补配对后形成至少一个由1至4个核苷酸构成的3’突出端。这些突出能够提高的核酸序列的敲低效率。
进一步优选地,所述3’突出端末尾为连续的2个脱氧胸腺嘧啶核苷酸dTdT或尿嘧啶核苷酸UU。dT或UU能够促进核酸序列形成RISC复合物,提高敲低效率。
优选的,所述正义链的修饰为5’-C-s-U-s-GAGUCUUGGAAGTGGAUCCUU-3′,所述反义链的修饰为-5′fA-s-fA-s-fG-fG-fA-fU-fC-CACUUCCAAGACUCAG-3’,其中所述小写字母f表示该字母右侧的一个核苷酸的核糖基团为2’-O-核糖氟代修饰;小写字母s表示字母两侧的脱氧核糖核苷酸之间磷酸酯基为硫代磷酸酯基。硫代修饰可以提高核酸的稳定性。
优选的,所述正义链和/或反义链的3’末端连接有1至4个额外的核苷酸,在所述正义链和反义链互补配对后形成至少一个由1至4个核苷酸构成的3’突出端。这些突出能够提高的核酸序列的敲低效率。
进一步优选地,所述3’突出端末尾为连续的2个脱氧胸腺嘧啶核苷酸dTdT或2个尿嘧啶核苷酸UU。dT或UU能够促进核酸序列形成RISC复合物,提高敲低效率。
本发明的优点如下:本发明将针对NOX1靶基因表达的siRNA,在大鼠心肌缺血再灌注造模前48h,通过舌下静脉注射1次药物,在灌注120min后,腹主动脉采血,制备血清、并取出心脏,行TTC染色。结果显示注射本发明siRNA药物,心肌梗死面积下降了约一半,且具有统计学意义。将本发明的siRNA修饰后用于上述大鼠心肌缺血再灌注模型后,结果显示大鼠心肌梗死面积明显降低,并能显著降低模型大鼠血清中LDH水平。实验结果表明,针对NOX1靶基因表达的siRNA有望能为治疗心肌缺血再灌注损伤的药物。
附图说明
下面结合附图对本发明的具体实施方式作进一步详细的说明。
图1TTC染色测定各组大鼠心肌梗死情况,其中:A:假手术组;B:溶媒模型组;C:空白模型组;D:NOX1 siRNA低剂量组(0.05mg/kg),E:NOX1 siRNA中剂量组(0.25mg/kg);F:NOX1 siRNA高剂量组(0.50mg/kg)
图2NOX1 siRNA对心肌缺血再灌注模型大鼠心肌梗死面积的影响
图3TTC染色测定各组大鼠心肌梗死情况,A:假手术组;B:模型组;C:NOX1 siRNAm2-低剂量组(0.05mg/kg);D:NOX1 siRNA m2-中剂量组(0.25mg/kg);E:NOX1 siRNA m2-高剂量组(0.50mg/kg);F:NOX1 siRNA m8-中剂量组(0.25mg/kg);G:阳性药组组(10mg/kg)
具体实施方式
实施例1:NOX1 siRNA对于大鼠心肌缺血再灌注模型的保护作用实验
一、实验药品及试剂
NC及NOX1 siRNA,invivo-jetPEI(polyplus),水合氯醛(批号:L10J10G90315,上海源叶生物科技有限公司),4%***组织固定液(批号:2009170725,北京益利精细化学品有限公司),TTC粉末(批号:BN30765,北京百瑞极生物科技有限公司)
siRNA:针对NOX1靶基因表达的siRNA合成如下序列,同时在序列的3’末端连接连续的2个脱氧胸腺嘧啶核苷酸dTdT(序列中未标注)。均由苏州贝信生物技术有限公司合成。
正义链:5’-CUGAGUCUUGGAAGTGGAUCCUU-3’;(SEQ ID NO.2)
反义链:5’-AAGGAUCCACUUCCAAGACUCAG-3’;(SEQ ID NO.3)
二、实验方法:
(一)RNA药物配制方法
1)siRNA溶液:配制含siRNA的无酶无菌水、10%葡萄糖溶液。
2)PEI溶液:将100mM invivo-jetPEI溶液加入到无酶无菌水中,轻轻涡旋并瞬离。
3)将上述2)PEI溶液滴加入到1)siRNA溶液中,轻轻涡旋瞬离。
4)室温孵育15min。
5)标记样品名称、配制日期等信息,用封口膜封住容器盖,置于4℃冰箱中备用。
(二)实验动物分组及给药
雄性SD大鼠适应性饲养一周后,根据体质量随机分为假手术组11只、溶媒模型组16只、空白模型组15只、NOX1 siRNA低剂量组16只、NOX1 siRNA中剂量组16只、NOX1siRNA高剂量组16只。各组于造模前48h舌下静脉注射1次相应的药物,假手术组和模型组大鼠注射NC溶液(5mL/kg),NOX1 siRNA低、中、高剂量组注射不同剂量NOX1siRNA溶液(0.05mg/kg、0.25mg/kg、0.50mg/kg,5mL/kg)。
(三)大鼠心肌缺血再灌注模型制备
大鼠称重后水合氯醛麻醉(0.35mL/100g),仰卧位固定于可移动鼠板上,气管插管,连接BL-420I信息化集成化信号采集与处理***,选择小动物呼吸机功能(潮气量6.0ml,呼吸比值5:4,呼吸频率80次/分)。连接心电图,剪开胸部皮肤、肌肉,在第2~3肋间,打开胸腔,暴露心脏,用6-0手术线,在主动脉圆锥与左心耳交界处下1mm处(冠状动脉左前降支)穿线,结扎,心电图QRS波幅加大、ST段抬高或降低,T波高耸或倒置作为缺血是否成功的判断标准。结扎45min后,剪开结扎线,实现再灌注,再灌注120min后,腹主动脉采血,制备血清、并取出心脏,行TTC染色。取结肠及肝组织冻存,用于后续检测。
(四)心肌梗死面积百分比的测定
取4g氯化三苯基氯化四氮唑(TTC)粉末溶于100mL生理盐水中,配制成4% TTC溶液一,另取2.2822g磷酸氢二钾晶体溶于生理盐水配成10mL的溶液二,取90mL溶液一、6mL溶液二混合,加生理盐水稀释至300mL,配成溶液三,避光4℃储存,待用。
梗死面积测定:摘取心脏,用生理盐水洗净余血,滤纸吸去多余水分,置-20℃冰箱20~30min,待心脏达到一定硬度后,自心室至心尖分切成4~5片,每片厚约1mm,用配制好的TTC溶液进行染色,置于37℃水浴锅中,避光孵育10~15min,梗死区呈灰白色或者白色,未梗死区因为含有完整的脱氢酶呈红色。染色完成后,将切片置于4%多聚甲醛中,24h后进行扫描拍照。经Image J软件分析记录每片心肌面积及梗死区面积、计算心肌梗死区面积占心肌总面积百分比。
三、实验结果:
(一)TTC染色结果
如图1所示。
(二)心肌梗死面积计算结果
如图2所示。图2为NOX1 siRNA对心肌缺血再灌注模型大鼠心肌梗死面积的影响。与NC对照组相比,空白模型组和NC模型组的大鼠有约15%的心肌梗死面积,而在NOX1siRNA的治疗下干预下,心肌梗死面积下降了约一半,且具有统计学意义。
实施例2:NOX1 siRNA治疗大鼠心肌缺血再灌注模型药效学研究
一、实验目的
考察受试样品对于大鼠心肌缺血再灌注模型下的药理药效。
二、实验材料
2.1实验仪器
信息化集成化信号采集与处理***(型号:BL-420I,成都泰盟软件有限公司),分析天平(型号:AEG-220,日本岛津公司),恒温水浴锅(型号:02810136,北京长安科学仪器厂)。
2.2实验药品及试剂
invivo-jetPEI(polyplus),无菌无酶水(批号:PWL071-Nov-10G),乐坦注射液(批号:2005227,浙江永宁药业股份有限公司),水合氯醛(批号:L10J10G90315,上海源叶生物科技有限公司),4%***组织固定液(批号:2009170725,北京益利精细化学品有限公司),TTC粉末(批号:BN30765,北京百瑞极生物科技有限公司),乳酸脱氢酶(LDH)测定试剂盒(批号:A020-2,南京建成生物科技有限公司)。
siRNA:针对实施例1中的NOX1的siRNA,做了如下2种修饰
第一种修饰序列NOX1 siRNA m2:
正义链(Sense):5’-CsUsGAGUCUUGGAAGTmGmGmAmUmCmCmUmUTT-3’
反义链(Antisense):5’-mA-s-mA-s-mGmGmAmUmCCACUUCCAAGACUCAGTT-3’
其中,小写字母m表示该字母右侧的一个核苷酸的核糖基团为2’-甲氧基核糖基;小写字母s表示字母两侧的脱氧核糖核苷酸之间磷酸酯基为硫代磷酸酯基。
第二种修饰序列NOX1 siRNA m8:
正义链(Sense):5’-C-s-U-s-GAGUCUUGGAAGTGGAUCCUUTT-3′
反义链(Antisense):5’-fA-s-fA-s-fG-fG-fA-fU-fC-CACUUCCAAGACUCAGTT-3’
小写字母f表示该字母右侧的一个核苷酸的核糖基团为2’-O-核糖氟代修饰;小写字母s表示字母两侧的脱氧核糖核苷酸之间磷酸酯基为硫代磷酸酯基。
2.3实验动物
SPF级雄性SD大鼠143只,体质量180~200g,购于北京斯贝福实验动物技术有限公司,动物许可证编号:SCXK(京)2019-0010,动物合格证号:No.110324221103517485。大鼠自购入起饲养温度为22±2℃,自由获取食物和饮用水,适应性饲养3d。实验过程中因手术造模死亡原因,剩余大鼠数量为119只。
三、实验方法
3.1 RNA药物配制方法
3.1.1 NC冻干粉(批号:NC-220530)配制方法:
1)配制RNA溶液:取3瓶20mg/瓶的NC-siRNA,使用10%海藻糖溶液溶解并转移至烧杯中,加10%海藻糖至150ml,测得RNA浓度为378.795ng/微升;
2)配制PEI溶液:称取PEI 59.6mg于烧杯中,加150ml10%海藻糖溶解备用;
3)将RNA溶液与PEI溶液混合后冻干。
3.1.2 NOX1 siRNA冻干粉(批号:NOX1 siRNA-220613)配制方法:
1)配制RNA溶液:称取142.71mg siRNA于烧杯中,加入300ml10%海藻糖溶解备用;
2)配制PEI溶液:称取PEI 121.58mg于烧杯中,加300ml10%海藻糖溶解备用;
3)将RNA溶液与PEI溶液混合后冻干。
3.1.3 NOX1 siRNA-m8配制方法
1)siRNA溶液:配制含2000μg NOX1 siRNA-m8 siRNA的无酶无菌水、10%葡萄糖溶液20ml。
2)PEI溶液:将480μl的100mM invivo-jetPEI溶液加入到9520μl无酶无菌水中,轻轻涡旋并瞬离。
3)将上述2)PEI溶液滴加入到1)siRNA溶液中,轻轻涡旋瞬离。
4)室温孵育15min。
5)标记样品名称、配制日期等信息,用封口膜封住容器盖,置于4℃冰箱中备用。
3.2实验动物分组及给药
实验动物随机分为假手术组16只、模型组21只、NOX1 siRNA m2-低剂量组21只、NOX1 siRNA m2-中剂量组22只、NOX1 siRNA m2-高剂量组21只、NOX1 siRNA m8-中剂量组21只,阳性药组21只。除阳性药组外,其余各组于造模前48h舌下静脉注射1次相应的药物,假手术组和模型组大鼠注射NC溶液(5mL/kg),NOX1 siRNA m2-低、中、高剂量组注射不同剂量NOX1 siRNA m2溶液(0.05mg/kg、0.25mg/kg、0.50mg/kg,5mL/kg),NOX1 siRNA m8-中剂量组注射NOX1 siRNA m8溶液(0.25mg/kg,5mL/kg),阳性药组大鼠于造模前连续3天腹腔注射乐坦注射液(10mg/kg,1mL/100g)。
3.3大鼠心肌缺血再灌注模型制备
大鼠称重后水合氯醛麻醉(0.35mL/100g),仰卧位固定于可移动鼠板上,气管插管,连接BL-420I信息化集成化信号采集与处理***,选择小动物呼吸机功能(潮气量6.0ml,呼吸比值5:4,呼吸频率80次/分)。连接心电图,剪开胸部皮肤、肌肉,在第2~3肋间,打开胸腔,暴露心脏,用6-0手术线,在主动脉圆锥与左心耳交界处下1mm处(冠状动脉左前降支)穿线,结扎,心电图QRS波幅加大、ST段抬高或降低,T波高耸或倒置作为缺血是否成功的判断标准。结扎45min后,剪开结扎线,实现再灌注,再灌注120min后,腹主动脉采血,制备血清,每组取4只心脏组织冻存,其余大鼠心脏组织,进行TTC染色。取结肠及肝组织冻存,用于后续检测。
3.4心肌梗死面积百分比的测定
取4g氯化三苯基氯化四氮唑(TTC)粉末溶于100mL生理盐水中,配制成4% TTC溶液一,另取2.2822g磷酸氢二钾晶体溶于生理盐水配成10mL的溶液二,取90mL溶液一、6mL溶液二混合,加生理盐水稀释至300mL,配成溶液三,避光4℃储存,待用。
梗死面积测定:摘取心脏,用生理盐水洗净余血,滤纸吸去多余水分,置-20℃冰箱20~30min,待心脏达到一定硬度后,自心室至心尖分切成4~5片,每片厚约1mm,用配制好的TTC溶液进行染色,置于37℃水浴锅中,避光孵育10~15min,梗死区呈灰白色或者白色,未梗死区因为含有完整的脱氢酶呈红色。染色完成后,将切片置于4%多聚甲醛中,24h后进行扫描拍照。经Image J软件分析记录每片心肌面积及梗死区面积、计算心肌梗死区面积占心肌总面积百分比。
3.5检测大鼠血清LDH、CK-MB水平
采用大鼠LDH和CK-MB试剂盒,按照说明书分别测定血清LDH、CK-MB水平。
四、实验结果
4.1 NOX1 siRNA对心肌梗死面积的影响
TTC染色结果如表1、图3所示,与假手术组比较,模型组大鼠心肌梗死面积明显增大(P<0.01);与模型组相比,NOX1 siRNA m2各剂量组大鼠心肌梗死面积均有明显的降低,以NOX1 siRNA m2高剂量组降低最为显著(P<0.01),NOX1 siRNA m8剂量组大鼠心肌梗死面积明显降低,但无统计学差异;阳性药组大鼠心肌梗死面积可见明显减少。
表1 NOX1 siRNA对心肌缺血再灌注模型大鼠心肌梗死面积的影响
注:与假手术组比较,*P<0.05,**P<0.01;与模型组比较,#P<0.05,##P<0.01
4.2 NOX1 siRNA对大鼠血清LDH和CK-MB的影响
结果见表2,血清乳酸脱氢酶的结果显示,与假手术比较,模型组大鼠血清中LDH活性显著升高(P<0.01);与模型组比较,NOX1 siRNA m2低、高剂量组和NOX1 siRNA m8中剂量组均能显著降低模型大鼠血清中LDH水平(P<0.01)。
血清肌酸激酶同工酶MB的结果显示,与假手术比较,模型组大鼠血清中CK-MB活性显著升高(P<0.01);与模型组比较,NOX1 siRNA m8中剂量组和阳性药均能显著降低模型大鼠血清中CK-MB水平(P<0.01)
表2 NOX1 siRNA对心肌缺血再灌注模型大鼠心肌梗死面积的影响
注:与假手术组比较,*P<0.05,**P<0.01;与模型组比较,#P<0.05,##P<0.01
上文所列出的一系列的详细说明仅仅是针对本发明的可行性实施方式的具体说明,它们并非用以限制本发明的保护范围,本领域技术人员可以设计出很多其他的修改和实施方式,这些修改和实施方式将落在本申请公开的原则范围和精神之内。更具体地说,在本申请公开、附图和权利要求的范围内,可以对主题组合布局的组成部件和/或布局进行多种变型和改进。除了对组成部件和/或布局进行的变型和改进外,对于本领域技术人员来说,其他的用途也将是明显的。
Claims (12)
1.一种针对NOX1靶基因表达的小干扰RNA在制备预防和治疗心肌缺血再灌注损伤药物中的用途。
2.根据权利要求1所述的用途,其特征在于,所述NOX1靶基因具有序列表SEQ ID NO.1所示的碱基序列、或其同源度不低于80%的序列。
3.根据权利要求1所述的用途,其特征在于,所述小干扰RNA还含有药学上可接受的载体,所述药学上可接受的载体为阳离子聚合物、脂质纳米粒、无机纳米粒、抗体、多肽及具备靶向递送功能的小分子化合物,其中所述抗体能靶向目标脏器中高表达的特异性抗原或受体,包括抗体和抗体片段(Fab,Fv,单链抗体,纳米抗体),所述小分子化合物为半乳糖、葡萄糖、甘露糖、叶酸、透明质酸、脂肪酸链、DUPA(2-[3-(1,3-二羧基丙基)-脲基]戊二酸)谷氨酸脲。
4.根据权利要求1至3中任何一项所述的用途,其特征在于,所述针对NOX1靶基因表达的小干扰RNA由正义链和与其反向互补的反义链组成,所述正义链具有序列表SEQ ID NO.2所示的核苷酸序列或其同源序列,所述反义链具有序列表SEQ ID NO.3所示的核苷酸序列或其同源序列,所述反义链与所述靶基因上的区域反向互补。
5.根据权利要求4所述的用途,其特征在于:所述正义链和反义链上分别自5’端起的前23个核苷酸进行2’-O-核糖修饰,和/或至少一个磷酸酯基为具有修饰基团的磷酸酯基;优选地,所述含有修饰基团的磷酸酯基为磷酸酯基中的磷酸二酯键中的至少一个氧原子被硫原子取代而形成的硫代磷酸酯基。
6.根据权利要求5所述的用途,其特征在于:所述2’-O-核糖修饰选自2’-O-核糖甲基修饰、2’-O-核糖氟代修饰和2’-O-MOE核糖修饰中的一种或几种。
7.根据权利要求5或6所述的用途,其特征在于:所述正义链的修饰为5’-CsUsGAGUCUUGGAAGTmGmGmAmUmCmCmUmU-3’,所述反义链的修饰为5’-mA-s-mA-s-mGmGmAmUmCCACUUCCAAGACUCAG-3’,其中小写字母m表示该字母右侧的一个核苷酸的核糖基团为2’-甲氧基核糖基;小写字母s表示字母两侧的脱氧核糖核苷酸之间磷酸酯基为硫代磷酸酯基。
8.根据权利要求7所述的用途,其特征在于:所述正义链和/或反义链的3’末端连接有1至4个额外的核苷酸,在所述正义链和反义链互补配对后形成至少一个由1至4个核苷酸构成的3’突出端。
9.根据权利要求8所述的用途,其特征在于:所述3’突出端末尾为连续的2个脱氧胸腺嘧啶核苷酸dTdT或尿嘧啶核苷酸UU。
10.根据权利要求5或6所述的用途,其特征在于,所述正义链的修饰为5’-C-s-U-s-GAGUCUUGGAAGTGGAUCCUU-3′,所述反义链的修饰为-5′fA-s-fA-s-fG-fG-fA-fU-fC-CACUUCCAAGACUCAG-3’,其中小写字母f表示该字母右侧的一个核苷酸的核糖基团为2’-O-核糖氟代修饰;小写字母s表示字母两侧的脱氧核糖核苷酸之间磷酸酯基为硫代磷酸酯基。
11.根据权利要求10所述的用途,其特征在于:所述正义链和/或反义链的3’末端连接有1至4个额外的核苷酸,在所述正义链和反义链互补配对后形成至少一个由1至4个核苷酸构成的3’突出端。
12.根据权利要求11所述的用途,其特征在于:所述3’突出端末尾为连续的2个脱氧胸腺嘧啶核苷酸dTdT或尿嘧啶核苷酸UU。
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