CN1860228B - 订书钉型寡核苷酸及包含该订书钉型寡核苷酸的药物 - Google Patents
订书钉型寡核苷酸及包含该订书钉型寡核苷酸的药物 Download PDFInfo
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Abstract
本发明提供寡核苷酸和药物,该寡核苷酸改善了下述问题点,即以往型寡核苷酸由于两端是开放的,因此不稳定;另外,虽然通过硫代磷酸酯化(S化)修饰来提高对分解酶的稳定性,但由硫代磷酸酯而引起毒性的产生等。详细地说,提供订书钉型寡核苷酸以及将其作为有效成分的药物。具体地说,为转录因子抑制剂、反义寡核苷酸或siRNA,更具体地说,为炎症、自身免疫性疾病、中枢性疾病、缺血性疾病的再灌注障碍、器官移植或器官手术后的预后恶化或PTCA后的再狭窄的预防、治疗或改善剂,进一步具体地说,为关节炎、皮炎、肾炎、肝炎、肾衰竭、膀胱炎、前列腺炎、尿道炎、溃疡性结肠炎或克罗恩氏病、慢性类风湿性关节炎或变形性关节炎、特应性皮炎、接触性皮炎、干癣、皮肤溃疡或者褥疮的预防、治疗或改善剂。
Description
技术领域
本发明涉及新型的订书钉(staple)型寡核苷酸以及将其作为有效成分的药物。
背景技术
以往的寡核苷酸作为转录因子抑制剂、反义寡核苷酸、siRNA等而被广泛利用。
其中,例如作为转录因子抑制剂,具体地可以列举出分子诱饵(诱饵寡核苷酸,以下称为诱饵)型核酸,其特异性地阻碍控制基因表达的转录因子的活性。
这里的转录是指生物体内的遗传信息表达时,以DNA为模板合成信使RNA的过程,以通过转录而制成的信使RNA的信息为基础合成蛋白质。将控制该转录的因子称为转录调节因子。
具体地说,已知的有NF-κB、STAT-1、STAT-2、STAT-3、STAT-4、STAT-5、STAT-6、GATA-3、AP-1、E2F、Ets、CRE等54种。
作为反义寡核苷酸,具体地可列举出具有与目标基因相对合的序列并抑制该基因表达的药物。
作为siRNA,具体地可列举出通过RNA干涉(RNA interferance;RNAi)来阻碍目标基因表达的药物。
另外,这些寡核苷酸的特征为在结构上构成双链。
成为本发明背景的现有文献为:Biochem Biophys Res Commun.2003 Sep 5;308(4):689-97、Gene Ther.2002 Dec;9(24):1682-92以及CircRes.2002 Jun 28;90(12):1325-32。
发明内容
本发明所要解决的问题点为:以往型的寡核苷酸由于两端是开放(open)的,因此不稳定;另外,虽然通过硫代磷酸酯化(S化)修饰来提高对核酸外切酶(exonuclease)等分解酶的稳定性,但由硫代磷酸酯而引起毒性的产生。
本发明具体为下述的物质和药物。
(1)订书钉型寡核苷酸,其为单链寡核苷酸,其中,5’端序列具有与中间部序列反方向的互补性,3’端序列也具有与中间部序列反方向的互补性,中间部的两端具有在分子内不形成互补结合且包含3~10个碱基序列的环部。
(2)如(1)所述的订书钉型寡核苷酸,其中,单链寡核苷酸为30~70碱基长。
(3)如(1)或(2)所述的订书钉型寡核苷酸,其中,单链寡核苷酸为34~64碱基长。
(4)如(1)~(3)任一项所述的订书钉型寡核苷酸,其中,单链寡核苷酸为38~58碱基长。
(5)如(1)~(4)任一项所述的订书钉型寡核苷酸,其中单链寡核苷酸为42~54碱基长。
(6)如(1)~(5)任一项所述的订书钉型寡核苷酸,其中环部为4~6碱基长。
(7)如(1)~(6)任一项所述的订书钉型寡核苷酸,其中单链寡核苷酸为42~54碱基长、环部为4~6碱基长。
(8)如(1)~(7)任一项所述的订书钉型寡核苷酸,其中寡核苷酸为DNA或DNA衍生物。
(9)如(1)~(8)任一项所述的订书钉型寡核苷酸,其特征在于,磷酸基没有被硫代磷酸酯化。
(10)如(1)~(9)任一项所述的订书钉型寡核苷酸,其为选自以序列表的序列号1~3所示的寡脱氧核苷酸中的1种。
(11)包含(1)~(10)任一项所述的订书钉型寡核苷酸的药物。
(12)如(11)所述的药物,该药物为转录因子抑制剂、反义寡核苷酸或siRNA。
(13)如(12)所述的药物,其中转录因子抑制剂为拮抗型抑制剂。
(14)如(12)或(13)所述的药物,其中转录因子为选自NF-κB、STAT-1、STAT-2、STAT-3、STAT-4、STAT-5、STAT-6、GATA-3、AP-1、E2F、Ets和CRE中的1种。
(15)如(12)~(14)任一项所述的药物,该药物为炎症、过敏性疾病、自身免疫性疾病、中枢性疾病、缺血性疾病的再灌注障碍、器官移植或器官手术后的预后恶化、经皮冠状动脉成形术(percutaneoustransluminal coronary angioplasty;PTCA)后的再狭窄的预防、治疗或改善剂。
(16)如(12)~(15)任一项所述的药物,其中炎症为关节炎、皮炎、肾炎、肝炎、肾衰竭、膀胱炎、前列腺炎、尿道炎、溃疡性结肠炎或克罗恩氏病(Crohn disease)。
(17)如(16)所述的药物,其中关节炎为慢性类风湿性关节炎或变形性关节炎。
(18)如(16)所述的药物,其中皮炎为特应性皮炎、接触性皮炎、干癣、皮肤溃疡或者褥疮。
(19)将(1)~(10)任一项所述的订书钉型寡核苷酸用于制造转录因子抑制剂、反义寡核苷酸或siRNA的用途。
(20)通过给患者服用药理学上有效量的(1)~(10)任一项所述的订书钉型寡核苷酸,从而预防、治疗或改善转录因子抑制剂、反义寡核苷酸或者siRNA对其有效的疾病的方法。
本发明中的订书钉型寡核苷酸为单链,其具有下述订书钉型结构(挤压后的订书钉型的形状),即5’端序列具有与中间部序列反方向的互补性,3’端序列也具有与中间部序列反方向的互补性,中间部的两端具有在分子内不形成互补结合且包含3~10个碱基序列的环部,具体地,例如具有下述化学式的结构。
式中,竖线表示非结合部[5’端和3’端]。
另外,其链长没有限定,通常为30~70碱基长,优选为34~64碱基长,更优选为38~58碱基长,进一步优选为42~54碱基长。
环部为3~10碱基长,优选为4~6碱基长。
5’端和3’端的折回部序列(从5’端或3’端开始到环部为止的、具有互补性的序列)的链长也没有限定,通常为4~20碱基长,优选为6~18碱基长,更优选为8~16碱基长。
5’端和3’端的折回部序列的链长可以相同(对称形),也可以不同(非对称形)。
本发明中的寡核苷酸没有限定,可以是DNA、DNA衍生物、RNA或RNA衍生物,但更优选为DNA或DNA衍生物。
作为本发明的订书钉型寡核苷酸的具体例,例如可列举出以序列表的序列号1~3所示的寡脱氧核苷酸。
本发明的订书钉型寡核苷酸可按照常法,通过利用DNA合成机等合成目标单链序列后,在溶剂中加热而得到。
本发明中的硫代磷酸酯是指磷酸基中的氧原子的一部分或全部被硫原子取代的结构。
本发明中的订书钉型寡核苷酸的药物用途没有限定,具体地说,例如为转录因子抑制剂、反义寡核苷酸、siRNA等,更具体地说,例如为炎症、自身免疫性疾病、中枢性疾病、缺血性疾病的再灌注障碍、器官移植或器官手术后的预后恶化、PTCA后的再狭窄等的预防、治疗或改善剂。
这里的炎症,更具体地可列举出例如关节炎、皮炎、肾炎、肝炎、肾衰竭、膀胱炎、前列腺炎、尿道炎、溃疡性结肠炎、克罗恩氏病等。
其次,这里的关节炎,更具体地可列举出例如慢性类风湿性关节炎(RA)或变形性关节炎(OA)等。
再者,作为皮炎,更具体地可列举出例如特应性皮炎、接触性皮炎、干癣、皮肤溃疡、褥疮等。
本发明中的订书钉型寡核苷酸的给药量或者给药途径也根据疾病的种类或程度、症状、患者年龄、性别、并发症、联用药等的不同而不同,并没有限定,但通常为每次10μg~10g、优选为100μg~5g、更优选为1mg~1g,通过经皮、皮下、关节内、肌肉内、静脉内或者口服给药。
除本发明之外,例如还有WO03/091432号公报等中公开的环状诱饵(哑铃型诱饵),但本发明的订书钉型具有开环部,结构上完全不同。
通过本发明的实施,以往型寡核苷酸所具有的不稳定性被改善,可以降低给药量,且还能够提高安全性。
附图说明
图1为表示LPS刺激24小时后,培养上清中的IL-1β量的图。
图2为表示LPS刺激24小时后,滑膜上清中的IL-1β量的图。
图3为表示订书钉型诱饵的稳定性的电泳图。
具体实施方式
以下,列举实施例进一步详细说明本发明,当然本发明不受这些实施例的限定。
实施例1
订书钉型寡核苷酸的抗炎效果研究
A.细胞因子的定量
1.滑膜组织的处理
(1)将手术时采集的类风湿性关节炎患者的滑膜组织均质后,每100mg接种于24孔板中(无血清培养基500μl/孔)。
NF-κB诱饵、Scramble诱饵的转染(HVJ envelope法)
(2)在HVJ 1.1×104HAU/1.1ml BSS[平衡盐溶液(137mM NaCl,5.4mM KCl,10mM Tris-HCl,pH为7.6)]的状态下以99mJ/cm2进行紫外线处理。
(3)每1ml分装于1.5ml管中,在4℃,15000rpm的条件下离心处理15min。
(4)在200μg的诱饵中加入BSS,使其为92μl。
(5)添加8μl 3%TritonX-100/TE缓冲溶液。
(6)在4℃,15000rpm的条件下离心15min后,除去上清。
(7)添加1ml BSS混合后,在15000rpm的条件下离心分离15min。
(8)除去上清后,悬浮于200μl的PBS中。
(9)在滑膜组织中加入诱饵-HVJ envelope混合体,使其为15μM,并在37℃的二氧化碳培养箱中培养30分钟。
所加诱饵的序列
双链NF-κB诱饵
5’-CCTTGAAGGGATTTCCCTCC-3’/5’-GGAGGGAAATCCCTTCAAGG-3’(双链)
Scramble诱饵
5’-CATGTCGTCACTGCGCTCAT-3’/5’-ATGAGCGCAGTGACGACATG-3’(双链)
订书钉型寡核苷酸(i)
5-ATTTCCCTCCAAAAGGAGGGAAATCCCTTCAAGGAAAACCTTGAAGGG-3’(在1处连接)
哑铃型寡核苷酸(ii)
5’-ATTTCCCTCCAAAAGGAGGGAAATCCCTTCAAGGAAAACCTTGAAGGG-3’(在2处连接)
2.LPS刺激
(10)除去诱饵-HVJ envelope混合体,加入500μl含有10%FBS的培养液(培养基),添加LPS,使其为0.01μg/ml。
3.培养液、滑膜组织的回收、IL-1β的测定
(11)24小时后回收培养液和滑膜组织。在滑膜组织中加入500μlPBS,使用均质机进行均质化。
5000rpm的条件下离心10min后,收集上清。测定IL-1β前在-20℃保存。
(12)使用IL-1βELISA试剂盒(ENDOGEN公司、目录号:EH21L1B)测定培养上清、滑膜上清。
4.结果
NT:未处理组
SC:scramble诱饵给药组
NF:NFκB诱饵给药组
R1:订书钉型寡核苷酸(1处连接)
R2:哑铃型寡核苷酸(2处连接)
在订书钉型寡核苷酸作用组中,抑制了培养上清、滑膜上清中的IL-1β的产生。1处连接的订书钉型寡核苷酸的抑制效果更强。(双链NFκB作用组在此次实验中的抑制效果弱。)
上述细胞因子定量的整个流程如下所示。
实施例2
B.哑铃型诱饵的稳定性试验(参照图3)
目的:在关节液(原液)中的诱饵的耐性比较
序列、实验条件:
1)S化双链诱饵
2)S化订书钉型诱饵
3)非S化订书钉型诱饵(无S化)
4)单链诱饵(订书钉型寡核苷酸的连接前阶段)
5)末端S化单链诱饵(订书钉型寡核苷酸的连接前阶段、仅两个末端S化的诱饵)
分别向其中加入0%、50%或100%的关节液(原液),利用电泳比较稳定性。
结果:在关节液中,1)S化双链诱饵、2)S化订书钉型诱饵和3)非S化订书钉型诱饵稳定,4)单链诱饵基本稳定,5)末端S化单链诱饵被分解。
详细地说,1)S化双链诱饵和2)S化订书钉型诱饵,即使在100%关节液中也都与0%(无添加)同样地稳定。
另外,3)非S化订书钉型诱饵的稳定性与关节液的浓度依赖性地减少,但即使在100%关节液中,也存在有足可检出的稳定的诱饵。
另一方面,4)单链诱饵和5)末端S化单链诱饵,与1)~3)相比较,在关节液中的稳定性低。
但是,如果比较4)单链诱饵和5)末端S化单链诱饵,可知4)单链诱饵即使在100%关节液中也存在有微量的稳定的诱饵,而5)末端S化单链诱饵即使在50%关节液中也没有稳定的诱饵。
序列表
<110>安琪士摩奇 株式会社
<120>订书钉型寡核苷酸及包含该订书钉型寡核苷酸的药物
<130>04075pCT
<150>JP 2003-341419
<151>2003-09-30
<160>5
<170>PatentIn version 3.1
<210>1
<211>48
<212>DNA
<213>Artificial Sequence
<220>
<223>Synthetic DNA
<400>1
atttccctcc aaaaggaggg aaatcccttc aaggaaaacc ttgaaggg 48
<210>2
<211>53
<212>DNA
<213>Artificial Sequence
<220>
<223>Synthetic DNA
<400>2
atttccctcc tggatcccag gagggaaatc ccttcaagga aaaccttgaa ggg 53
<210>3
<211>48
<212>DNA
<213>Artificial Sequence
<220>
<223>Synthetic DNA
<400>3
atttcccttt ttttaaaggg aaatcccttc aagatttttc ttgaaggg 48
<210>4
<211>20
<212>DNA
<213>Artificial Sequence
<220>
<223>NF-kB decoy
Represents the sequence of NF-kB decoy by paired with the sequence
No.5.
<400>4
ccttgaaggg atttccctcc 20
<210>5
<211>20
<212>DNA
<213>Artificial Sequence
<220>
<223>NF-kB decoy
Represents the sequence of NF-kB decoy by paired with the sequence
No.4.
<400>5
ggagggaaat cccttcaagg 20
<210>6
<211>20
<212>DNA
<213>Artificial Sequence
<220>
<223>Scramble decoy
Represents the sequence of Scramble decoy by paired with the
sequence No.7.
<400>6
catgtcgtca ctgcgctcat 20
<210>7
<211>20
<212>DNA
<213>Artificial Sequence
<220>
<223>Scramble decoy
Represents the sequence of Scramble decoy by paired with the
sequence No.6.
<400>7
atgagcgcag tgacgacatg 20
Claims (10)
1.一种订书钉型寡核苷酸,其为选自以序列表的序列号1~3所示且具有下述结构式所示结构的寡脱氧核苷酸中的1种,
A A
A GGAGGGAAATCCCTTCAAGG A
SEQ.1 A CCTCCCTTTA|GGGAAGTTCC A
A A
C A
C CAGGAGGGAAATCCCTTCAAGG A
SEQ.2 T GGTCCTCCCTTTA|GGGAAGTTCC A
A A
T T
SEQ.3 T AAAGGGAAATCCCTTCAAGA T
T TTTCCCTTTA|GGGAAGTTCT T
T T
式中,竖线表示非结合部,即5’端和3’端。
2.包含权利要求1所述的订书钉型寡核苷酸的药物。
3.如权利要求2所述的药物,该药物为转录因子抑制剂。
4.如权利要求3所述的药物,其中,转录因子抑制剂为拮抗型抑制剂。
5.如权利要求3或4所述的药物,其中转录因子为NF-κB。
6.如权利要求3~5任一项所述的药物,该药物为炎症、过敏性疾病、自身免疫性疾病、中枢性疾病、缺血性疾病的再灌注障碍、器官移植或器官手术后的预后恶化、经皮冠状动脉成形术后的再狭窄的预防、治疗或改善剂。
7.如权利要求6所述的药物,其中,炎症为关节炎、皮炎、肾炎、肝炎、肾衰竭、膀胱炎、前列腺炎、尿道炎、溃疡性结肠炎或克罗恩氏病。
8.如权利要求7所述的药物,其中,关节炎为慢性类风湿性关节炎或变形性关节炎。
9.如权利要求7所述的药物,其中,皮炎为特应性皮炎、接触性皮炎、干癣、皮肤溃疡或者褥疮。
10.将权利要求1所述订书钉型寡核苷酸用于制造转录因子抑制剂的用涂。
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JP341419/2003 | 2003-09-30 | ||
PCT/JP2004/014694 WO2005030960A1 (ja) | 2003-09-30 | 2004-09-29 | ステイプル型オリゴヌクレオチドおよびそれからなる医薬 |
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GB0404209D0 (en) * | 2004-02-25 | 2004-03-31 | Uws Ventures Ltd | Materials and methods for treatment of allergic disease |
US20080311552A1 (en) * | 2005-09-20 | 2008-12-18 | London Health Sciences Centre Research, Inc. | Use of Sirnas in Organ Storage/Reperfusion Solutions |
JP5601777B2 (ja) * | 2008-02-13 | 2014-10-08 | 株式会社ジーンデザイン | 新規オリゴヌクレオチド誘導体及びそれから成るNF−κBデコイ |
GB2459922A (en) * | 2008-05-13 | 2009-11-18 | Univ Dundee | Treatment for keratinizing dermatological disorders by reduction in keratin expression |
EP2415869A4 (en) | 2009-04-03 | 2013-06-19 | Biomics Biotechnologies Co Ltd | MODIFIED OLIGO-NUCLEIC ACID MOLECULE, PROCESS FOR PREPARATION AND USES THEREOF |
EP2558578B1 (en) | 2010-04-13 | 2015-10-28 | Life Technologies Corporation | Compositions and methods for inhibition of nucleic acids function |
US8785121B2 (en) | 2010-07-08 | 2014-07-22 | Bonac Corporation | Single-stranded nucleic acid molecule for controlling gene expression |
HUE026520T2 (en) * | 2010-07-08 | 2016-06-28 | Bonac Corp | Single chain nucleic acid molecule for gene expression control |
EP2674494B1 (en) * | 2010-08-03 | 2014-12-17 | Bonac Corporation | Single-stranded RNA molecule having nitrogen-containing alicyclic skeleton |
US8691782B2 (en) | 2010-08-03 | 2014-04-08 | Bonac Corporation | Single-stranded nucleic acid molecule having nitrogen-containing alicyclic skeleton |
US9528111B2 (en) | 2012-01-07 | 2016-12-27 | Bonac Corporation | Single-stranded nucleic acid molecule having amino acid backbone |
US9663784B2 (en) | 2012-05-26 | 2017-05-30 | Bonac Corporation | Single-stranded nucleic acid molecule for regulating expression of gene having delivering function |
WO2014170441A1 (en) * | 2013-04-19 | 2014-10-23 | Dna Therapeutics | Inhibition of dna damage repair by artificial activation of parp with oligonucleotide molecules |
EP3088524A4 (en) | 2013-12-26 | 2017-08-09 | Tokyo Medical University | Artificial mimic mirna for controlling gene expression, and use of same |
JP6425142B2 (ja) | 2013-12-27 | 2018-11-21 | 株式会社ボナック | 遺伝子発現制御のための人工マッチ型miRNAおよびその用途 |
MX2017008587A (es) | 2014-12-27 | 2017-10-20 | Bonac Corp | Mirna natural para controlar la expresion de gen y uso del mismo. |
CN108064289A (zh) | 2015-03-27 | 2018-05-22 | 株式会社博纳克 | 具有递送功能和基因表达调控能力的单链核酸分子 |
CN113004362B (zh) * | 2021-02-26 | 2023-08-15 | 南方科技大学 | 一种订书钉核酸、dna纳米机器人及其制备方法和应用 |
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FR2703053A1 (fr) * | 1993-03-26 | 1994-09-30 | Genset Sa | Oligonucléotides agrafes et semi-agrafes, procédé de préparation et applications . |
CN1346363A (zh) * | 1999-04-08 | 2002-04-24 | 朴钟九 | 具有更好稳定性和反义效果的新颖的反义寡核苷酸 |
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FR2675803B1 (fr) * | 1991-04-25 | 1996-09-06 | Genset Sa | Oligonucleotides fermes, antisens et sens et leurs applications. |
GB2273932A (en) | 1992-11-24 | 1994-07-06 | Stiefel Laboratories | Stable oligonucleotides |
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FR2703053A1 (fr) * | 1993-03-26 | 1994-09-30 | Genset Sa | Oligonucléotides agrafes et semi-agrafes, procédé de préparation et applications . |
CN1346363A (zh) * | 1999-04-08 | 2002-04-24 | 朴钟九 | 具有更好稳定性和反义效果的新颖的反义寡核苷酸 |
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CN1860228A (zh) | 2006-11-08 |
ATE532865T1 (de) | 2011-11-15 |
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US7595301B2 (en) | 2009-09-29 |
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