CN102888404A - siRNA缀合物及其制备方法 - Google Patents
siRNA缀合物及其制备方法 Download PDFInfo
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- CN102888404A CN102888404A CN2012103015512A CN201210301551A CN102888404A CN 102888404 A CN102888404 A CN 102888404A CN 2012103015512 A CN2012103015512 A CN 2012103015512A CN 201210301551 A CN201210301551 A CN 201210301551A CN 102888404 A CN102888404 A CN 102888404A
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Abstract
本发明提供一种siRNA-聚合物缀合物及其制备方法,更具体地,涉及一种通过siRNA和用于提高siRNA生物稳定性的聚合物的共价结合而形成的杂合缀合物,以及制备该杂合缀合物的方法。本发明的缀合物能提高siRNA的生物稳定性,从而实现治疗性siRNA向细胞的有效传递,且即使相对低浓度的小剂量也具有siRNA的活性。因此,该缀合物不仅可有利地用作癌症和其它传染性疾病的siRNA治疗工具,也可用作一种新型的siRNA传递系统。
Description
本申请要求申请日为2009年5月14日的韩国专利申请KR10-2009-0042297的优先权;本申请是申请日为2010年5月13日、发明名称为“siRNA缀合物及其制备方法”的中国专利申请No.201080021324.3(原申请,即第一次提出的申请)的分案申请。
技术领域
本发明涉及一种缀合物、制备该缀合物的方法以及利用该缀合物传递siRNA的方法,其中,在癌症和其它传染性疾病的基因疗法中用于促进siRNA传递的聚合物通过可降解键(degradable bond)或不可降解键缀合到siRNA。
背景技术
RNA干扰是指这样一种机制,其为在基因表达过程中,双链RNA(dsRNA)通过核苷酸序列特异性方式启动基因转录后沉默,并且这种机制首先在线虫(C.elegans)中被发现,并常见于植物、果蝇和脊椎动物(Fire等,自然(Nature),391:806-811,1998;;Novina &Sharp,自然(Nature),430:161-164,2004)。已知以如下方式发生RNA干扰:进入到细胞内的19~25bp的dsRNA与RISC(RNA-诱导的沉默复合物)结合,且仅反义(向导)链与mRNA结合而与mRNA的核苷酸序列互补,从而通过存在于RISC的核酸内切酶结构域降解靶mRNA(Rana,T.M.,Nat.Rev.Mol.Cell Biol.,8:23-36,2007;Tomari,Y.和Zamore,P.D.,Genes Dev.,19:517-529,2005)。
当dsRNA被传递到细胞中,它特异性地结合靶基因序列以降解mRNA,因此,它被认为是一种可调控基因表达的新型工具。然而,对于人类来说,由于向人体细胞引入dsRNA会诱导抗病毒干扰素途径,因此难以获得RNAi效果。在2001年,Elbashir和Tuschl等发现向人体细胞导入21nt长度(核苷酸长度)的小dsRNA不会引起干扰素途径,但特异性地降解靶mRNA(Elbashir,S.M.,Harborth,J.,Lendeckel,W.,Yalcin,A.,Weber,K.,Tuschl,T.,自然(Nature),411,494-498,2001;;Elbashir,S.M.,Lendeckel,W.,Tuschl,T.,基因(Genes)&Dev.,15,188-200,2001;;Elbashir,S.M.,Martinez,J.,Patkaniowska,A.,Lendeckel,W.,Tuschl,T.,EMBO J.,20,6877-6888,2001)。因此,21nt长度的dsRNA已经作为一种新型的功能基因组工具引起了公众的注意,并命名为小干扰RNA(siRNA)。
自从siRNA被报道在动物细胞中抑制特定基因表达上具有极好的效果,siRNA作为一种基因治疗的药物引起了广泛关注。实际上,由于它的高活性和精确的基因选择性,根据20年的研究结果,siRNA有望成为一种目前用作治疗药物的反义寡核苷酸(ODN)的替代治疗药物(Dana J.Gary等,控释杂志(Journal of Controlled Release)121:64-73,2007)。用于治疗的siRNA技术具有显著的优势,与其它药物相比,它易于设计并具有高目标选择性和抑制特定基因表达的性能。此外,由于RNA干扰利用生命系统中天然存在的机制抑制基因表达,所以它毒性较低。目前OPKO Inc.开发的湿性老年黄斑变性病的治疗药物“贝伐西尼(Bevasiranib)”为一种siRNA,其可选择性地作用于血管内皮生长因子(VEGF),诱导新血管形成以抑制VEGF的表达,并通过了三期临床试验(Dejneka NS等,Mol Vis.,28(14):997-1005,2008)。此外,目前正在研发含有靶向多种基因的siRNA的治疗药物(Ryan P.Million,Nature Reviews Drug Discovery 7:115-116,2008)。
尽管各种结果表明体内通过RNA干扰诱导特异性的表达抑制,但siRNA在体内的传递还有许多问题需要解决,如在血液中被酶降解、与血液中组分的相互作用以及非特异性地传递给细胞(ShigeruKawakami和Mitsuru Hashida,Drug Metab.Pharmacokinet.22(3):142-151,2007)。正在通过部分地利用抗核酸酶的核苷酸类似物或改良传递技术,尝试克服这些问题。
改良的传递技术的实例包括:利用病毒如腺病毒、逆转录病毒等的基因传递技术,以及利用脂质体、阳离子脂质和阳离子聚合物的非病毒载体的基因传递技术。然而,由于传递的基因有可能整合到宿主的染色体中,从而诱导宿主基因的正常功能发生异常并激活致癌基因,因此病毒载体存在安全性方面的问题,此外,即使以较小的量连续表达病毒基因也可能引起自身免疫性疾病,或在由病毒载体诱导的修饰的病毒感染情况下,可能不会引起有效的保护性免疫。同时,非病毒载体的有效性低于病毒载体,但考虑到体内安全性和经济可行性,其具有副作用低和生产成本低的优势(Lehrman S.,自然(Nature).401(6753):517-518,1999)。此外,非病毒传递方法要求有效地保护酶或非酶的降解,以便传递包括siRNA的RNA分子,其中一种方法为利用编码短发夹RNA(shRNA)的DNA表达质粒。通过DNA的系统的优势在于,仅当表达载体存在时进行siRNA表达。此外,目前siRNA化学修饰的研究提出一种用于改良针对核酸酶的稳定性以及低胞内摄取的方法(Shigery Kawakami和Mitsuru Hashida.Drug Metab.Parmacokinet.22(3):142-151,2007)。
在一种siRNA的化学修饰中,作为被核酸酶降解部位的磷酸二酯键用硫代磷酸连接进行修饰,或戊糖的2’位被2'-O-meRNA、2’-脱氧-2’-氟尿嘧啶核苷或通过2’位和4’位连接形成的锁核酸(LNA)修饰,结果提高了血清中的稳定性((Braasch D.A.等,Bioorg.Med.Chem.Lett.14:1139-1143,2003;;Chiu Y.L.和Rana T.M.,RNA,9:1034-1048,2003;;Amarzguioui M.等,Nucleic Acid Res.31:589-595,2003)。在另一种化学修饰中,官能团连接到正义(反向导)链的3’端区域,结果与对照相比,药动力学特性得到了改良,体内通过siRNA的亲水性和疏水性的平衡在施用时诱导出较高的效率(Soutschek J.等,Nature 432:173-1782004)。
然而,上述方法在保护siRNA免于核酸酶的消化以及改良细胞膜透性效率上仍然有许多有待改进之处。
基于这一原因,发明人发现了一种缀合物,其中亲水性或疏水性聚合物通过可降解或不可降解键缀合到siRNA,改良了siRNA的体内稳定性,并基于此完成了本发明。
发明内容
技术问题
本发明的一个目的在于提供一种缀合物,其中作为生物相容性聚合物的亲水性或疏水性聚合物通过可降解或不可降解键缀合到siRNA的正义链或反义链的末端,以提高siRNA在细胞内传递的效率。
本发明的另一个目的在于提供一种含有聚合物的固相支持物,具体地,施用到人体时稳定性得到证实的聚合物,例如聚乙二醇(PEG),以及提供一种有效地制备包括RNA、DNA、RNA-DNA嵌合体及其类似物的寡核苷酸的方法,其中,通过支持物将PEG结合到它的3’端。
本发明又一目的在于提供制备siRNA缀合物的方法,以及利用siRNA缀合物传递siRNA的方法。
技术方案
为实现上述的目的,首先,本发明提供一种siRNA-聚合物缀合物,其结构如下:
A-X-R-Y-B
(其中,A和B独立地为亲水性聚合物或疏水性聚合物;X和Y独立地为简单的共价键或接头介导的(linker-mediated)共价键;且R为siRNA)。
其次,本发明提供一种siRNA-聚合物缀合物,其结构如下:
A-X-R
(其中,A为疏水性聚合物;X为简单的共价键或接头介导的共价键;R为siRNA)
第三,本发明提供一种缀合物,其中siRNA(R)的单链含有19~31个核苷酸。
第四,本发明提供一种缀合物,其中疏水性聚合物(A)的分子量为250~1,000。
第五,本发明提供一种缀合物,其中疏水性聚合物(A)为C16~C50烃或胆固醇。
第六,本发明提供一种缀合物,其中共价键(X,Y)为不可降解键或可降解键。
第七,本发明提供一种缀合物,其中不可降解键为酰胺键或磷酸键。
第八,本发明提供一种缀合物,其中可降解键选自二硫键、酸裂解键、酯键、酸酐键、生物降解键和酶裂解键。
第九,本发明提供一种缀合物,其中亲水性聚合物(A或B)为分子量为1,000~10,000的非离子聚合物。
第十,本发明提供一种缀合物,其中亲水性聚合物选自下组:聚乙二醇(PEG)、聚乙烯吡咯烷酮和聚噁唑啉。
第十一,本发明提供一种结合聚乙二醇的固相支持物,其结构如下:
[其中,R为烷基、烯基、炔基、芳基、芳烷基、杂烷基或杂芳基;m为2~18的整数;n为5~120的整数;且X为氢、4-单甲氧基三苯甲基、4,4’-二甲氧基三苯甲基或4,4’,4”-三甲氧基三苯甲基]。
第十二,本发明提供一种结合聚乙二醇的固相支持物,其中固相支持物为可控孔度玻璃(CPG)。
第十四,本发明提供一种结合聚乙二醇的固相支持物,其为具有下述结构式IV的3'-PEG-CPG:
[结构式IV]
第十五,本发明提供一种制备3’-PEG-CPG的方法,该方法包括:
1)将CPG与3-氨丙基三乙氧基硅烷反应,形成长链烷基胺可控孔度玻璃(LCAA-CPG);
2)将聚乙二醇与4,4'-二甲氧基三苯甲基氯化物反应,形成2-[双-(4-二甲氧基三苯甲基)-聚(乙二醇)];
3)将步骤2)形成的化合物与下述化学式1的化合物反应,形成下述结构式I的化合物;
4)将形成的下述结构式I的化合物与4-硝基苯基氯甲酸酯反应,形成下述结构式II的化合物;
5)将步骤3)形成的下述结构式I的化合物与N-琥珀酰亚胺基三氟乙酸反应,形成下述结构式III的化合物;以及
6)将步骤1)形成的LCAA-CPG化合物分别与步骤3)至5)形成的下述结构式I、II和III的化合物反应。
[化学式1]
[结构式I]
[结构式II]
[结构式III]
[结构式IV]
[其中,R为烷基、烯基、炔基、芳基、芳烷基、杂烷基或杂芳基;且n为不小于5且不大于120的整数]。
第十六,本发明提供一种制备siRNA缀合物的方法,该方法包括:
1)利用本发明第十一项所述的结合聚乙二醇的固相支持物制备针对靶基因的siRNA;以及
2)通过共价键将siRNA的末端基团与聚乙二醇连接。
第十七,本发明提供由本发明第一项或第二项所述的siRNA缀合物组成的纳米微粒。
第十八,本发明提供一种基因治疗方法,该方法包括:
1)制备本发明第十七项所述的纳米微粒;以及
2)向动物的体内施用该纳米微粒。
第十九,本发明提供一种基因治疗方法,其中通过口服给药或静脉注射向体内施用所述纳米微粒。
第二十,本发明提供一种药物组合物,其包括药学有效量的本发明第一项或第二项所述的siRNA缀合物。
第二十一,本发明提供一种药物组合物,其包括药学有效量的本发明第十七项所述的纳米微粒。
接下来,将对本发明进行详细说明。
本发明提供一种siRNA-聚合物缀合物,其结构如下:
A-X-R-Y-B。
其中,A和B独立地为亲水性聚合物或疏水性聚合物;X和Y独立地为简单的共价键或接头介导的共价键;且R为siRNA。
此外,本发明提供一种siRNA-聚合物缀合物,其结构如下:
A-X-R。
其中,A为疏水性聚合物;X为简单的共价键或接头介导的共价键;且R为siRNA。
在本发明的缀合物中,siRNA的寡核苷酸链可包括19~31个核苷酸。任何源自用于或可用于基因疗法或研究的基因的siRNA均可用作本发明中可用的siRNA。
疏水性聚合物可为分子量为250~1,000的疏水性聚合物。疏水性聚合物的实例包括烃,优选C16~C50烃,以及胆固醇。本申请中,疏水性聚合物不限于仅为烃和胆固醇。
疏水性聚合物引起疏水性互作,使得形成由siRNA-疏水性聚合物缀合物组成的微团(micelle)。在这些疏水性聚合物中,具体地,饱和烃的优势在于,它在siRNA生产过程中易于缀合到siRNA,因此,它非常适合用于生产本发明的缀合物。
同样,共价键(即X,Y)可以是任何不可降解或可降解键。在本申请中,不可降解键可以是酰胺键或磷酸键,可降解键可以是二硫键、酸裂解键、酯键、酸酐键、生物降解键和酶裂解键。然而,不可降解键或可降解键不限于此。
接头介导的键能共价结合亲水性聚合物(或疏水性聚合物)和siRNA残基的末端,只要必要时它能够在某一环境中提供可降解键,对接头介导的键没有特别的限定。因此,接头(linker)可包括任何能被siRNA和/或亲水性聚合物(或疏水性聚合物)结合以在缀合物的生产过程中激活它们的化合物。
同样,亲水性聚合物可以是分子量为1,000~10,000的非离子聚合物。例如,亲水性聚合物可包括聚乙二醇、聚乙烯吡咯烷酮、聚噁唑啉等的非离子亲水性聚合物,但不限于此。
必要时,亲水性聚合物的官能团可被另一个官能团取代。在亲水性聚合物中,特别地,PEG非常适合用于生产本发明的缀合物,因为它具有各种分子量,并具有能引入官能团的末端,具有优异的生物相容性,不诱导免疫反应,且能增加水溶性从而提高体内基因传递效率。
此外,本发明提供下述结构的结合聚乙二醇的固相支持物:
其中,固相支持物包括,例如CPG、聚苯乙烯、硅胶、纤维素纸等,但不限于此;R为烷基、烯基、炔基、芳基、芳烷基、杂烷基或杂芳基;m为2~18的整数;n为5~120的整数(摩尔质量282~5300);且X为4-单甲氧基三苯甲基、4,4’-二甲氧基三苯甲基或4,4’,4”-三甲氧基三苯甲基,其经酸处理后被去除而变成氢。在其中固相支持物为CPG的情况下,它的直径可为40~180μm,孔径可为
本发明还提供一种结合聚乙二醇的固相支持物,其中结合有下述结构式IV的3'-PEG-CPG:
[结构式IV]
此外,本发明提供一种制备3’-PEG-CPG的方法,该方法包括:
1)将CPG与3-氨丙基三乙氧基硅烷反应,形成LCAA-CPG;
2)将聚乙二醇与4,4'-二甲氧基三苯甲基氯化物反应,形成2-[双-(4-二甲氧基三苯甲基)-聚(乙二醇)];
3)将步骤2)形成的化合物与下述化学式1的化合物反应,形成下述结构式I的化合物;
4)将形成的下述结构式I的化合物与4-硝基苯基氯甲酸酯反应,形成下述结构式II的化合物;
5)将步骤3)形成的下述结构式I的化合物与N-琥珀酰亚胺基三氟乙酸反应,形成下述结构式III的化合物;以及
6)将步骤1)形成的LCAA-CPG化合物分别与步骤3)至5)形成的下述结构式I、II和III的化合物反应。
[式1]
[结构式I]
[结构式II]
[结构式III]
[结构式IV]
[其中,R为烷基、烯基、炔基、芳基、芳烷基、杂烷基或杂芳基;且n为不小于5且不大于120的整数]。
本发明还提供利用结合聚乙二醇的固相支持物制备含有siRNA和PEG的缀合物的方法。更具体地,提供一种制备siRNA缀合物的方法,该方法包括:
1)利用结合聚乙二醇的固相支持物制备针对靶基因的siRNA;以及
2)通过共价键将siRNA的末端基团与聚乙二醇连接。通过这种方式,可有效制备包括RNA、DNA、RNA-DNA嵌合体及其类似物的寡核苷酸。
根据本发明的一个优选的实施方案,可通过利用β-氰乙基亚磷酰胺连接磷酸二酯键构建RNA骨架结构而制备siRNA(Shina等.核酸研究(Nucleic Acids Research),12:4539-4557,1984)。例如,利用RNA合成器重复地在吸附核苷酸的固相支持物上进行由去封闭、偶联、氧化和加帽组成的一系列步骤,以获得含有所需长度RNA的反应物。然而,本发明不限于此。
本发明还提供由siRNA缀合物组成的纳米微粒。
本发明的siRNA-聚合物缀合物可通过相互之间的反应形成纳米微粒结构,且siRNA-聚合物缀合物以及所获得的由siRNA-聚合物缀合物组成的纳米微粒改良了siRNA的胞内传递,并且可用于疾病模型的治疗。缀合物的制备、由该缀合物组成的纳米微粒的特性和胞内传递效率以及作用将在下面的实施例中详细描述。
本发明还提供一种利用所述纳米微粒进行基因治疗的方法。
更具体地,该基因治疗的方法包括:制备各自由siRNA-聚合物缀合物组成的纳米微粒,以及向动物的体内施用该纳米微粒。
本发明还提供一种药物组合物,其包括药学有效量的各自由siRNA缀合物组成的纳米微粒。
本发明的组合物还可以制备成,除上述的活性组分外,包括一种或多种药用载体,以便给药。药用载体需要与本发明的活性组分相兼容。药用载体可与盐水溶液、无菌水、林格氏溶液、缓冲盐水溶液、葡萄糖溶液、麦芽糖糊精溶液、甘油和乙醇中的一种或多种混合使用,必要时,还可加入常规的添加剂,如抗氧化剂、缓冲液、抑菌剂等。此外,还可辅助加入稀释剂、分散剂、表面活性剂、粘合剂和润滑剂制备成用于注射的制剂,如水溶液、悬浮液、乳液等。此外,本发明的组合物可优选根据特定的疾病或组分,利用本领域的适合的方法或雷明顿氏制药科学(Remington's pharmaceuticalScience)(Mack Publishing company,Easton PA)中公开的方法进行制备。
本领域技术人员可基于患者的综合征和疾病严重性确定本发明的药物组合物。同样,本发明的药物组合物可制备成各种类型,如粉剂、片剂、胶囊、液体、注射剂、膏剂、糖浆等,并可在单剂或多剂容器中提供,如密封的安瓿、小瓶等。
本发明的药物组合物可口服或胃肠外给药。本发明药物组合物的给药途径可包括但不限于口服、静脉内、肌内、髓内、鞘内、心脏内、真皮下、皮下、腹膜内、肠内、舌下或外用。
为了这种临床施用,可使用本领域公知的方法将本发明的药物组合物制成适合的制剂。本发明的药物组合物的剂量根据体重、年龄、性别、健康状况、饮食、给药时间和方法、排泄率以及患者疾病的严重性而有所不同,对此本领域技术人员很容易确定。
有益效果
由本发明缀合物组成的纳米微粒可改善体内siRNA的稳定性,从而有效地将治疗性siRNA传递到细胞中,并且即使没有转染试剂,相对低剂量浓度也具有siRNA的活性,因此其可在生物技术和医药行业的基础研究中用作一种新型的siRNA传递系统以及癌症和其它传染性疾病的siRNA治疗工具。
附图说明
图1所示为制备的3'-PEG-CPG的结构式;
图2所示为实施例1获得的化合物的1H NMR数据;
图3所示为与实施例1的LCAA-CPG结合的3’-PEG试剂[化合物A]的1H NMR数据;
图4所示为与实施例1的LCAA-CPG结合的3’-PEG试剂[化合物B]的1H NMR数据;
图5所示为与实施例1的LCAA-CPG结合的3’-PEG试剂[化合物C]的1H NMR数据;
图6所示为实施例1~3制备的3'-PEG-CPG和寡核苷酸(siRNA)的Maldi-Tof分子量数据;
图7所示为实施例1~4制备的3'-PEG-CPG和寡核苷酸(siRNA)的Maldi-Tof分子量数据;
图8所示为无聚合物缀合的裸siRNA与缀合亲水性或疏水性聚合物的siRNA-聚合物缀合物的电泳照片(siRNA是裸siRNA,各自的缀合物表示表1中所示的siRNA-聚合物缀合物。同样,19mer、23mer、27mer和31mer分别是指含有19、23、27和31个核苷酸的siRNA,并且它们都用于以siRNA缀合物4的结构制备siRNA-聚合物缀合物。);
图9所示为存在血清蛋白的条件下,siRNA随时间的降解度的电泳照片,用于评价无聚合物缀合的裸siRNA和缀合亲水性聚合物PEG的siRNA-聚合物缀合物在血液中的稳定性;
图10所示为siRNA-聚合物缀合物形成的纳米微粒的示意图;
图11所示为通过电动电位测定仪器测定的由无聚合物缀合的裸siRNA组成的纳米微粒的粒径的结果;
图12所示为通过电动电位测定仪器测定的各自由siRNA-聚合物缀合物9组成的纳米微粒的粒径结果;
图13所示为通过电动电位测定仪器测定的各自由siRNA-聚合物缀合物10组成的纳米微粒的粒径结果;
图14所示为通过电动电位测定仪器测定的各自由siRNA-聚合物缀合物11组成的纳米微粒的粒径结果;
图15所示为通过电动电位测定仪器测定的各自由siRNA-聚合物缀合物12组成的纳米微粒的粒径结果;
图16所示为通过电动电位测定仪器测定的各自由siRNA-聚合物缀合物13组成的纳米微粒的粒径结果;
图17所示为与转染试剂共同转染后的存活素基因的mRNA表达水平的比较,用于分析裸siRNA和各自缀合亲水性聚合物PEG的siRNA-聚合物缀合物的RNAi效果;
图18所示为与转染试剂共同转染后的存活素基因的mRNA表达水平的比较,用于分析裸siRNA和各自转化长序列siRNA的siRNA-聚合物缀合物4的RNAi效果;以及
图19所示为无转染试剂下转染后的存活素基因的mRNA表达水平的比较,用于分析裸siRNA和siRNA-聚合物缀合物1~5以及9~14的RNAi效果。
最佳实施方式
下文将详细描述本发明的示例性实施方式。然而,以下示例性实施方式仅以举例方式说明,不用来限制本发明。
实施例1:用于制备3’-PEG寡核苷酸的固相支持物的制备
实施例1-1:用于结合LCAA-CPG的3’-PEG试剂(化合物A、B和
C)的制备
在下面的实施例中,3'-PEG-CPG按如下反应式制备得到。
实施例1-1-1:2-[双-(4-二甲氧基三苯甲基)-聚(乙二醇)]的制备
将30g(15mmol)聚乙二醇2000(Alfa Aesar GmbH & Co.KG,德国)作为起始物,溶解于270ml吡啶(Sigma Aldrich,美国)中,然后加入3.55ml(25.5mmol)三乙胺(Sigma Aldrich,美国)和7.12g(21mmol)4,4’-二甲氧基三苯甲基氯(GL biochem,中国),然后所得物在室温下反应20小时。反应完成后,浓缩反应混合物,用450ml乙酸乙酯和450ml水萃取,然后真空蒸发并真空干燥,获得2-[双-(4-二甲氧基三苯甲基)-聚(乙二醇)]23g(66%)。
该化合物的1H NMR数据如图2所示。
1H NMR(CDCl3);δ1.93(br,1,OH),3.20-3.80(m,186,PEG,DMT-OCH3),6.80-6.83(m,4,DMT),7.19-7.47(m,9,DMT)。
实施例1-1-2:琥珀酸2-[双-(4-二甲氧基三苯甲基)-聚(乙二醇)][化合
物A]的制备
将3.9g(1.672mmol)实施例1-1-1中获得的2-[双-(4-二甲氧基三苯甲基)-聚(乙二醇)]溶解于20ml吡啶中,然后冷却至0℃。向反应物中加入351mg(3.512mmol)琥珀酸酐(Acros Organics,美国)和42.5mg(0.334mmol)DMAP(4-二甲氨基吡啶,Sigma Aldrich,美国),50℃搅拌3天后反应结束。反应完成后,真空蒸发反应混合物,获得琥珀酸2-[双-(4-二甲氧基三苯甲基)-聚(乙二醇)][化合物A]3.65g(90%,白色固体)。
该化合物的1H NMR数据如图3所示。.
1H NMR(CDCl3);δ2.65(m,2,CH2CO),3.20-3.88(m,186,PEG,DMT-OCH3),4.25(m,2,CH2CO),6.80-6.82(m,4,DMT),7.19-7.47(m,9,DMT)。
实施例1-1-3:对硝基苯基琥珀酸2-[双-(4-二甲氧基三苯甲基)-聚(乙
二醇)][化合物B]的制备
将1g(0.411mmol)实施例1-1-2中获得的化合物A溶解于20ml二氯甲烷(DaeYeon Chemicals,Co.Ltd.,韩国)中,并冷却至0℃。向反应物中加入143μl(1.03mmol)三乙胺,并向其中加入149mg(0.740mmol)4-硝基氯甲酸苯酯。然后,将温度升至室温,搅拌所得物4小时,然后反应结束。反应完成后,反应混合物用20ml水饱和的NaHCO3和冷却至0℃~4℃的20ml 1M柠檬酸(Sigma Aldrich,美国)洗涤一次,然后用Na2SO4(Samchum Chemical Co.,韩国)干燥。用抽滤瓶、布氏漏斗或抽吸器过滤所得物,然后真空蒸发,获得对硝基苯基琥珀酸2-[双-(4-二甲氧基三苯甲基)-聚(乙二醇)][化合物B]1.0g(94%,奶油状固体)。
该化合物的1H NMR数据如图4所示。
1H NMR(CDCl3);δ2.80-2.90(m,2,CH2CO),3.20-3.87(m,186,PEG,DMT-OCH3),4.25(m,2,CH2CO),6.80-6.82(m,4,DMT),7.19-7.47(m,9,DMT)。
实施例1-1-4:2,5-二氧代-吡咯烷-1-基琥珀酸酯2-[双-(4-二甲氧基三
苯甲基)-聚(乙二醇)][化合物C]的制备
将500mg(0.206mmol)实施例1-1-2中获得的化合物A溶解于10ml二氯甲烷中,然后向其中加入83.14μl(1.03mmol)吡啶。向其中加入165mg(0.781mmol)N-琥珀酰亚胺三氟乙酸(Sigma Aldrich,美国),室温下搅拌7小时后反应结束。反应完成后,真空蒸发反应混合物,获得2,5-二氧代-吡咯烷-1-基琥珀酸酯2-[双-(4-二甲氧基三苯甲基)-聚(乙二醇)][化合物C]490mg(94%,白色固体)。
该化合物的1H NMR数据如图5所示。.
1H NMR(CDCl3);δ2.72-2.97(m,6,CH2CO,CH2CH2),3.20-3.87(m,186,PEG,DMT-OCH3),4.27-4.28(m,2,CH2CO),6.80-6.83(m,4,DMT),7.20-7.47(m,9,DMT)。
实施例1-2:LCAA-CPG和3'-PEG试剂(化合物A)的结合
在下面的实施例中,CPG和3′-PEG试剂按如下反应式结合:
将2g实施例1-2-1中获得的LCAA-CPG(2000)在20ml二氯甲烷中润湿。此外,将该LCAA-CPG(2000)溶液与含有80mg化合物A、14μL TEA(三乙胺,Sigma Aldrich,美国)的溶液均匀混合。将15mg BOP(苯并三唑-1-基氧代三(二甲氨基)磷鎓六氟磷酸盐,TCIOrg.Chem,日本)和5mg HOBT(无水1-羟基苯并三氮唑,TCI Org.Chem,日本)溶解于2ml二氯甲烷中,所得物回流反应8小时。反应完成后过滤该混合物,并依次用甲醇、水和亚甲二醇洗涤,然后真空干燥。
将1g所得物在10ml吡啶中润湿,然后向其中加入1ml 1-甲基咪唑(Sigma Aldrich,美国)和1.6ml醋酸酐(Sigma Aldrich,美国)。将该所得物均匀混合,在室温反应8小时。将反应完成后获得的完成加帽的CPG(capping-completed CPG)依次用甲醇、水、甲醇和二氯甲烷洗涤,然后真空干燥,获得1g的3'-PEG-CPG。
具体地,将1g实施例1-2-1中获得的LCAA-CPG(2000)在8ml吡啶中充分润湿。此外,将溶解于2ml吡啶的205mg(2eq)化合物B和55μL三乙胺的溶液与该LCAA-CPG溶液均匀混合。所得物在50~60℃反应8小时,反应完成后过滤该混合物。过滤后的偶联-CPG依次用甲醇、水和二氯甲烷洗涤,然后真空干燥。将1g干燥后的偶联-CPG在10ml吡啶中润湿,然后加入500μL 1-甲基咪唑和800μL醋酸酐。均匀混合所得物,然后在室温反应8小时。反应完成后过滤该混合物,然后偶联-CPG依次用甲醇、水和二氯甲烷洗涤,然后真空干燥,获得1g的3'-PEG-CPG。
图6显示了使用3'-PEG-CPG作为起始物制备的siRNA的Maldi-Tof分子量测定结果,如下文描述的实施例2中所示。
3'-PEG-CPG制备序列;
正义5'-AAGGAGAUCAACAUUUUCA(dTdT)-PEG(6664.96Da+2000Da)(SEQ ID No.1)
反义5'-UGAAAAUGUUGAUCUCCUU(dTdT)-PEG(6592.84Da+2000Da)(SEQ ID No.5)
可以发现,Maldi-Tof分子量随PEG的分子量(2000Da)增加。
具体地,将1g实施例1-2-1中获得的LCAA-CPG(2000)在8ml吡啶中充分润湿。此外,将溶解于2ml吡啶中的200mg化合物C和55μL三乙胺的溶液与LCAA-CPG溶液均匀混合。所得物在50~60℃反应8小时,反应完成后过滤该混合物。过滤后的偶联-CPG依次用甲醇、水和二氯甲烷洗涤,然后真空干燥。将1g干燥后的偶联-CPG在10ml吡啶中润湿,然后向其中加入500μL 1-甲基咪唑和800μL醋酸酐。均匀混合所得物,然后在室温反应8小时。反应完成后完全加帽的CPG依次用甲醇、水和二氯甲烷洗涤,然后真空干燥,获得1g的3'-PEG-CPG。
图7显示了使用3'-PEG-CPG作为起始物制备的siRNA结果,如下文描述的实施例2中所示。
3'-PEG-CPG制备序列;
正义5'-AAGGAGAUCAACAUUUUCA(dTdT)-PEG(6664.96Da+2000Da)(SEQ ID No.1)
反义5'-UGAAAAUGUUGAUCUCCUU(dTdT)-PEG(6592.84Da+2000Da)(SEQ ID No.5)
可以发现,Maldi-Tof分子量随PEG的分子量(2000Da)增加。
实施例2:siRNA-聚合物缀合物的制备
在下面的实施例中,使用存活素(survivin)siRNA以抑制存活素。存活素是一种到目前为止检测到的仅在大多数肿瘤或转化细胞系中普遍表达的蛋白,因此它有望成为抗癌治疗的重要靶标(AbbrosiniG.等,Nat.Med.3(8)917-921,1997)。本发明的存活素siRNA序列,当包含19个核苷酸时,由SEQ ID No.1的一条正义链和一条完全与该正义链互补的反义链组成,此外,当它包含23、27或31个核苷酸时,碱基序列为SEQ ID No.2、3或4。
(SEQ ID No.1)5'-AAGGAGAUCAACAUUUUCA-3'
(SEQ ID No.2)5'-AGGAAAGGAGAUCAACAUUUUCA-3'
(SEQ ID No.3)5'-AGGAAAGGAGAUCAACAUUUUCAAAUU-3'
(SEQ ID No.4)5'-AAAGGAGAUCAACAUUUUCAAAUUAGAUGUU-3'
使用β-氰乙基磷酰胺,通过连接磷酸二酯键构建RNA骨架结构制备siRNA(Shina等,核酸研究,12:4539-4557,1984)。具体地,采用RNA合成仪(384合成仪,BIONEER,韩国),在附着核苷酸的固相支持物上重复进行由去封闭、偶联、氧化和加帽组成的一系列步骤,获得含所需长度RNA的反应物。
此外,通过将PEG连接到5'-端区域,或通过疏水性聚合物十二烷接头将十六烷(C16)或十八烷(C18)饱和烃连接到5'-端区域,制备siRNA-聚合物缀合物。此外,采用实施例1中制备的3'PEG-CPG作为支持物进行上述反应,获得3'-端区域带有PEG的siRNA-聚合物缀合物。
通过HPLC(LC-20A Prominence,SHIMADZU,日本)从反应物中分离RNA,确定反应物是否与待制备的核苷酸序列一致,并采用MALDI-TOF质谱仪(MALDI TOF-MS,SHIMADZU,日本)测定其分子量。之后,正义RNA链和反义RNA链等量混合,并加入1×退火缓冲液(30mM HEPES,100mM醋酸钾,2mM醋酸镁,pH值7.0~7.5)。所得物在90℃恒温浴中反应3分钟,然后再于37℃反应,制备双链siRNA-聚合物缀合物。制得的siRNA-聚合物缀合物具有表1中所示的结构。制得的siRNA-聚合物缀合物的退火通过电泳照片证实(图8)。
【表1】
siRNA-聚合物缀合物的结构和末端修饰类型
*在缀合结构中,“ss”表示二硫键,“C16”或“C18”表示C16或C18烃。因此,“C18-C6-ss-C6”和“C16-C6-ss-C6”表示疏水聚合物。
实施例3:在体内条件下siRNA-聚合物缀合物稳定性的评价
确定实施例2中制备并分离的siRNA-聚合物缀合物与未结合聚合物的裸siRNA相比,稳定性是否提高。将未修饰的裸siRNA和实施例2中制得的siRNA-聚合物缀合物1-5在模拟体内条件的含10%胎牛血清(FBS)的培养基中孵育0、1、3、6、9、12、24、36或48小时,然后通过电泳对siRNA的降解度进行评价。
结果表明,引入PEG的siRNA-聚合物缀合物显示出长达48小时的siRNA稳定性(图9)。甚至在100%血清的条件下显示出12~24小时的siRNA稳定性。
实施例4:siRNA-疏水聚合物缀合物纳米微粒大小的测定
在siRNA-聚合物缀合物9~14的各情况下,由siRNA-聚合物缀合物组成的纳米微粒,也就是说,通过siRNA-末端提供的疏水聚合物之间的疏水性相互作用形成的胶质粒子(micelle)(图10)。使用Zeta电位测量仪测量疏水纳米微粒的大小。测定由实施例2中分别制备的siRNA-聚合物缀合物9~13和siRNA组成的纳米微粒的大小。
具体地,将2nmol siRNA和siRNA-聚合物缀合物溶解于1ml蒸馏水中,然后用超声波匀浆仪(Wiseclean,DAHAN,韩国)进行纳米微粒匀浆(200W,40kHz,5s)。使用Zeta电位测量仪(Nano-ZS,MALVERN,英国)测量匀浆纳米微粒的大小。此处,将材料的折射指数和吸收指数分别设定为1.454和0.001,然后输入作为溶剂的水的温度25℃,并输入其粘度和折射指数。一次测量包括20个重复大小的测定,这种测量进行三次。
图11显示了采用Zeta电位测量仪测得的裸siRNA纳米微粒的大小结果。它表明142~295nm(最高点:164nm)的微粒占各自由siRNA组成的总纳米微粒的73.5%。
图12显示了采用Zeta电位测量仪测得的各自由siRNA-聚合物缀合物9组成的纳米微粒的大小结果。它表明4.19~7.53nm(最高点:6.50nm)的微粒占各自由siRNA-聚合物缀合物9组成的总纳米微粒的59.1%。
图13显示了采用Zeta电位测量仪测得的各自由siRNA-聚合物缀合物10组成的纳米微粒的大小结果。它表明5.61~10.1nm(最高点:8.72nm)的微粒占各自由siRNA-聚合物缀合物10组成的总纳米微粒的58.9%。
图14显示了采用Zeta电位测量仪测得的组成各siRNA-聚合物缀合物11的纳米微粒的大小结果。它表明5.61~10.1nm(最高点:8.72nm)的微粒占各自由siRNA-聚合物缀合物11组成的总纳米微粒的45.6%。
图15显示了采用Zeta电位测量仪测得的组成各siRNA-聚合物缀合物12的纳米微粒的大小结果。它表明4.85~5.61nm的微粒占各自由siRNA-聚合物缀合物12组成的总纳米微粒的23.6%,21.0~32.7nm的微粒占各自由siRNA-聚合物缀合物12组成的总纳米微粒的23.5%,68.1~78.8nm的微粒占各自由siRNA-聚合物缀合物12组成的总纳米微粒的23.1%。
图16显示了采用Zeta电位测量仪测得的各自由siRNA-聚合物缀合物13组成的纳米微粒的大小结果。它表明4.85~8.72nm(最高点:5.61nm)的微粒占各自由siRNA-聚合物缀合物13组成的总纳米微粒的84.6%。
除siRNA-聚合物缀合物12之外,在siRNA-聚合物缀合物9~13的情况下,纳米微粒的大小大多是4~8nm。在siRNA-聚合物缀合物12的情况下,测得不同的纳米微粒大小,认为即使用超声波匀浆仪进行同质化,由于在测定过程中随着时间的推移,纳米微粒也会各自聚集。如图12~16所示,测得的各自由siRNA缀合物组成的纳米粒径为100nm或以下,其大小足以通过胞饮作用内吞进入胞内(KennethA.Dawson等,自然纳米技术4:84-85,2009)。
实施例5:通过使用siRNA-聚合物缀合物和转染试剂对肿瘤细胞系
中靶基因表达的抑制
人宫颈癌细胞系,其为肿瘤细胞系,分别用实施例2中制备的siRNA-聚合物缀合物1~8转染,分析转染的肿瘤细胞系中存活素的表达水平。
实施例5-1:肿瘤细胞系的培养
将从美国菌种保藏中心(ATCC)获得的人宫颈癌细胞(HeLa)于添加有10%(v/v)胎牛血清、青霉素100单位/ml和链霉素100μg/ml的RPMI 1640培养基(GIBCO,Invitrogen,美国)中,在37℃、5%(v/v)CO2条件下培养。
实施例5-2:使用siRNA-聚合物缀合物抑制靶基因的表达
用实施例2中制备的SEQ ID No.1的siRNA-聚合物缀合物1~8转染HeLa肿瘤细胞系,分析转染的肿瘤细胞系中存活素的表达。
实施例5-2-1:用siRNA-聚合物缀合物转染肿瘤细胞系
将实施例5-1中培养的1.3×105个肿瘤细胞系于盛有RPMI 1640培养基的6孔板中,在37℃、5%(v/v)CO2条件下培养18小时,然后去除培养基,然后每孔分配800μL Opti-MEM培养基(GIBCO,美国)。
同时,将2μL脂质体TM2000(LipofectamineTM 2000)(Invitrogen,美国)和198μL Opti-MEM培养基混合,然后在室温反应5分钟,然后向其中加入实施例2制备的各siRNA-聚合物缀合物(25pmole/μL)0.8或4μL(终浓度为20或100nM)。然后,所得物在室温下再反应20分钟,制备溶液。
之后,向已用Opti-MEM培养基分配的各孔中加入200μL该转染液,培养肿瘤细胞6小时,然后去除Opti-MEM培养基。向其中加入2.5ml RPMI 1640培养基,然后将肿瘤细胞置于37℃、5%(v/v)CO2条件下培养24小时。
实施例5-2-2:存活素基因mRNA的相对定量分析
从实施例5-2-1中的转染的细胞系中提取总RNA以制备cDNA,然后通过实时PCR相对定量存活素基因mRNA的含量。
实施例5-2-2-1:从转染细胞中分离RNA和制备cDNA
用RNA提取试剂盒(AccuPrep细胞总RNA提取试剂盒,BIONEER,韩国)从实施例5-2-1的转染细胞系中提取总RNA,用RNA反转录酶(AccuPower CycleScript RT Premix/dT20,BIONEER,韩国)从提取的RNA中制备cDNA,如下。
具体地,将1μg提取的RNA加入到含有AccuPower CycleScriptRT Premix/dT20(BIONEER,韩国)的各0.25ml Eppendorf管中,向其中加入经焦碳酸二乙酯(DEPC)处理的蒸馏水,使总体积为20μL。通过使用PCR仪(MyGenieTM96梯度热块(Gradient Thermal Block),BIONEER,韩国),将在30℃杂交RNA引物1分钟和在52℃合成cDNA4分钟的两个步骤重复6次。然后在95℃使酶失活5分钟以结束扩增反应。
实施例5-2-2-2:存活素基因mRNA的相对定量分析
通过实时PCR,以实施例5-2-2-1中制备的cDNA为模板,对存活素mRNA的相对含量进行定量,如下。
就是说,用蒸馏水对96-孔板各孔中由实施例5-2-2-1制备的cDNA进行1/5稀释,然后加入3μL稀释的cDNA、10μL 2×GreenStarTM PCR混合物(master mix)(BIONEER,韩国)、6μL蒸馏水和1μL存活素qPCR引物(各10pmole/μL,BIONEER,韩国),制备混合液以分析存活素表达水平。另一方面,使用下述管家基因(以下简称为“HK基因”):HMBS(羟甲基胆色烷合成酶)、HPRT1(次黄嘌呤磷酸核糖转移酶1)、UBC(泛素C)和YWHAZ(酪氨酸3-单加氧酶/色氨酸5-单加氧酶激活蛋白,zeta多肽),作为对照基因,为了使mRNA表达水平标准化,对实施例5-2-2-1中制备的cDNA进行1/5稀释,然后向96-孔板的各孔中加入3μL稀释的cDNA、10μL 2×GreenStarTM PCR混合物(master mix)(BIONEER,韩国)、6μL蒸馏水和1μL各HK基因的qPCR引物(各10pmole/μL,BIONEER,韩国),在96-孔板的每孔中制备HK基因实时PCR混合物液体。采用ExicyclerTM96实时定量加热块(Real-Time Quantitative Thermal Block)(BIONEER,韩国),在含有该混合液的96-孔板上进行以下反应。
通过在95℃反应15分钟灭活酶和破坏cDNA的二级结构。然后,在94℃变性30秒,58℃退火30秒,72℃延伸30秒和SYBR Green扫描,这四个步骤重复进行42次,最后72℃延伸3分钟。然后,55℃保持1分钟,分析55℃~95℃的融解曲线。
PCR结束后,通过HK基因标准化的mRNA值(标准化因子,NF)纠正各自获得的存活素Ct(循环阈值)值,然后获得了介于仅用转染试剂处理的对照组的Ct值和纠正的Ct值之间的ΔCt值。用ΔCt值和2(-ΔCt)×100(图17)的计算公式相互比较存活素mRNA的表达率。在图17中,模拟(mock)表示仅用转染试剂处理的对照组。
如图17所示,结果表明siRNA的RNAi效果是不同的,这取决于PEG、疏水聚合物缀合的siRNA-聚合物缀合物的末端修饰类型。特别是缀合物6~8,各自具有PEG缀合至3'-端区域的末端修饰类型,其表现出类似于裸siRNA的表达抑制度。因此,基于siRNA的RNAi机制,预计缀合物6~8在与RNA-诱导的沉默复合物(RISC)形成的复合物中具有小的空间位阻。此外,大多数的siRNA-PEG缀合物在低浓度(20nM)处理条件比在高浓度(100nM)处理条件下,表现出对靶基因mRNA表达的较高抑制,并因此预期由于形成siRNA-PEG缀合的PEG浓度条件较高,从而防止了siRNA与RISC的结合。
实施例5-3:使用长序列siRNA-聚合物缀合物对靶基因表达的抑制
当用siRNA-亲水聚合物缀合物与转染试剂一起转染细胞时,分析对靶基因mRNA表达的抑制。此处,使用siRNA-聚合物缀合物4的结构中引入末端修饰的siRNA,各siRNA碱基序列为SEQ ID No.1~4。
实施例5-3-1:用siRNA-聚合物缀合物转染肿瘤细胞系
将实施例5-1中培养的1.3×105个肿瘤细胞系于盛有RPMI 1640培养基的6-孔板中,在37℃、5%(v/v)CO2的条件下培养24小时,然后去除培养基,向各孔中加入800μL Opti-MEM培养基。
同时,将2μL脂质体TM2000和198μL Opti-MEM培养基混合,然后在室温反应5分钟,然后向其中加入实施例2中制备的各siRNA-聚合物缀合物(25pmole/μL)0.8或4μL(终浓度为20或100nM)。然后,所得物在室温下再反应20分钟,制备溶液。
之后,向已用Opti-MEM培养基分配的各孔中加入200μL该转染液,培养肿瘤细胞6小时,然后去除Opti-MEM培养基。向其中加入2.5ml RPMI 1640培养基,然后将肿瘤细胞置于37℃、5%(v/v)CO2条件下培养24小时。
实施例5-3-2:存活素基因mRNA的相对定量分析
从实施例5-3-1中转染的细胞系中提取总RNA以制备cDNA,然后通过实时PCR相对定量存活素基因mRNA的含量。
实施例5-3-2-1:从转染细胞中分离RNA和制备cDNA
用RNA提取试剂盒(AccuPrep细胞总RNA提取试剂盒,BIONEER,韩国)从实施例5-3-1的转染细胞系中提取总RNA,用RNA反转录酶(AccuPower CycleScript RT Premix/dT20,BIONEER,韩国)从提取的RNA中制备cDNA,如下。
具体地,将1μg提取的RNA加入到含有AccuPower CycleScriptRT Premix/dT20(BIONEER,韩国)的各0.25ml Eppendorf管中,向其中加入经焦碳酸二乙酯(DEPC)处理的蒸馏水,使总体积为20μL。通过使用PCR仪(MyGenieTM96梯度热块,BIONEER,韩国),将在30℃杂交RNA引物1分钟和在52℃制备cDNA4分钟的两个步骤重复6次。然后在95℃使酶失活5分钟以结束扩增反应。
实施例5-3-2-2:存活素基因mRNA的相对定量分析
通过实时PCR,以实施例5-3-2-1中制备的cDNA为模板,对存活素基因mRNA的相对含量进行定量,如下。
就是说,用蒸馏水对96-孔板各孔中由实施例5-3-2-1制备的cDNA进行1/5稀释,然后加入3μL稀释的cDNA、10μL 2×GreenStarTM PCR混合物(BIONEER,韩国)、6μL蒸馏水和1μL存活素qPCR引物(10pmole/μL,BIONEER,韩国),制备混合液以分析存活素表达水平。另一方面,使用如下HK基因:HMBS、HPRT1、UBC和YWHAZ,作为对照基因,为了使mRNA表达水平标准化,对实施例5-3-2-1中制备的cDNA进行1/5稀释,然后向96-孔板的各孔中加入3μL稀释的cDNA、10μL 2×GreenStarTM PCR混合物(BIONEER,韩国)、6μL蒸馏水和1μL各HK基因qPCR引物(10pmole/μL,BIONEER,韩国),在96-孔板的每孔中制备HK基因实时PCR混合物液体。采用ExicyclerTM96实时定量加热块(BIONEER,韩国),在含有该混合液的96-孔板上进行以下反应。
通过在95℃反应15分钟灭活酶和破坏cDNA的二级结构。然后,在94℃变性30秒,58℃退火30秒,72℃延伸30秒和SYBR Green扫描,这四个步骤重复进行42次,最后72℃延伸3分钟。然后,55℃保持1分钟,分析55℃~95℃的融解曲线。
PCR结束后,通过HK基因标准化的mRNA值(标准化因子,NF)纠正各自获得的存活素Ct(循环阈值)值,然后获得了介于仅用转染试剂处理的对照组的Ct值和纠正的Ct值之间的ΔCt值。用ΔCt值和2(-ΔCt)×100(图18)的计算公式相互比较存活素mRNA的表达率。在图18中,模拟(mock)表示仅用转染试剂处理的对照组,19mer、23mer、27mer和31mer分别代表SEQ ID No.1~4。5'P+P代表siRNA-聚合物缀合物4的结构。分别用20nM和40nM处理细胞,相互比较对靶基因表达的抑制程度。
结果,如图18所示,与裸siRNA相比,以siRNA-聚合物缀合物4形式的转化的长链裸siRNA,对靶基因mRNA表达的抑制表现出较小差异。因此,可以发现,与短链相比,转化的长链siRNA由于PEG而降低了空间位阻现象。
也就是说在长链siRNA的情况下,在RNAi的工作机制中,siRNA在19+2结构中被dicer酶断裂,裂解的siRNA与RISC复合物结合从而引发RNAi的工作机制。出于这个原因,两个末端带有PEG的长链siRNA,导致了大量未结合PEG的siRNA的存在,因此与SEQ IDNo.1相比,其具有与PESC复合物相对较强的相互作用,这被认为是保持了RNAi诱导作用。
实施例6:仅使用siRNA-聚合物缀合物,而不使用转染试剂对靶基
因表达的抑制
用实施例2中制备的siRNA-聚合物缀合物1~14转染HeLa肿瘤细胞系,分析转染的肿瘤细胞系中存活素基因的表达。
实施例6-1:肿瘤细胞系的培养
将从美国菌种保藏中心(ATCC)获得的人宫颈癌细胞(HeLa)于添加有10%(v/v)胎牛血清、青霉素100单位/ml和链霉素100μg/ml的RPMI 1640培养基(GIBCO,Invitrogen,美国)中,在37℃、5%(v/v)CO2条件下培养。
实施例6-2:用siRNA-聚合物缀合物转染肿瘤细胞系
将实施例6-1中培养的1.3×105个肿瘤细胞系于盛有RPMI 1640培养基的6孔板中,在37℃、5%(v/v)CO2条件下培养24小时,然后去除培养基,然后每孔加入900μL Opti-MEM培养基。
同时,向其中加入100μL Opti-MEM培养基,5或10μL实施例2制备的各siRNA-聚合物缀合物1~5(1nmole/μL)(终浓度为500nM或1μM),所得物在室温下再反应20分钟,制备溶液。
同时,向其中加入100μL Opti-MEM培养基,5或10μL实施例2制备的各siRNA-聚合物缀合物9~14(1nmole/μL)(终浓度为500nM或1μM),通过高频超声对由siRNA-聚合物缀合物组成的胶质粒子进行匀浆,制备溶液。
之后,向已用Opti-MEM培养基分配的各孔中加入100μL该转染液,培养肿瘤细胞24小时,然后加入含20%FBS的RPMI 1640培养基1ml。进一步将肿瘤细胞于37℃、5%(v/v)CO2条件下培养24小时,用siRNA-聚合物缀合物处理,然后总计培养48小时。
实施例6-3:存活素基因mRNA的相对定量分析
从实施例6-2中转染的细胞系中提取总RNA以制备cDNA,然后通过实时PCR相对定量存活素基因mRNA的含量。
实施例6-3-1:从转染细胞中分离RNA和制备cDNA
用RNA提取试剂盒(AccuPrep细胞总RNA提取试剂盒,BIONEER,韩国)从实施例6-2的转染细胞系中提取总RNA,用RNA反转录酶(AccuPower CycleScript RT Premix/dT20,BIONEER,韩国)从提取的RNA中制备cDNA,如下。
具体地,将1μg提取的RNA加入到含有AccuPower CycleScriptRT Premix/dT20(BIONEER,韩国)的各0.25ml Eppendorf管中,向其中加入经焦碳酸二乙酯(DEPC)处理的蒸馏水,使总体积为20μL。通过使用PCR仪(MyGenieTM96梯度热块,BIONEER,韩国),将在30℃杂交RNA引物1分钟和在52℃合成cDNA4分钟的两个步骤重复6次。然后在95℃使酶失活5分钟以结束扩增反应。
实施例6-3-2:存活素基因mRNA的相对定量分析
通过实时PCR,以实施例6-3-1中制备的cDNA为模板,对存活素基因mRNA的相对含量进行定量,如下。
就是说,用蒸馏水对96-孔板各孔中由实施例6-3-1制备的cDNA进行1/5稀释,然后加入3μL稀释的cDNA、10μL 2×GreenStarTM PCR混合物(master mix)(BIONEER,韩国)、6μL蒸馏水和1μL存活素qPCR引物(10pmole/μL,BIONEER,韩国),制备混合液以分析存活素表达水平。另一方面,使用如下管家基因(以下简称为“HK基因”):HMBS(羟甲基胆色烷合成酶)、HPRT1(次黄嘌呤磷酸核糖转移酶1)、UBC(泛素C)、YWHAZ(酪氨酸3-单加氧酶/色氨酸5-单加氧酶激活蛋白,zeta多肽),作为对照基因,为了使mRNA表达水平标准化,对实施例6-3-1中制备的cDNA进行1/5稀释,然后向96-孔板的各孔中加入3μL稀释的cDNA、10μL 2×GreenStarTM PCR混合物(BIONEER,韩国)、6μL蒸馏水和1μL各HK基因qPCR引物(10pmole/μL,BIONEER,韩国),在96-孔板的每孔中制备HK基因实时PCR混合物液体。采用ExicyclerTM96实时定量加热块(Real-Time Quantitative Thermal Block)(BIONEER,韩国),在含有该混合液的96-孔板上进行以下反应。
通过在95℃反应15分钟灭活酶和破坏cDNA的二级结构。然后,在94℃变性30秒,58℃退火30秒,72℃延伸30秒和SYBR Green扫描,这四个步骤重复进行42次,最后72℃延伸3分钟。然后,55℃保持1分钟,分析55℃~95℃的融解曲线。PCR结束后,通过HK基因标准化的mRNA值(标准化因子,NF)纠正各自获得的存活素Ct(循环阈值)值,然后获得了介于仅用转染试剂处理的对照组的Ct值和纠正的Ct值之间的ΔCt值。用ΔCt值和2(-ΔCt)×100(图19)的计算公式相互比较存活素mRNA的表达率。
结果,如图19所示,与siRNA-聚合物缀合物1相比,siRNA PEG的缀合物2~5高度抑制存活素mRNA水平,而不同于用转染试剂进行转染的结果。siRNA-聚合物缀合物1~5在低浓度(500nM)比在高浓度(100nM),表现出更高的RNAi作用。此外,当用相同浓度(500nM)处理时,与siRNA缀合物1~5相比,siRNA-疏水聚合物缀合物9~14表现出对存活素mRNA表达水平的较低抑制。然而,当在高浓度条件下(1μM)处理时,特别是siRNA-聚合物缀合物14的末端修饰导致对存活素mRNA表达水平的较高抑制。
序列表
见序列表
Claims (12)
1.存活素特异性siRNA和聚合物的缀合物,其结构如下:
A-X-R-Y-B
其中,A和B之一为亲水性聚合物且其另一为疏水性聚合物;X和Y各自独立地为简单的共价键或接头介导的共价键;且R为存活素特异性siRNA。
2.根据权利要求1所述的缀合物,其中所述存活素特异性siRNA(R)的单链由19-31个核苷酸组成。
3.根据权利要求1所述的缀合物,其中所述存活素特异性siRNA(R)的单链为核苷酸序列SEQ ID No.1~4之一。
4.根据权利要求1所述的缀合物,其中所述存活素特异性siRNA(R)具有化学修饰。
5.根据权利要求4所述的缀合物,其中所述化学修饰包括至少下述之一:
将磷酸二酯键修饰为硫代磷酸酯连接;
将戊糖2’-位上的-OH修饰为2’-OCH3或2’-脱氧-2’氟尿嘧啶核苷;和
通过连接戊糖的2’-位和4’-位而将戊糖2’-位上的-OH修饰为锁核酸型式。
6.根据权利要求1所述的缀合物,其中所述疏水性聚合物的分子量为250~1,000;所述亲水性聚合物为分子量为1,000~10,000的非离子聚合物;且所述共价键为不可降解键或可降解键。
7.根据权利要求6所述的缀合物,其中所述疏水性聚合物为C16~C50饱和烃或胆固醇。
8.根据权利要求6所述的缀合物,其中所述不可降解键为酰胺键或磷酸键;且所述可降解键选自二硫键、酸裂解键、酯键、酸酐键、生物降解键和酶裂解键。
9.根据权利要求6所述的缀合物,其中所述亲水性聚合物包括至少下述之一:聚乙二醇、聚乙烯吡咯烷酮和聚噁唑啉。
10.一种纳米微粒,其包含权利要求1所述的缀合物。
11.一种组合物,其包含权利要求1所述的缀合物作为药物有效成分。
12.一种组合物,其包含权利要求10所述的纳米微粒作为药物有效成分。
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Families Citing this family (37)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR101224828B1 (ko) | 2009-05-14 | 2013-01-22 | (주)바이오니아 | siRNA 접합체 및 그 제조방법 |
BR112014014730B1 (pt) * | 2011-12-15 | 2021-05-18 | Bioneer Corporation | estrutura de droga polímero terapêutica, métodos para preparar uma estrutura de oligo rna de dupla hélice, nanoparticula e composição farmacêutica |
KR101722948B1 (ko) * | 2012-01-05 | 2017-04-04 | (주)바이오니아 | 리간드가 결합된 이중나선 올리고 rna 구조체 및 그 제조방법 |
AU2013206989B2 (en) * | 2012-01-05 | 2015-05-28 | Bioneer Corporation | High-efficiency nanoparticle-type double-helical oligo-RNA structure and method for preparing same |
EP2805713B1 (en) * | 2012-01-18 | 2018-10-10 | Bioneer Corporation | Magnetic nanoparticle-samirna complex and method for preparing same |
RU2015114330A (ru) | 2012-09-17 | 2016-11-10 | У.Р. Грейс Энд Ко.-Конн. | Хроматографические среды и устройства |
US20150259690A1 (en) * | 2012-10-05 | 2015-09-17 | Bioneer Corporation | Amphiregulin-specific double-helical oligo-rna, double-helical oligo-rna structure comprising double-helical oligo-rna, and composition for preventing or treating respiratory diseases containing same |
WO2014088087A1 (ja) * | 2012-12-06 | 2014-06-12 | 協和発酵バイオ株式会社 | アジュバント用二重鎖リボ核酸 |
PL3018208T3 (pl) * | 2013-07-05 | 2020-09-21 | Bioneer Corporation | Ulepszona struktura oligonukleotydowa typu nanocząstki o wysokiej wydajności i sposób jej wytwarzania |
CN110592082A (zh) * | 2013-07-05 | 2019-12-20 | 柏业公司 | 呼吸疾病相关基因特异性siRNA、含有siRNA的双螺旋寡RNA结构及其用途 |
SG11201507571TA (en) * | 2013-07-05 | 2015-10-29 | Bioneer Corp | Dengue virus-specific sirna, double helix oligo-rna structure comprising sirna, and composition for suppressing proliferation of dengue virus comprising rna structure |
KR20150006742A (ko) * | 2013-07-09 | 2015-01-19 | (주)바이오니아 | 간암 연관 유전자 특이적 siRNA, 그러한 siRNA를 포함하는 이중나선 올리고 RNA 구조체 및 이를 포함하는 암 예방 또는 치료용 조성물 |
KR20150006743A (ko) * | 2013-07-09 | 2015-01-19 | (주)바이오니아 | 간암 연관 유전자 특이적 siRNA, 그러한 siRNA를 포함하는 이중나선 올리고 RNA 구조체 및 이를 포함하는 암 예방 또는 치료용 조성물 |
CN106459974A (zh) * | 2014-04-04 | 2017-02-22 | 柏业公司 | 新颖的双链寡rna和包含它的用于预防或治疗纤维化或呼吸系统疾病的药物组合物 |
EP3137209B1 (en) | 2014-05-02 | 2022-10-05 | W.R. Grace & CO. - CONN. | Functionalized support material and methods of making and using functionalized support material |
EP3207069B1 (en) | 2014-10-15 | 2021-03-10 | University Of Connecticut | Bio-reducible self-assembled liquid crystalline block copolymer for drug delivery |
PL3302784T3 (pl) | 2015-06-05 | 2022-01-17 | W.R. Grace & Co.-Conn. | Adsorbentowe środki klarujące do bioprzetwarzania oraz sposoby ich wytwarzania i stosowania |
WO2016204515A1 (ko) * | 2015-06-15 | 2016-12-22 | (주)바이오니아 | STAT3 유전자 특이적 siRNA, 그러한 siRNA를 포함하는 이중나선 올리고 RNA 구조체, 이를 포함하는 조성물 및 이의 용도 |
KR101861738B1 (ko) | 2016-08-24 | 2018-05-29 | (주)바이오니아 | 마이크로 rna를 포함하는 이중나선 올리고 rna 구조체 |
EP3600439A4 (en) * | 2017-03-22 | 2021-01-13 | The Regents of the University of California | MODIFIED OLIGONUCLEOTIDES AND THEIR THERAPEUTIC USES |
US11414665B2 (en) | 2017-12-01 | 2022-08-16 | Suzhou Ribo Life Science Co., Ltd. | Nucleic acid, composition and conjugate comprising the same, and preparation method and use thereof |
CN118291456A (zh) | 2017-12-01 | 2024-07-05 | 苏州瑞博生物技术股份有限公司 | 一种核酸、含有该核酸的组合物与缀合物及制备方法和用途 |
WO2019105418A1 (zh) | 2017-12-01 | 2019-06-06 | 苏州瑞博生物技术有限公司 | 双链寡核苷酸、含双链寡核苷酸的组合物与缀合物及制备方法和用途 |
JP7360716B2 (ja) * | 2017-12-01 | 2023-10-13 | スーチョウ リボ ライフ サイエンス カンパニー、リミテッド | 核酸、当該核酸を含む組成物および複合体ならびに調製方法と使用 |
AU2018394875B2 (en) | 2017-12-29 | 2023-08-03 | Suzhou Ribo Life Science Co., Ltd. | Conjugates and preparation and use thereof |
KR102141124B1 (ko) | 2018-01-30 | 2020-08-04 | (주)바이오니아 | 이중 가닥 miRNA를 포함하는 이중나선 올리고뉴클레오타이드 구조체 및 이의 용도 |
US20210189398A1 (en) | 2018-05-25 | 2021-06-24 | Bioneer Corporation | Amphiregulin gene-specific double-stranded oligonucleotide and composition for preventing and treating fibrosis-related diseases and respiratory diseases, comprising same |
EP3842534A4 (en) | 2018-08-21 | 2022-07-06 | Suzhou Ribo Life Science Co., Ltd. | NUCLEIC ACID, COMPOSITION AND CONJUGATE CONTAINING NUCLEIC ACID AND METHOD OF USE THEREOF |
CN111655297A (zh) * | 2018-09-30 | 2020-09-11 | 苏州瑞博生物技术有限公司 | 一种siRNA缀合物及其制备方法和用途 |
KR102473989B1 (ko) | 2018-11-28 | 2022-12-07 | (주)바이오니아 | 안드로젠 수용체 특이적 서열을 포함하는 이중나선 올리고뉴클레오티드 구조체, 및 이를 포함하는 탈모 예방 및 발모용 조성물 |
WO2020149644A1 (ko) | 2019-01-15 | 2020-07-23 | (주)바이오니아 | Dkk1 유전자를 표적으로 하는 이중나선 올리고뉴클레오티드, 이를 포함하는 구조체 및 이를 포함하는 탈모 예방 또는 발모용 조성물 |
KR20210063137A (ko) | 2019-11-22 | 2021-06-01 | (주)바이오니아 | Ctgf 유전자 특이적 이중가닥 올리고뉴클레오티드 및 이를 포함하는 섬유증 관련 질환 및 호흡기 관련 질환 예방 및 치료용 조성물 |
US20230348912A1 (en) | 2020-05-14 | 2023-11-02 | Bioneer Corporation | Composition for preventing or treating obesity-related disease containing amphiregulin-specific double-stranded oligonucleotide structure |
JP2023533124A (ja) | 2020-05-22 | 2023-08-02 | バイオニア コーポレーション | 二本鎖オリゴヌクレオチド及びこれを含むコロナウイルス感染症-19(covid-19)治療用組成物 |
KR102272800B1 (ko) | 2020-06-30 | 2021-07-05 | 국방과학연구소 | 코로나바이러스 특이적 이중가닥 올리고뉴클레오티드 및 이를 포함하는 코로나바이러스 감염증-19 예방 및 치료용 조성물 |
CA3206861A1 (en) | 2021-02-25 | 2022-09-01 | Han-Oh Park | Composition for alleviating hair graying, promoting hair growth and/or preventing or alleviating hair loss, comprising double-stranded mirna as active ingredient |
WO2022191567A1 (ko) | 2021-03-08 | 2022-09-15 | (주)바이오니아 | Covid-19를 포함하는 호흡기 바이러스 감염증, 바이러스 감염에 의한 폐섬유증, 또는 호흡기 질환 예방 또는 치료를 위한 초음파 방식 연무식 흡입기를 이용한 이중가닥 올리고뉴클레오티드 구조체 투여용 조성물 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004087931A1 (en) * | 2003-04-03 | 2004-10-14 | Korea Advanced Institute Of Science And Technology | Conjugate for gene transfer comprising oligonucleotide and hydrophilic polymer, polyelectrolyte complex micelles formed from the conjugate, and methods for preparation thereof |
KR20070061770A (ko) * | 2005-08-17 | 2007-06-14 | (주)바이오니아 | siRNA의 세포내 전달을 위한 siRNA와 친수성 고분자 간의접합체 및 그의 제조방법 |
Family Cites Families (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5451463A (en) * | 1989-08-28 | 1995-09-19 | Clontech Laboratories, Inc. | Non-nucleoside 1,3-diol reagents for labeling synthetic oligonucleotides |
US5141813A (en) * | 1989-08-28 | 1992-08-25 | Clontech Laboratories, Inc. | Multifunctional controlled pore glass reagent for solid phase oligonucleotide synthesis |
KR100270195B1 (ko) * | 1991-09-06 | 2001-03-02 | 와일러 제임스 에프. | 겔로닌 폴리펩타이드를 암호화하는 dna 서열 |
US6221959B1 (en) * | 1994-11-18 | 2001-04-24 | Supratek Pharma, Inc. | Polynucleotide compositions |
US6348583B1 (en) * | 1999-08-30 | 2002-02-19 | Bio-Rad Laboratories, Inc. | Poly(ether-thioether), poly(ether-sulfoxide) and poly(ether-sulfone) nucleic acids |
US7491805B2 (en) * | 2001-05-18 | 2009-02-17 | Sirna Therapeutics, Inc. | Conjugates and compositions for cellular delivery |
DK2284266T3 (da) | 2002-11-14 | 2014-01-13 | Thermo Fisher Scient Biosciences Inc | sIRNA-MOLEKYLE MOD TP53 |
US7851615B2 (en) * | 2003-04-17 | 2010-12-14 | Alnylam Pharmaceuticals, Inc. | Lipophilic conjugated iRNA agents |
CN1984921B (zh) * | 2003-06-03 | 2010-06-16 | Isis药物公司 | 存活蛋白表达的调节 |
KR101147147B1 (ko) * | 2004-04-01 | 2012-05-25 | 머크 샤프 앤드 돔 코포레이션 | Rna 간섭의 오프 타겟 효과 감소를 위한 변형된폴리뉴클레오타이드 |
WO2007051303A1 (en) | 2005-11-02 | 2007-05-10 | Protiva Biotherapeutics, Inc. | MODIFIED siRNA MOLECULES AND USES THEREOF |
EP2046954A2 (en) * | 2006-07-31 | 2009-04-15 | Curevac GmbH | NUCLEIC ACID OF FORMULA (I): GIXmGn, OR (II): CIXmCn, IN PARTICULAR AS AN IMMUNE-STIMULATING AGENT/ADJUVANT |
EP2025348A1 (en) * | 2007-08-13 | 2009-02-18 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Targeted block copolymer micelles |
WO2009039173A2 (en) * | 2007-09-19 | 2009-03-26 | Applied Biosystems Inc. | SiRNA SEQUENCE-INDEPENDENT MODIFICATION FORMATS FOR REDUCING OFF-TARGET PHENOTYPIC EFFECTS IN RNAi, AND STABILIZED FORMS THEREOF |
TW200927177A (en) * | 2007-10-24 | 2009-07-01 | Nat Inst Of Advanced Ind Scien | Lipid-modified double-stranded RNA having potent RNA interference effect |
KR101224828B1 (ko) | 2009-05-14 | 2013-01-22 | (주)바이오니아 | siRNA 접합체 및 그 제조방법 |
-
2009
- 2009-05-14 KR KR1020090042297A patent/KR101224828B1/ko active IP Right Grant
-
2010
- 2010-05-13 CN CN201210301551.2A patent/CN102888404B/zh active Active
- 2010-05-13 BR BRPI1012141A patent/BRPI1012141B1/pt active IP Right Grant
- 2010-05-13 RU RU2011150787/10A patent/RU2558258C2/ru active
- 2010-05-13 AU AU2010248239A patent/AU2010248239B2/en active Active
- 2010-05-13 EP EP10775118.2A patent/EP2463371B1/en active Active
- 2010-05-13 EP EP13159602.5A patent/EP2626427A3/en not_active Withdrawn
- 2010-05-13 WO PCT/KR2010/003039 patent/WO2010131916A2/ko active Application Filing
- 2010-05-13 CN CN201310147988.XA patent/CN103233003B/zh active Active
- 2010-05-13 JP JP2012510752A patent/JP5758381B2/ja active Active
- 2010-05-13 CA CA2871888A patent/CA2871888A1/en not_active Abandoned
- 2010-05-13 CN CN201080021324.3A patent/CN102439148B/zh active Active
- 2010-05-13 US US13/319,885 patent/US8779114B2/en active Active
- 2010-05-13 CA CA2761749A patent/CA2761749C/en active Active
- 2010-05-13 EP EP13185358.2A patent/EP2682466B1/en active Active
-
2012
- 2012-09-13 US US13/613,071 patent/US8772472B2/en active Active
-
2013
- 2013-05-08 JP JP2013098798A patent/JP5797688B2/ja active Active
- 2013-11-21 RU RU2013151977/10A patent/RU2571218C2/ru active
-
2014
- 2014-05-30 US US14/291,656 patent/US20140350233A1/en not_active Abandoned
- 2014-05-30 US US14/291,540 patent/US9211343B2/en active Active
- 2014-12-02 JP JP2014244541A patent/JP2015107115A/ja active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004087931A1 (en) * | 2003-04-03 | 2004-10-14 | Korea Advanced Institute Of Science And Technology | Conjugate for gene transfer comprising oligonucleotide and hydrophilic polymer, polyelectrolyte complex micelles formed from the conjugate, and methods for preparation thereof |
KR20070061770A (ko) * | 2005-08-17 | 2007-06-14 | (주)바이오니아 | siRNA의 세포내 전달을 위한 siRNA와 친수성 고분자 간의접합체 및 그의 제조방법 |
Non-Patent Citations (2)
Title |
---|
ALSHAMSAN A ET AL: "Formulation and delivery of siRNA by oleic acid and stearic acid modified polyethylenimine", 《MOLECULAR PHARMACEUTICS》 * |
SORIM CHOUNG ET AL: "Chemical modification of siRNAs to improve serum stability without loss of efficacy", 《BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS》 * |
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