CN110251682A - 多肽序列设计及其在多肽介导的siRNA传递中的应用 - Google Patents
多肽序列设计及其在多肽介导的siRNA传递中的应用 Download PDFInfo
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Abstract
本发明提供一种多肽以及其与药物分子复合的复合物,所述多肽与所述药物分子通过非共价键连接。本发明所述的多肽可以促进siRNA分子传递进入细胞,并对siRNA介导的细胞内部靶点沉默起作用。
Description
本申请是申请人于2014年6月19日申请的名称为多肽序列设计及其在多肽介导的siRNA传递中的应用,申请号为201280057898.5的中国发明专利申请的分案申请。该中国发明专利申请来自于2012年11月23日申请的名称为PEPTIDE SEQUENCE DESIGN AND USETHEREOF FOR PEPTIDE-MEDIATED siRNA DELIVERY(多肽序列设计及其在多肽介导的siRNA传递中的应用),申请号为PCT/CA2012/50843的国际申请。
相关申请的参考
依照巴黎公约,本申请要求一篇美国专利申请的优先权,该美国专利申请号为61563591,申请日为2011年11月24日。本申请包括该美国专利申请所有的内容。
技术领域
本发明涉及多肽序列以及短干扰RNA(siRNA)传递应用领域。
背景技术
在过去的十年中,我们已经见证了对RNA分子在基因表达调节领域的的巨大进步。而这种进步的主要贡献来自于RNA干扰(RNAi)的发现。自从法耶和梅洛首次发现秀丽隐杆线虫[Fire,et al.1998],RNAi已成为一个革命性的保守机制,其通过使用短RNAs带来了序列特异性以及使转录后的基因发生沉默(PTGS)。RNAi背后的基本理念是一种被称为短干扰RNA或siRNA的短RNA双链体,其包括21~23个核苷酸并与mRNA的片段互补。这种短RNA双链体可以外源合成并被引入细胞。这会触发最终降低同源的mRNA并抑制产生相应的蛋白质。几种类型的短RNA可能参与了RNAi的过程,它们包括SiRNA(siRNA),微RNA(miRNA)的,微小的非编码RNA(tncRNA),和短发夹RNA(shRNA)[Novina,et al.2004,Paddison,et al.2008,Engelke,et al.2005]。
siRNA在体内和体外的应用的主要局限于裸的siRNA在生理条件下不稳定,其很快从血液中清除,并且不能穿越细胞膜以进入细胞内的环境。由于它们尺寸小并且亲水,管理裸的siRNA的一个重要部分是通过网状内皮系统(RES)将其排出体外[Moghimi,etal.1999]。另据报道,相比于中性的或者低带电的粒子,高带电的粒子可以被RES更快的识别[Benoit,et al.2006,Mahato.2005]。此外,核酸(NA)为基础的药物在循环中以及细胞内进行酶分解,导致对靶位点的药效不足。对siRNA的糖、核碱基以及磷酸酯主链的修饰已经被应用到提高其核酸酶抗性,同时不影响其沉默效率[Manoharan2004,Verma,et al.2003,Zhang,et al.2006]。与疏水功能基团结合也可以增强其细胞摄取能力[Oliviera et al.,2006]。
与费时而且昂贵的核酸的化学修饰相比,载体介导的策略正成为一种简单而快速的可以制备核酸治疗剂并保护它们免受降解的方法。这些载体包括病毒载体、脂质、聚合物和肽,它们与siRNA共组装或共价缀合,可以增强细胞靶向,延长药物的循环时间,并改善对细胞膜的穿透。
由于通过不同物理化学性质的氨基酸的应用,使得肽设计具有多样性和多功能性,肽已经被用于合成药物、小分子、生物活性肽、治疗性蛋白的传递,但是核酸机理尚未完全清楚。这些肽可以包括源于蛋白质的细胞穿透肽(CPP)[Langel 2007],阳离子肽,经过设计得到的双性肽[Oehlke,et al.1998],融合肽[Mok,et al.2008],细胞靶向肽(CTP)[vives 2005]以及包含核定位信号的肽(NLS)[Cartier,et al.2002]等。阳离子肽富含氨基酸,可以与小的核酸或者浓缩的核酸相互作用形成稳定的小颗粒。细胞穿透肽可以促进复合物通过细胞膜。富含组氨酸并且对pH敏感的或者膜融合的肽可以加强内涵体逃逸能力和基因复合物向细胞质的释放。基因传递系统中所包含的细胞靶向肽可以介导细胞或者组织特异性靶向。核定位细胞肽的连接可以提高基因复合物的核定位能力[Jarvert,etal.2007]。
在细胞穿透肽中,只有少数可以展示高转染效率、低细胞毒性和免疫力。Tat andPenetratin是蛋白质多肽中研究最广泛的肽。源自人体免疫病毒1(HIV-1)的反式激活转录活化剂,由Frankel and Pabo发现于1988,其可以在培养液中通过多种细胞类型被高效采用。
目前已经发现了,能够被认为是核酸的有效载体的特异性细胞穿透肽(CPPs),如Brock等的公开号为WO2007/076904的国际专利申请以及Divita等的公开号为WO2007/069090的国际专利申请,虽然其不是全都描述siRNA的传递,见Mixson的美国专利7163695以及Rice等的美国专利7112442。
在许多载体介导传递系统中,核酸(NA)与具有特异性序列的载体肽呈共价连接,如Langel等的公开号为WO2008/063113的国际专利申请和Troy等的公开号为US2005/0260756的美国专利申请。特异性多肽与核酸通过化学连接部连接,如WO2008/033285和WO2007/069068。Karas的美国专利7420031报道了一种可以传递核酸到细胞内部的多肽;多肽与药物分子的形成的复合物通过活血交联或者桥接形成。Piwinica-Worms的美国专利7306784描述了细胞膜穿透肽结合物和具有靶细胞特异性的共价复合物的应用。
Hallbrink等的公开号为US2008/0234183的美国专利申请描述了预测、设计、检测和验证CPPs的方法。其中,CPP-药物复合物包含一个连接CPP与药物分子的共价连接部。
Kariem等的公开号为WO2010/039088的国际专利申请描述了一些硬脂酰化的线性或者枝状的CPP的应用,尤其核酸的传递与穿透效果。
Holmes等的美国专利6800481描述了一种自组装的双性多肽,其具有交互的亲水和疏水残基,进入肉眼可见的薄膜。
Langel等的公开号为WO2003/106491的国际专利申请描述了预测、设计、检测和验证CPPs的方法,以及应用其提高细胞对连接有CPP的细胞受动器的吸收。
发明内容
在一个实施例中,本发明提供了一种多肽,其包含的氨基酸序列选自SEQ.ID.NO 1至SEQ.ID.NO 46。
在另一个实施例中,本发明提供了一种多肽与药物分子的复合物,所述多肽包含的氨基酸序列选自SEQ.ID.NO 1至SEQ.ID.NO 46。
在一个优选实施例中,本发明的药物分子为核酸;在一个更优选的实施例中,所述药物分子为siRNA。
在另一个实施例中,本发明提供了一种包含有上述复合物的药物组合物,用于输送一定量的具有药物疗效的siRNA。
在另一个实施例中,本发明提供了一种降低动物细胞或者组织内的基因产品含量水平的方法,该方法包括控制一定含量的具有药物疗效的siRNA。
在另一个实施例中,本发明提供了一种降低用于控制凋亡的基因产品含量水平的方法。
附图说明
图1为多肽C6M和C6M1的螺旋轮图。
图2为GAPDH基因在CHO细胞内的沉默效果示意图。
图3A/3B为多肽-siRNA复合物的沉默效率示意图,多肽与siRAN的摩尔比率20/1和40/1;图3A和图3B展示了不同的比率。
图4A/4B为多肽-siRNA复合物对CHO-K1细胞的毒性示意图,图4A为18个多肽-siRNA复合物的示意图,图4B为6个多肽-siRNA复合物的示意图。
图5为C8M1-siRNA复合物在PBS中随时间推移的尺寸变化示意图。
图6为C6M1-siRNA复合物在不同的摩尔比下荧光光谱的结果,C6M1为固定浓度。
图7为C6M1的荧光光谱结果和标记的siRNA复合物在不同的摩尔比下的荧光光谱结果,C6M1为固定浓度。
图8A/8B/8C为多肽-siRNA复合物在体内的效果。图8A显示了肿瘤分离前小鼠由于颈脱位而死亡的肿瘤组织的照片。图8B显示了在小鼠肿瘤模型中siRNA/C6M1复合物的抗肿瘤活性;其中该复合物被施加于小鼠模型A549的肿瘤细胞内,每天测量皮肤下肿瘤大小。图8C显示了治疗期间每天测量的小鼠体重。
具体实施方式
本发明的多肽-药物复合物通过多肽与siRNA之间的弱相互作用力产生的非共价分子缔合形成,这是一种简单快速的形成siRNA治疗剂的方法。
相比较与交联或者桥接的方法,本发明复合物的分子缔合方法显得廉价而且简单。通过这种组装,可以方便地产生经常以纳米粒子形式出现的多肽-siRNA复合物或者组装物。
考虑到细胞膜两亲性,所设计的多肽同时拥有疏水基团和亲水部分。亲水侧与亲水的药物或者基因以及脂质双分子层的亲水头通过静电作用相互作用,疏水侧则锚定于脂质双分子层的疏水核,辅助多肽-药物转运到胞质溶液中。
i.C1多肽
在一个实施例中,本发明提供了具有以下氨基酸序列的多肽:
表1:C1家族多肽序列
这组多肽是设计用来探索β-链二级结构的多肽作为传递载体的可行性。位于这些序列的相对的两端的亲水部和疏水部通过一个连接部相连。在公开号为WO2009/026729的国际专利申请里,陈等人描述了包含在多肽组装中的静电作用、氢键、疏水键以及π-π堆积作用的不同机理。这些策略被用于设计这种可以通过诸如氢键和疏水作用力等弱相互作用自组装的多肽的疏水部。通过将疏水部氨基酸的个数从8个增加到12个来改变C1M2到C1M4疏水部的长度,以此来评估疏水部长度的作用。
GGG为连接部,用于将疏水部和亲水部连接在一起,同时保持多肽主链的灵活性。C1M3中的连接部变为WSQP,以评价不同连接部的影响[Deshayes,et al.2004]。
在带正电荷的亲水性链段是负责与siRNA的共组装以及通过静电相互作用接近细胞膜。静电作用产生于胍基基团和磷酸基团,其中胍基基团来自多肽中精氨酸和赖氨酸,磷酸基团来自siRNA主链以及细胞的磷脂双层。多肽C1M中核定位序列(NLS),PKKKRKV,取代了多肽C1中的富含精氨酸链段。多肽C1序列为FQFNFQFNGGGHRRRRRRR,最初由Chen等人的公开号为WO2011/020188的国际专利申请中揭示。(其全部公开内容在此引入作为参考)。该序列通过NLS中的一个赖氨酸残基的单突变进一步被修饰,并转变为脯氨酸残基,以限制其核转运以及C1M1、C1M3和C1M4中的药物在细胞质的快速释放。在C1M5和C1M6中,亲水性链段也分别被替换为CHHRRRRRRHC和CPKPKRKVC,以评估胱氨酸在提高由于在内涵体和细胞质中的pH变化而对复合物中的siRNA的释放效果的影响。
II.E3多肽
在另一个实施例中,本发明提供的多肽具有以下氨基酸序列:
表2:E3家族多肽序列
名称 | 序列 | 序列代号 |
E3M | RLTLHLRLELTLHLE | (SEQ.ID.NO 8) |
E3M1 | RWTWHWRWEWTWHWE | (SEQ.ID.NO 9) |
这组多肽的设计也是基于陈等人的公开号为WO2009/026729的国际专利申请中提及的氨基酸配对方法。然而,与C1家族的多肽不同的是,这类多肽的亲水性氨基酸,R和H,分布于疏水性氨基酸残基中。多肽E3,含有序列RFTFHFRFEFTFHFE,最初报道于陈等人的公开号为WO2011/020188的国际专利申请。这里,我们进一步修饰了这个序列。R和E之间的静电作用,T和H之间的氢键,以及E3中的F、E3M中的L、E3M中的W之间的疏水作用,以上的这些作用有助于这些序列的自组装。组氨酸在低pH时容易质子化,因而也被用于提高复合物的内涵体逃逸。当一个包含组氨酸的多肽在内吞过程中进入内涵体,组氨酸可以作为一个质子海绵,打破内涵体的PH平衡,从而释放内涵体中的复合物。利用疏水氨基酸,E3M和E3M1中的L和W,各自取代E3中的F残基,以评估多肽中疏水基团长度的影响。进一步,色氨酸残基被报道也可以促进多肽对透过细胞膜的迁移。
III.A7多肽:
在另一个实施例中,本发明提供以具有以下氨基酸序列的多肽:
表3:A7家族多肽序列
A7M | RHALAHLLHKLKHLLHALAHR | (SEQ.ID.NO 10) |
A7M1 | RHALAHLLHRLRHLLHALAHR | (SEQ.ID.NO 11) |
这些主要包含精氨酸、赖氨酸、组氨酸、亮氨酸和丙氨酸的基团,经过设计拥有一个α螺旋的二级结构,并且从上部观察具有三个不同的部分,每个部分由亮氨酸和/或丙氨酸,精氨酸和/或赖氨酸,以及组氨酸组成。疏水性残基如亮氨酸可以通过与细胞的脂质双层的疏水尾部相互作用,对细胞穿透有所帮助,同时也有助于形成细胞膜上孔的形成[Langel 2007]。多个研究已经研究过不同长度的富含精氨酸多肽的转移效率。已经发现含有7-9个精氨酸的多肽具有最高的转移效率,且至少需要5个精氨酸或者赖氨酸残基才能使迁移发生。正如上面提及,组氨酸是一种对pH敏感的氨基酸,因为其在低pH下将被质子化。当一种包含组氨酸的多肽经过内吞进入内涵体时,组氨酸将如一个质子海绵一样吸收质子,以打破内涵体pH的平衡。这可以导致内涵体的泄漏,从而将siRNA复合物释放到细胞质中。具有序列HRLRHALAHLLHKLKHLL-HALAHRLRH的多肽A7,最初报道于陈等人的公开号为WO2011/020188的国际专利申请。这里,我们评估了序列的长度影响,也评估了A7M和A7M1中利用赖氨酸取代精氨酸而产生的影响。
IV.C6多肽:
在另一个实施例中,本发明提供了具有以下氨基酸序列的多肽:
表4:C6家族多肽序列
这组多肽的设计基于多肽C6。多肽C6报道于陈等人的公开号为
WO2009/026729的国际专利申请(其全部公开内容在此引入作为参考)。设计的多肽C6包含7个带正电的精氨酸残基。在至少需要五个精氨酸残基保持转染效率的基础上,富含精氨酸的多肽被报道可以高效低毒地将siRNA传递入细胞[Futaki,et al.,2001;Wang,et al.,2006]。疏水性残基如赖氨酸,也包含C6序列。这些疏水性氨基酸可以通过与脂质双层中的疏水尾部相互作用提高多肽的转移,或者帮助在细胞膜上形成孔[Langel.2007]。已经发现丙氨酸、赖氨酸和组氨酸大量存在于蛋白质的螺旋区域中[Chou and Fasman1973]。研究指出赖氨酸是他们所研究的蛋白中可以形成残基最强力的结构。
为了提高C6的溶解性和转染效率,将C6序列进行修饰产生一系列的衍生物。这些衍生物被设计用来允许将亲水和疏水的残基定位在分子的相对的面,以适应于螺旋结构。图1显示了氨基酸被预置为非极性和极性氨基酸定位在螺旋的两个不同面,可以促进自组装或者共组装。一些疏水赖氨酸残基选择性地被疏水基团较少的色氨酸取代,以提高在水中的溶解性。同时,据报道含有芳香基团的色氨酸在细胞摄取多种细胞穿透多肽的过程中具有关键的作用,因为它们能够与细胞膜的脂质或者胆固醇相互作用[Derossi,etal.1994;Heitz,et al.2004]。组氨酸是一种对pH敏感的氨基酸,其适合包含在一个多肽序列中,展示出能够与siRNA分子相互作用的能力,并且由于其咪唑环在pH为6时发生的质子化作用,组氨酸有助于siRNA从内涵体中逃逸[Lo&S.Wang,2008]。这导致内涵体包含物的泄露,从而将siRNA释放到发生RNA干扰的细胞质中。在多肽序列的不同位置加入色氨酸和组氨酸,以结合它们对细胞穿透以及内涵体逃逸的效力。C6多肽衍生物纳入了多种不同的共组装多肽、细胞穿透肽以及可以破坏内涵体的多肽的功能部分。
V.MW多肽:
在另一个实施例中,本发明提供了具有以下氨基酸序列的多肽:
表5:MW家族多肽序列
名称 | 序列 | 序列代号 |
MW1 | MWKSKIGSWILVRWAMWSKKRPKP | (SEQ.ID.NO 22) |
MW2 | MWKSHIGSWILVRWAMWSHKRPKP | (SEQ.ID.NO 23) |
MW3 | MWKSKISWILVSKPGLCKKRPKP | (SEQ.ID.NO 24) |
MW4 | MHKSKISWHLVSKPGLCHKRPKP | (SEQ.ID.NO 25) |
这组多肽衍生自牛朊蛋白(bPrPp)的N末端序列(1-30个氨基酸残基)。具有序列MVKSKIGSWILVLFVAMWSDVGLCKKRPKP的牛朊蛋白主要采用a-螺旋结构[etal.2004]。牛朊蛋白的基本序列(23-28个残基)类似于一个核定位序列,通过脂质筏依赖和细胞巨吞刺激细胞摄取[Wadia,et al.2008]。据报道牛朊蛋白的转染效率为48%[Endoh&Ohtsuki,2009]。因此,一些色氨酸和组氨酸残基加入到了序列中,以增加多肽与细胞膜的亲和力,并且提高内涵体逃逸能力。
VI.D-型多肽:
在另一个实施例中,本发明提供了具有以下氨基酸序列的多肽:
表6:D-型多肽序列
名称 | 序列 | 序列代号 |
C6-Dr | rLLrLLLrLWrrLLrLLr | (SEQ.ID.NO 26) |
C6-D | rllrlllrlwrrllrllr | (SEQ.ID.NO 27) |
C6M1-Dr | rLWrLLWrLWrrLWrLLr | (SEQ.ID.NO 28) |
C6M1-D | rlwrllwrlwrrlwrllr | (SEQ.ID.NO 29) |
C6M3-Dr | rLWHLLWrLWrrLHrLLr | (SEQ.ID.NO 30) |
C6M3-D | rlwhllwrlwrrlhrllr | (SEQ.ID.NO 31) |
C6M6-Dr | GLWHLLLHLWrrLLrLLr | (SEQ.ID.NO 32) |
C6M6-D | glwhlllhlwrrllrllr | (SEQ.ID.NO 33) |
对于多肽介导的药物传递系统,多肽不仅仅负责对siRNA的传递,还对保护siRNA免受血清中的核酸酶降解起到很大的作用。siRNA降解将影响基因的沉默效率。这给我们一些对多肽序列进行修饰的线索。
在转染过程中有一个障碍是细胞膜。据报道,D型的R(r)可以加强细胞穿透能力并且加强其在血清中的稳定性。在序列C6、C6M1、C6M3和C6M6中的L-型的R被D型R(r)替代。
使用这种D型R(r)也会影响多肽的二级结构,并且相应地可能影响多肽-siRNA复合物的形态和尺寸。多肽序列C6、C6M1、C6M3和C6M6被转化为D-型以改变转染效率、稳定性和毒性。
VII.GL多肽:
在另一个实施例中,本发明提供具有以下氨基酸序列的多肽:
表7:GL多肽序列
这组序列包括附加的W和H氨基酸,它们可以提高多肽与细胞膜的亲和性,并且提供内涵体逃逸能力。同样n-末端域被GLW代替,这在CADY多肽[Karidia et al.2009]和MPG多肽[Simeoni,et al.2003]中报道过。
序列HA2,GLFGAIAGFIENGWEGMIDGWYG,已经加入到多肽中,以提高CPPs的内涵体逃逸能力,CPPs,比如穿膜肽和HA2穿膜肽。序列HA2也被证明可以提高穿膜肽的转染效率。此外,一种附加的核定位序列(NLS),即带有富含赖氨酸片段的PKKKRKV,已经展现出对细胞核和细胞质的高定位性质[Simeoni,et al.2003]。
VIII.硬脂酸(Stearic acid,简称STR)修饰的多肽
在另一个实施例中,本发明提供具有以下氨基酸序列的多肽:
表8:STR修饰的多肽序列
名称 | 序列 | 序列代号 |
STR-C1 | CH<sub>3</sub>(CH<sub>2</sub>)<sub>16</sub>-GGGPKPKRKV | (SEQ.ID.NO 40) |
STR-HK | CH<sub>3</sub>(CH<sub>2</sub>)<sub>16</sub>-HHHPKPKRKV | (SEQ.ID.NO 41) |
STR-HKC | CH<sub>3</sub>(CH<sub>2</sub>)<sub>16</sub>-HHHCPKKKRKVC | (SEQ.ID.NO 42) |
硬脂酸是大量存在于人体的一种脂肪酸,并且与细胞膜具有高相互作用力。据报道,在N-端具有硬脂酸修饰的细胞穿透肽[Tanaka,et al.2010],可以提高对siRNA-多肽复合物的细胞摄取能力。这是因为,通过硬脂酸介导可以提高细胞膜的亲和性。同时,相比于母体PEI,硬脂酸修饰的PEI可以提保护siRNA免受小牛血清(FBS)降解[Alshamsan,etal.2009]。
根据一些报道,可以将硬脂酸纳入到C1家族多肽中,以增加疏水域的膜亲和性和加强siRNA在血清中的稳定性。因为C1家族多肽的疏水部与亲水部是分开的,所以比较容易用硬脂酸替代疏水部,以评估不同疏水部的影响。从C1多肽家族中选出三种多肽,其与细胞膜具有不同程度的疏水作用,并且使用硬脂酸进行修饰。
同时,在STR-HK和STR-HKC中的连接部GGG被改为HHH,以增强复合物的内涵体逃逸,这是由于其在低pH中被质子化导致的,因此也可以评估不同连接部的作用。
IX.半胱酰胺(Cysteamide,简称CYSt)修饰的多肽
在另一个实施例中,本发明提供了具有以下任一氨基酸序列的多肽:
表9:半胱酰胺修饰的多肽序列
这组多肽在C端修饰了半胱酰胺基团。半胱胺是最简单稳定的氨基硫醇以及半胱氨酸的降解产物。已有报道,在多肽的碳端连接半胱酰胺基团可以提升DNA-多肽复合物的高效转染[Simeoni,et al.2003]。也有研究表明,在多肽的碳端连接半胱酰胺基团可以比最初的多肽显现更高穿过细胞膜的能力[Crombez,et al.2009]。
这个假设已经延伸到siRNA-多肽传递系统。为了研究半胱酰胺基团对增强复合物转染效率的影响,选择不同转染效率水平的C6家族的多肽在C-端修饰半胱酰胺。
实施例1:多肽-siRNA组装体/纳米颗粒:制备和体外转染
利用GAPDH作为目标基因,这些富有潜力的多肽传递siRNA的能力在CHO细胞上进行了评估。中国仓鼠卵巢细胞CHO-K1(ATCCCCL-61)源自母体CHO细胞系的亚克隆,母体细胞系最初取自一个成年中国仓鼠的活组织切片检查。磷酸甘油醛脱氢酶(GAPDH)是一种管家基因,广泛表达于不同组织和细胞类型中,并且在细胞变化过程中具有多种不同的作用。。相应的siRNA,也就是沉默剂GAPDHsiRNA(人,小鼠,大鼠),买自Ambion公司。siRNA的摩尔浓度通过吸收光谱测定,消光系数为385103L/mol cm。
一个由N端乙酰化碳端氨基酸的天然多肽组成多肽阵列,购买于Pepscan系统公司(莱斯特拉德,荷兰)。
制备方案:
筛选试验中,siRNA浓度为50nM,多肽/siRNA摩尔比率为20/1和40/1。
转染方案:
转染前,将35000个细胞/孔板的F12K培养基与含有10%FBS的无抗菌剂汇合进行细胞培种24小时。
第二天,转染前利用PBS缓冲液洗涤细胞,并加入200μL Opti-MEM培养基。每个孔板中加入100μL复合物溶液(负电控制的siRNA-多肽和GAPDH siRNA-多肽)。
细胞与复合物在37℃的培养皿中培养。4个小时后,加入300μL含有20%PBS的F12-K培养基,无需除去转染混合物。
在转染开始48小时后,将细胞进行洗涤并且溶解。
实时逆转录聚合酶链反应(RT-PCR)是当前检测低含量mRNA最为灵敏的方法。细胞内所有的RNA通过TRIzol试剂(Invitrogen,Carlsbad,CA,USA)萃取,然后如生产厂家推荐,用氯仿(西格玛,奥克维尔,安大略,加拿大)和2-丙醇(西格玛,奥克维尔,安大略,加拿大)处理。RNA浓度由Nanodrop测试(Nanodrop分光光度计ND-1000,热科技,渥太华,加拿大)。所有的RNAs根据本协议与Bio-Rad公司的iScript cDNA合成试剂盒进行反转录。cDNA合成用一个独特的混合寡核苷酸(DT)和随机引物预先准备。用于鼠GAPDH基因的引物的序列为5’-TTGCTGTTGAAGTCGCAGGAG-3’(引物1,SEQ ID NO:47)和5’-TGTGTCCGTCGTGGATCTGA-3’(引物2,SEQ ID NO:48)(西格玛,奥克维尔,安大略,加拿大)。为了避免存在偏差,实时RT-PCR通常涉及一个或者多个内部控制基因,这应该不会在测试中产生波动[Nicot,et al.2005]。管家基因亲环蛋白,被用来内部控制GAPDH基因基因的标准化。标准化通过小鼠亲环蛋白mRNA使用下列引物进行的扩增实现:5'-AGGGTTTCTCCACTTCGATCTTGC-3'(引物3,SEQ IDNO:49)和5'-AGATGGCACAGGAGGAAAGAGCAT-3'(引物4,SEQ ID NO:49)(Sigma公司,奥克维尔,安大略省,加利福尼亚州)。
不同肽与GAPDH siRNA在摩尔比率为20和40时复合的RT-PCR结果如图2所示。图2显示了GAPDH基因在CHO细胞中的沉默,其中多肽与siRNA分别以20:1和40:1的摩尔比率进行复合。mRNA的水平在转染48小时后进行测试。结果显示在图2中,对应至少三次并行的试验的平均值。
实施例2:制备方案参照上述实施例
转染方案
对于CHO-K1细胞系,96孔板并进行无血清处理。
转染前,将5000个细胞/孔板的F12K培养基与含有10%FBS的无抗菌剂汇合进行细胞培种24小时。转染当天细胞汇合40%-60%。
用PBS洗涤细胞,并加入50μL Opti-MEM培养基,然后每个孔板加入50μL复合物溶液(siRNA-多肽或者对比空白)。
细胞与复合物在37℃下二氧化碳培育器中培育3-6个小时;一般4个小时足够。培育之后,加入50μL含有30%FBS的F12K培养基,无需移出转染混合物。
在转染开始48小时后,将细胞进行洗涤并且溶解。
分别在处理过的细胞和对比空白细胞中,用KDalertTMGAPDH试剂盒测试磷酸甘油醛脱氢酶(GAPDH)的酶活性。
KDalertTMGAPDH试剂盒是一种基于荧光的快速简便的方法,其可以测试源自人体或大鼠或小鼠的培养细胞中磷酸甘油醛脱氢酶(GAPDH)酶活性。KDalertTMGAPDH试剂盒是设计用来通过评估在蛋白水平的GAPDH的表达以及敲打,促进识别最佳的siRNA传递条件。
GAPDH是一种四聚物酶,由36kD蛋白亚单元组成。其将氧化磷酸化的甘油醛-3-磷酸(G-3-P)催化为二磷酸甘油酸的异构酯(BPG)。
KDalertTMGAPDH试剂盒通过在磷酸和G-3-P存在时的GAPDH,测试了NAD+向NADH的转变过程。在厂家推荐的阵列条件下,NADH产物的产率与实时的GAPDH酶是成比例的。因此该阵列可以被用于准确检测样品中GAPDH蛋白的含量。
实验结果:
图3A显示了多肽-siRNA复合物的沉默效率,多肽和siRNA复合的摩尔比率为20/1和40/1。图3B显示了同样摩尔比率不同实施的试验多肽-siRNA复合物的沉默效率结果。图4A显示了18组多肽-siRNA复合物在CHO-K1细胞中的毒性。图4B显示了6组多肽-siRNA复合物在CHO-K1细胞中的毒性。
实施例3:多肽-siRNA复合物的尺寸
应用动态光散射(DLS)方法在Zeta纳米系列尺寸仪(马尔文)上测试复合物的尺寸。图5显示了C6M1多肽以及siRNA的复合物的尺寸,多肽/siRNA摩尔比率随时间变化为1:1至60:1。如图所示,通过增加多肽/siRNA的摩尔比率,复合物的尺寸随时间增加。当尺寸范围在100-250nm可能比较适合体外和体内测试,即当摩尔比率为20:1并培育至少30min可能是复合物制备的最佳条件。
实施例4:荧光测试
C6M1多肽被用于本实验,以通过荧光光谱的方法研究多肽与siRNA之间的相互作用。C6M1具有四个色氨酸,这些色氨酸可以用作荧光探针。如图8所示,通过向固定浓度的C6M1样品中加入更多的siRNA,其荧光强度减弱,表明多肽与siRNA之间发生了相互作用。当多肽与siRNA的摩尔比率低于40:1时,尤其明显为20:1和10:1时,可以观察到一个从355到330nm的蓝移。这可能是由于加入更多的亲水性的siRNA分子使得色氨酸的环境发生改变。
通过跟踪荧光标记的siRNA与C6M1相互作用时的荧光性质进行一个相似的试验。如图7所示,在低摩尔比2:1和4:1时,siRNA的荧光光谱没有明显的变化。然而通过加入更多的多肽,当摩尔比率在10:1和20:1时荧光强度降低,表明多肽分子与siRNA相互作用并且围绕在siRNA周围。红移也可以表明siRNA的环境发生了变化,或者通过与多肽作用siRNA结构的构象发生了变化。
实施例5:C6M1/siRNA复合物抑制无胸腺老鼠的癌细胞增殖
在一个动物癌细胞样品上研究了C6M1/siRNA复合物对阻止癌细胞增殖的作用。这里使用的是靶向Bcl-2基因产物的siRNA。Bcl-2蛋白控制线粒体介导的凋亡路径,以及多种细胞死亡的刺激物,包括化学疗法试剂。因此期望一种降低这种蛋白的药物来提高癌细胞的凋亡,并由此作为一种有前景的治疗剂。
通过在裸鼠的右腋下的皮下方接种5×106A549个细胞,以建立动物模型。当肿瘤的体积达到100-200mm3,注入复合物。C6M1/siRNA复合物的制备如上所述,并注入到肿瘤内。每过3天进行一次药物治疗,总共治疗9次,每只鼠的剂量为4μg siRNA。每天测试鼠的体重以及肿瘤直径。肿瘤体积由下述公式计算:
肿瘤体积=0.5×(宽度)2×长度
老鼠在第一次注射药物后第27天死亡。结果对应由两个不同的动物试验的平均水平。
结果如图8所示。这些结果说明Bcl-2siRNA与多肽C6M1的复合物明显阻止了肿瘤的生长,因为在经过治疗后肿瘤尺寸有一个明显的减小。此外,复合物显示了低毒性。在治疗过程中,如果接受治疗的鼠的平均体重(肿瘤组织除外)降低超过15%,这说明药物具有毒性反应,药物剂量需要降低并重新进行测试。在我们的试验中,每组中有6个老鼠在测试过程中体重不会明显变化。所有这些结果表明C6M1是一种体内传递siRNA的有效安全的工具。
鼠因脱臼而死亡,分离出肿瘤并且称重。我们计算出每组的平均重量以及肿瘤的抑制率。siRNA组的抑制率为37%,siRNA-C6M1的抑制率为53%,说明siRNA-C6M1可以有效明显地一直肿瘤的生长。
虽然已经参考一些特定的实施例描述了本发明,在不脱离本发明目的以及附加的权利要求范围内的任何改进,均对本领域技术人员而言是显而易见的。本文所提供的任何实施例仅用于说明本发明的目的,而不是为了以任何方式限制本发明。本文所提供的任何附图仅用于为了说明本发明各个方面,而不旨在按比例绘制或以任何方式限制本发明。在此叙述的所有现有技术中的公开内容,均通过参考文献全部引用在本文中。
Claims (16)
1.一种多肽与药物分子的复合物,其特征在于:所述多肽的氨基酸序列选自序列SEQID NOs:1-12,22-29,34-46中的任意一个序列。
2.根据权利要求1所述的复合物,其特征在于:所述药物分子为核酸。
3.根据权利要求2所述的复合物,其特征在于:所述核酸为siRNA。
4.根据权利要求3所述的复合物,其特征在于:所述多肽与所述siRNA复合的摩尔比率的范围为1:1至60:1。
5.根据权利要求3所述的复合物,其特征在于:所述多肽与所述siRNA复合的摩尔比率的范围为5:1至60:1。
6.根据权利要求3所述的复合物,其特征在于:所述多肽与所述siRNA复合的摩尔比率为20:1。
7.根据权利要求3所述的复合物,其特征在于:所述多肽与所述siRNA复合的摩尔比率为40:1。
8.一种药物组合物,其特征在于:所述药物组合物包括如权利要求3-7任一所述的复合物,用于传递具有药物疗效的siRNA。
9.一种如权利要求3-7中任一所述的复合物,用于将siRNA传递入细胞。
10.根据权利要求9所述的复合物,其特征在于:所述细胞为中国仓鼠卵巢细胞。
11.根据权利要求9或者10所述的复合物,其特征在于:所述siRNA降低了所述细胞中内生蛋白的含量水平。
12.一种如权利要求8所述的用于降低动物的细胞或者组织内基因产物水平的药物组合物。
13.根据权利要求12所述的药物组合物,其特征在于:所述细胞为肿瘤细胞或者所述组织为肿瘤组织。
14.根据权利要求12所述的药物组合物,其特征在于:所述siRNA靶向一种控制凋亡的基因。
15.根据权利要求14所述的药物组合物,其特征在于:所述控制凋亡的基因为Bcl-2。
16.一种多肽,其特征在于:所述多肽的氨基酸序列选自序列SEQ ID NOs:1-1112-21,28-33,44-46中的任意一个序列。
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