CN103597075A - 用于选择性地杀伤细胞的生物活性核苷酸分子、其用途及应用试剂盒 - Google Patents
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Abstract
用于选择性地杀伤细胞的生物活性核苷酸分子、其用途以及应用试剂盒。目的在于,在广泛的应用领域内,尽可能有效、可靠和高效地杀伤生物体中的细胞,而不出现上文所述的已知的化学、物理、生物化学或分子生物学方法的缺点。根据本发明,生物活性核苷酸分子(8)经设计,其具有与多个基因的mRNA(7、9、10、11)有结合能力的核苷酸序列,从而通过与其结合,引发多个(特别是大量的)用于细胞杀伤应激状态的“脱靶(off-target)”效应,通过其,不依赖于用于减少单个基因的表达的分子的传统插入,而巨大地影响细胞(2),以至于杀死细胞或者在细胞(2)中启动程序性细胞死亡(凋亡)。特别地,发现了这些分子用于有针对性地杀死肿瘤细胞或病毒感染的细胞,以及植物细胞和真菌细胞的用途。
Description
本发明涉及可用于选择性地杀伤细胞的基于核苷酸的生物活性分子、所述生物活性分子的用途以及用于应用的应用试剂盒。
用于选择性地杀伤生物细胞的方法,利用物理学手段的常规方式,如UV辐射、热等(Hsie AW,Brimer PA,Mitchell TJ,Gosslee DG.Thedose-response relationship for ultraviolet-light-induced mutations at thehypoxanthine-guanine phosphoribosyltransferase locus in Chinese hamsterovary cells.Somatic Cell Genet.1975Oct;1(4):383-9.;Gillespie EH、GibbonsSA.Autoclaves and their dangers and safety in laboratories.J Hyg(Lond).1975Dec;75(3):475-87.),或化学物质,例如酸、碱、甲醛(National ToxicologyProgram.Final Report on Carcinogens Background Document forFormaldehyde.Rep Carcinog Backgr Doc.2010Jan;(10-5981):i-512.),其破坏细胞结构本身。这些手段通常对环境有害并且几乎不能在生物体内使用。为了在生物体中杀伤细胞,使用生物化学手段(蛋白质抑制剂、拮抗剂、细胞抑制剂等)(Tanaka S,Arii S.Current Status of molecularly targeted therapyfor hepatocellular Carcinoma:basic science.Int J Clin Oncol.2010Jun;15(3):235-41.Epub2010May27.),其可以大大地影响细胞的生理并因此可以导致细胞的死亡。然而,这些方法却没有一个可以选择性地杀伤生物体中的特定细胞类型,这是因为这些物质对所有细胞同样地起作用。
选择性地影响细胞的一种分子生物学手段是使用短的、双链RNA。这些所谓的siRNA(短干扰RNA)分子可以在被激活后以常规方式与目标基因的mRNA相互作用并与特定的核糖核酸内切酶一起形成一种名称为“RISC”(RNA诱导的沉默复合物)的RNA-蛋白质复合物。RISC复合物与靶mRNA结合,其中核酸内切酶切割目标mRNA。以这种方式抑制基因表达并由此阻止目标蛋白质的产生。
已经描述了通过将特异于目标基因mRNA序列片段的短的(19-23bp)、双链RNA分子(siRNA)引入真核细胞来抑制基因表达(Elbashir SM等人:Duplexes of21-nucleotide RNAs mediate RNA interference in culturedmammalian cells,Nature,2001May24,411(6836)、494-8;Liu Y等人:Efficient and isoform-selective Inhibition of cellular gene expression by peptidenucleic acids,Biochemistry,2004Feb24、43(7),1921-7;US5,898,031A;US7,056,704B2)。
借助于这样的分子,并未阻止基因的阅读和mRNA的产生,而是通过siRNA来起始降解靶mRNA的细胞自身机制。最后,如上文所述,压制特定蛋白质的形成,而不损害其他基因的表达(转录后基因沉默)。
对于siRNA的目前的应用,通常需要单独压制细胞中单个基因的表达。因此不希望其中多个基因同时地或非特异地被关闭的效应,因而需要对siRNA的序列进行设定,从而阻止这些效应。
还开发了这样的方法,用siRNA在体内转染目标组织经强化的细胞(Ikeda等人,“Ligand-Targeted Delivery of Therapeutic siRNA”,Pharmaceutical Research,Vol.23,No.8,August2006)或通过结合经细胞特异剪切以获得取得细胞特异性的短肽(WO2008/098569A2)。通过使用经修饰的siRNA分子,可以实现选择性地在特定细胞中降低或者压制基因的表达。
如果所使用的siRNA序列特异于对细胞存活很重要的基因,则由此可以导致细胞的死亡。此过程可以通过所述的机制(任选也是细胞特异性地)来进行应用。
然而,缺点是,单个基因或几个基因的关闭常常并不必然会导致这些细胞的死亡;为此通常必须特异性地同时关闭多个基因,以获得所希望的效应。例如,对于治疗性应用而言期望的会是,细胞是否可以被siRNA分子直接杀伤。借此可以非常特异性地杀伤肿瘤细胞或病毒感染的细胞,特别通过使用所述的方法。
此外,经常存在这样的问题,在肿瘤细胞或病毒感染的细胞的基因组中,常发生突变,因此所使用的siRNA分子可成为无活性的并由此无法对细胞产生所期望的影响或至少不能被有效利用。
本发明的目标在于,在广泛的应用领域内,尽可能有效、可靠且高效地杀伤生物体中的细胞,而不出现上文所述的已知的化学、物理、生物化学或分子生物学方法中本身的缺点。
根据本发明所提供的是生物活性核苷酸分子(例如基于RNA、siRNA、PNA、DNA或LNA的),其具有与多个其所靶向的基因的mRNA有结合能力的核苷酸序列,从而通过与这些基因的结合,引发多个(更特别是大量的)用于细胞杀伤应激状态的“脱靶(off-target)”效应。
术语“生物活性核苷酸分于”包含根据本发明的核苷酸分子,其在所有本文所述的条件和应用下发挥其功能。特别是,根据本发明的生物活性核苷酸分子通过引发所谓的“脱靶”效应来发挥其活性。引发细胞杀伤应激状态的“脱靶”效应,其在本发明的背景下理解为这样的生物活性或过程,其中一个核苷酸序列具有多个靶mRNA序列并潜在地影响多个基因的表达或者不依赖于对基因表达的影响而引发细胞应激。
通过该应激状态,不依赖于用于减少单个基因表达的核苷酸分子特别是siRNA的常规应用,而通过非特异性的核苷酸序列大大地影响细胞,从而杀伤细胞或在细胞中启动程序性细胞死亡(凋亡)。
虽然如本文开头所述,具有用于结合mRNA的核苷酸序列的核苷酸分子(例如基于siRNA的)是熟知的,然而,那些核苷酸序列每个均是特异性地针对于一个或几个基因的mRNA,以通过与所靶向的目标基因选择性地结合,而在细胞中实施确定的基因操纵并以这种方式实施基因定向的细胞影响。
通过本发明,对所述核苷酸序列进行有目的的设计,以便可以对接多个、更特别是大量的基因的mRNA,如果需要,也并不依赖于这些可结合的基因的mRNA目前是否实际上存在于细胞中。所提议要实现的mRNA-结合的主要目标因而并不是上文所述的针对细胞活性的基因操纵,而是要用大量的(实际上是任何的)核苷酸分子的mRNA-结合引发尽可能多的“脱靶”效应,其此前是为了尽可能地避免或减轻目标明确的基因影响。通过尽可能多的“脱靶”效应,产生对于细胞而言过大的应激状态,其是目标细胞不能承受的并且通过其可有目的性地杀伤上述的目标细胞(不是通过有目的性的基因表达操纵,而是通过普通的应激)。
用基因结合必然的和已知的方式产生的和作用于细胞活性的特定基因影响因此是附带的效应,并且如过需要,可以根据基因影响的作用,进一步促进细胞作用(在上述的根据本发明的有目的性的应激状态之外)。
因此,通过可结合目标基因的核苷酸序列的选择并不是或者至少并不主要是用作有目的地基因操纵以对细胞产生影响的目标效应,而是通过基因结合可达到的“脱靶”效应及用其在细胞中引起的应激状态的指定作用的目标效应。
为了产生上述的“脱靶”效应,对核苷酸序列进行选择,从而使这些核苷酸序列不像传统方式那样针对一个目标基因,而是针对尽可能多的目标基因。由此产生对于大量基因有毒性作用的核苷酸干扰并巨大地影响细胞的生理学。
在细胞中可以将上述应用与实现细胞特异性的已知机制组合应用,以及与已知的稳定性的可能性(例如siRNA的)组合应用,以改善核苷酸分子的吸收。
所述核苷酸序列不限于作为传统siRNA使用;也可以将短的(10-20bp)双链RNA、长的(20-300bp)双链或单链RNA、DNA或化学类似物,例如PNA,与上述核苷酸序列一起使用。
除了上述的“脱靶”效应的诱导,也可以通过已知的应激诱导的核苷酸序列依次促进生物活性核苷酸分子的细胞毒性作用(FEDOROV Y等人,Off-target effctsby siRNA can induce toxic phenotype.RNA(2006),12:1188-1196.)。
通过合适的转染系统,例如纳米粒子、聚乙烯亚胺或脂质,可以以已知的方式将活性物质分子引入细胞中。
为了更好地被转移入或转移至细胞,以及为了其稳定性或为了其检测,还可以将所述分子结构与其他物质(例如作为载体系统的纳米粒子或荧光色素)相连接。
生物活性核苷酸分子适合于选择性地杀伤真核细胞,特别是动物细胞、植物细胞或真菌细胞,以及病毒感染的细胞和原核细胞。
在生物活性核苷酸分子的应用中,也可以将它们与蛋白酶抑制剂组合使用。
有利地,用于生物活性核苷酸分子的应用和施用的应用试剂盒至少由下列组成:
-至少一个安剖瓶(安剖瓶A),其包含所述生物活性分子并且还可包含:
-至少一个其它的具有转染系统,例如细胞渗透性肽、纳米粒子、聚乙烯亚胺或脂质的安剖瓶(安剖瓶B),
-至少一个其它的安剖瓶(安剖瓶C),其包含用于与生物活性分子结合的其它成分,或转染系统,
-用于安剖瓶A、B和C的内容物的稀释缓冲液和反应缓冲液,
-一个或多个探针或注射器,其具有针头和其它所需材料,用于将安剖瓶内容物的混合物注射至包含目标细胞的培养基中,以及
-用于使用和施用的说明书。
下面,将根据附图中所述的实施例更详细地说明本发明。
其表明:
图1:一个已知的siRNA的示意图,其被引入细胞中,特异于一个mRNA并且压制目标基因的表达。
图2:一个根据本发明的siRNA的示意图,其被引入细胞中并在其中尽可能多地引发非特异性的RNAi效应(脱靶效应)。
图3:一个siRNA的示意图,其被引入细胞中且目的不是在其中减少基因的表达和降解mRNA,而是通过在细胞中经siRNA的特异性序列剪切而引起应激反应以导致细胞死亡。
在图1中描绘了通常的和已知的siRNA1的机制,所述siRNA被引入细胞2中(参见图标箭头的描绘)并且具有特异于与第一基因的mRNA3特异性结合的核苷酸序列(未作详尽描绘)。随后,该siRNA1被并入RNA诱导的沉默复合体(RISC)(未作详尽描绘)中,后者使siRNA分开为其两个单链并且siRNA的反义链与RISC一起附加至第一mRNA3。随后,基因特异的第一mRNA3被切割并片段化,由此压制了基于第一mRNA3的目标基因的表达(比较图1中第一mRNA7的降解)。之后,未被占据的并且装配在RISC中的siRNA1此时附加至距离最近的细胞2中存在的特异性的第一mRNA3并同样将其降解。因此,其目标为,每个siRNA1仅与特异性的第一mRNA3结合并将其降解。其它细胞2中还存在的另外的第二mRNA4、另外的第三mRNA5和另外的第四mRNA6则总是相对于siRNAl的第一mRNA3或未作详尽描绘的核苷酸序列完好地保留,从而mRNA4-6的基因其表达不被改变。这些方法是熟知的。
作为比较,图2表示根据本发明的siRNA8的机制,所述siRNA8被引入细胞2中(也参见图标箭头描绘),其中再次示例性地存在第一mRNA3、第二mRNA4、第三mRNA5以及第四mRNA6。
所提出的siRNA8包含由一个或多个下列核苷酸序列组成的链(出于清楚目的,未作详尽描绘):GGUA、CGUC、CGUU、CCAA、AAGG、GGUG、CUCG、CUCC、CUCU、CUUA、GGUC、GGUU、AAAG、AAAC、AAAU、AAGA、AAGC、AAGU、AACA、AACG、AACC、AACU、AAUA、CUUU、AAUG、AAUC、AAUU、AGGA、AGUG、AGUC、AGUU、ACAA、ACAG、ACAC、ACAU、ACGA、ACGG、ACGC、ACGU、ACCA、CAUU、CGAA、ACCG、ACCC、ACCU、ACUA、ACUG、ACUC、ACUU、AUAA、GGAG、GGAC、GGAU、GGGA、GGGC、GGGU、GGCA、GGCG、GGCC、GGCU、GCAA、GCAG、GCAC、GCAU、AUAG、AUAC、AU AU、AUGA、AUGG、AUGC、AUGU、AUCA、CGCG、CGCC、CGCU、AUCG、AUCC、AUCU、AUUA、AUUG、AUUC、AUUU、GAAA、GAAG、GAAC、GAAU、GAGA、GAGG、GAGC、GAGU、GACA、GACG、GACC、GACU、GAUA、GAUG、GAUC、GAUU、GGAA、GCGA、GCGG、GCGC、GCGU、GCCA、GCCG、GCCC、GCCU、GCUA、GCUG、GCUC、GCUU、GUAA、GUAG、GUAC、GUAU、GUGA、GUGG、GUGC、GUGU、GUCA、GUCG、GUCC、GUCU、GUUA、GUUG、GUUC、GUUU、CAAA、CAAG、CAAC、CAAU、CAGA、CAGG、CAGC、CAGU、CACA、CACG、CACC、CACU、CAUA、CAUG、CAUC、CGAG、CGAC、CGAU、CGGA、CGGG、CGGC、CGGU、CGCA、CGUA、CGUG、CCAG、CCAC、CCAU、CCGA、CCGG、CCGC、CCGU、CCCA、AGAA、AGAG、AGAC、AGAU、CCCG、CCCU、AGGG、AGGC、AGGU、AGCA、CCUA、CCUG、CCUC、CCUU、CUAA、CUAG、CUAC、CUAU、AGCG、AGUA、CUGA、GUGG、CUGC、CUGU、CUCA、CUUG、CUUC、AGCC、AGCU,从而链中结合的核苷酸序列的全体相对于图l而言不仅对单个的mRNA3-6,还对多个或大量mRNA起降解作用,并因此结合所有在图2中所描绘的mRNA分子(mRNA3-6)。因此,至少一个所选择的核苷酸序列可结合在多个或所有所描绘的mRNA分子上,或者每个所选择的核苷酸序列总是选择性地作用于特定的mRNA3-6。重要的是,使尽可能多的(最好的情况下,所有的)mRNA3-6通过siRNA8的链(所有核苷酸序列的全体)而被结合和降解(比较图2中降解的第一至第四mRNA7、9、10、11)。
通过上述的大量mRNA分子(在本实施例中简化描绘了仅四个mRNA分子)的降解,引发多个直至数目众多的非特异性RNAi效应(脱靶效应),通过具有(在最好的情况下)仅一个核苷酸序列的siRNA8压制多个至许多基因的表达(比较图2中降解的mRNA7、9、10、11),目标在于,以这种方式杀伤细胞2,其通过siRNA8的巨大作用而被杀伤。
作为例子,可以通过具有核苷酸序列(5′-3′)UUAACUGUAUCUGGAGCtt(SEQ ID NO:3)的siRNA8降解细胞因子信号抑制物-1(SOCS1,NM_003745.1)、N-乙酰神经氨酸磷酸酶(NANP,NMl52667.2)、跨膜蛋白215(TMEM215、NM_212558.2)和CD81分子(CD81,NM_004356.3)基因的mRNA。
siRNA8AACUGUAUCUGGAGCtt(SEQ ID NO:4)的核苷酸序列对于细胞因子信号抑制物-1(SOCS1,NM_003745.1)和N-乙酰神经氨酸磷酸酶(NANP,NM152667.2)基因的mRNA是特别有效的。核苷酸序列GGCUGAACAAAGGAGAtt(SEQ ID NO:6)特异性地作用于I类主要组织相容性复合物G(HLA-G,NM_002127.4)、甘油激酶5(假定的)(GK5,NM_001039547.1)和DIP2disco-相互作用蛋白2同源物C(NM_014974.2)。
相应地,具有序列GCUCACCAAUGGAGAtt(SEQ ID NO:5)的siRNA8特异性地作用于补体成分(3b/4b)受体1(Knops血型)(CR1,NM_000651.4)转录本变体S、补体成分(3b/4b)受体1(Knops血型)(CR1,NM_000573.3)转录本变体F和谷胱甘肽S-转移酶α4(GSTA4,NM_001512.3)。
作为对于siRNA8的核苷酸序列的其他例子,是序列UGGCUGGCUGGCUGGCtt(SEQ ID NO:7),有利地针对焦谷氨酸肽酶I(PGPEP1,NM_017712.2)、Rap鸟嘌呤交换因子(GEF)3(RAPGEF3,NM_006105.5)转录物变体2和类视觉X受体α(RXRA,NM_002957.4),以及针对信号转导子与转录激活子3(急性期反应因子)(STAT3,NM_213662.1)转录物变体3、信号转导子与转录激活子3(急性期反应因子)(STAT3、NM_003150.3)转录物变体2、信号转导子与转录激活子3(急性期反应因子)(STAT3、NM_139276.2)转录物变体1、原钙粘蛋白α9(PCDHA9,NM_014005.3)和分离蛋白(Secernin)3(SCRN3,NM024583.3)的序列GUCUAUCAGCACAAUtt(SEQ ID NO:1)。
作为上面提到的对于siRNA8(其针对具体的基因)的核苷酸序列的例子的另外选择,也可以使用这样的核苷酸序列,其不具有与人mRNA的同源性并因此没有直接的目标基因。在此可以使用这样的相应序列,从现有技术中已知的是,它们引发该细胞应激。一个这样的核苷酸序列可以具有序列GCUUAACUGUAUCUGGAGCtt(SEQ ID NO:2)。
如从上面列示的核苷酸序列可知的是,它们在3′末端由经修饰的核苷酸引领,其中“t”在本发明的范围内为2′-脱氧胸苷。在上面描述的序列中,在3′末端由两个2′-脱氧胸苷引领并且这些末端核苷酸用“tt”标示。但突出部分的结构不限于本文所述的“tt”-突出部分,这是因为突出部分本身的类型对于根据本发明在此描述的siRNA的效应并不重要。因此也可以使用本领域技术人员已知的其他的突出部分。
根据本发明的生物活性核苷酸分子也可以作为药物使用。例如对于治疗性应用,可以借助于根据本发明的siRNA分子直接杀伤细胞。因此可以例如选择性地杀伤非常特定的肿瘤细胞或病毒感染的细胞。因此可以在治疗和/或预防肿瘤疾病或病毒引起的疾病的情况下使用所提出的核苷酸序列,即应用上面描述的生物活性核苷酸分子。在本发明的范围内,病毒引起的疾病包括这样的疾病,其例如由疱疹病毒、乳头瘤状病毒或HIV病毒引起。因此病毒引起的疾病包括例如肝炎、宫颈癌或AIDS。
同样,本发明包括用于在治疗和/或预防肿瘤疾病的情况下应用的根据本发明的生物活性核苷酸分子,用根据本发明的药物治疗的肿瘤疾病包括乳腺癌、卵巢癌、支气管癌、结肠癌、黑素瘤、胆囊癌、胃癌、头/颈肿瘤、脑肿瘤、宫颈肿瘤、前列腺癌、睾丸癌、骨肿瘤、肾癌、胰腺肿瘤、食管肿瘤、恶性淋巴瘤、非霍奇金淋巴瘤、霍奇金淋巴瘤和甲状腺淋巴瘤。
在一个优选的实施方案中,根据本发明的生物活性核苷酸分子、核苷酸或核苷酸类似物可以如上文所述那样,与蛋白酶抑制剂相组合使用。相应的蛋白酶抑制剂是本领域技术人员已知的。可以提及的这些蛋白酶抑制剂的例子有,丙肝蛋白酶抑制剂或HIV蛋白酶抑制剂,其中本发明并不局限于此。
此外,在另一个优选的实施方案中,如果需要,可以将根据本发明的生物活性核苷酸分子、核苷酸或核苷酸类似物,与“药学上可接受的载体”和/或溶剂相组合配制。对于特别合适的药学上可接受的载体的例子是本领域技术人员已知的并且包括缓冲生理盐水、水、乳剂例如油/水乳剂、各种类型的洗涤剂、无菌溶液等。
可以借助于已知的常规方法来配制包含上面所列示的药学上可接受的载体的在本发明范围内的药物。可以以合适的剂量给个体施用这些药物。施用可以口服地或胃肠外地进行,例如静脉内、腹膜内、皮下、肌内、局部、鼻内、支气管内或真皮内,或经由动脉内一定位置处的导管。配量的方式由主治医生根据临床因素来确定。本领域技术人员已知的是,配量的方式取决于各种因素,例如患者的身体大小或体重、身体表面、年龄、性别或一般健康状况,但也取决于特定的待施用手段、持续时间和施用方式,和可能平行施用的其他药物。典型的剂量可以例如在0、01至10000μg之间的范围内,其中可设想的是低于或高于该示例范围的剂量,特别是在考虑上面提到的因素之下。通常,在根据本发明的药物制剂的定期施用情况下,所述剂量在10ng-10mg单位/日或/应用间隔的范围内。作为静脉内施用成分,所述剂量应当在1ng-0.1mg单位/千克体重/分钟的范围内。
本发明的药物可以局部地或全身地进行施用。对于胃肠外施用的制剂包括无菌的水性或非水性溶液、悬浮液和乳剂。对于非水性溶剂的例子为丙二醇、聚乙二醇、植物油例如橄榄油,和有机酯类化合物例如油酸乙酯,其适合于注射。水性载体包括水、醇水溶液、乳剂、悬浮液、盐溶液和缓冲介质。胃肠外载体包括氯化钠溶液、林格-右旋糖、右旋糖和氯化钠、林格-乳酸盐和结合油。静脉内载体包括例如液体介质、营养补充物质和电解质补充物质(如这样的,例如基于林格-右旋糖的)。此外,根据本发明的药物可以包含防腐剂和其他添加剂,例如抗微生物化合物、抗氧化剂、复合物组分和惰性气体。此外可以,取决于所打算的应用,包含化合物例如白介素、生长因子、分化因子、干扰素、趋化蛋白或非特异的免疫调节试剂。
此外,可以将单个的siRNA8序列也相组合地同时或在时间上分开地施用以及以相同的或不同的浓度使用,以便有效地切断大量基因或降解mRNA。
在图3中描绘了另外的效应,其可进一步促进上文所述的根据本发明的siRNA8的毒性作用。在siRNA12之中另外包括一个或多个核苷酸序列(例如AAA(SEQID NO:8)、UUU(SEQ ID NO:9)、GCCA(SEQ ID NO:10)、UGGC(SEQ ID NO:11)、GUCCUUCAA(SEQ ID NO:12)、UGUGU(SEQ ID NO:13)、AUUUG(SEQ ID NO:14)、GUUUU(SEQ ID NO:15)、AUUUU(SEQ ID NO:16)、CUUUU(SEQ IDNO:17)、UUUUU(SEQ ID NO:18)或GUUUG(SEQ ID NO:19)),已知其在细胞2中引起并非是由于siRNA8与一个或多个mRNA的结合而引起的应激反应。因此,在将siRNA12引入细胞2中后,具有所述作用的siRNA12的这些核苷酸序列不降低基因的表达和mRNA的降解(比较在图3中,所描绘的和不具有该核苷酸序列的降解的mRNA3-6),而是诱导细胞中的非特异性的应激反应,其在产生图2所描述的作用之外,还由此强烈地导致细胞2的死亡。
所使用的附图标记列表
1、8、12-siRNA
2-细胞
3、4、5、6-基因特异性mRNA
7、9、10、11-降解的基因特异性mRNA
Claims (13)
1.生物活性核苷酸分子,具有至少一个目的在于结合mRNA的核苷酸序列以用于选择性地影响细胞,其特征在于,所述核苷酸分子(8)的所述至少一个核苷酸序列针对多个基因的mRNA(3、4、5、6)结合能力进行设计,以通过所述核苷酸分子与所述基因的mRNA(3、4、5、6)的结合来引发多个、特别是大量的用于细胞杀伤应激状态的以毒性方式作用于细胞(2)的“脱靶”效应。
2.根据权利要求1的生物活性核苷酸分子,其特征在于,RNA、siRNA、PNA、DNA或LNA被用作核苷酸并且具有10-300bp的大小。
3.根据权利要求1或2的生物活性核苷酸分子,其特征在于,所述核苷酸分子(8)包含特定的序列,其本身且也未与mRNA相结合而引起细胞中的应激反应,如AAA、UUU、GCCA、UGGC、GUCCUUCAA、UGUGU、AUUUG、GUUUU、AUUUU、CUUUU、UUUUU或GUUUG。
4.根据权利要求1至3的生物活性核苷酸分子,其特征在于,所述(8),特别是为了将其引入细胞(2)中,与分子如细胞渗透性肽结合或者被整合至试剂如聚乙烯亚胺、纳米粒子或脂质中。
5.根据权利要求1至4中的一个或多个的生物活性核苷酸分子,其特征在于,所述(8)包含至少一个下述核苷酸序列:GGUA、CGUC、CGUU、CCAA、AAGG、GGUG、CUCG、CUCC、CUCU、CUUA、GGUC、GGUU、AAAG、AAAC、AAAU、AAGA、AAGC、AAGU、AACA、AACG、AACC、AACU、AAUA、CUUU、AAUG、AAUC、AAUU、AGGA、AGUG、AGUC、AGUU、ACAA、ACAG、ACAC、ACAU、ACGA、ACGG、ACGC、ACGU、ACCA、CAUU、CGAA、ACCG、ACCC、ACCU、ACUA、ACUG、ACUC、ACUU、AUAA、GGAG、GGAC、GGAU、GGGA、GGGC、GGGU、GGCA、GGCG、GGCC、GGCU、GCAA、GCAG、GCAC、GCAU、AUAG、AUAC、AUAU、AUGA、AUGG、AUGC、AUGU、AUCA、CGCG、CGCC、CGCU、AUCG、AUCC、AUCU、AUUA、AUUG、AUUC、AUUU、GAAA、GAAG、GAAC、GAAU、GAGA、GAGG、GAGC、GAGU、GACA、GACG、GACC、GACU、GAUA、GAUG、GAUC、GAUU、GGAA、GCGA、GCGG、GCGC、GCGU、GCCA、GCCG、GCCC、GCCU、GCUA、GCUG、GCUC、GCUU、GUAA、GUAG、GUAC、GUAU、GUGA、GUGG、GUGC、GUGU、GUCA、GUCG、GUCC、GUCU、GUUA、GUUG、GUUC、GUUU、CAAA、CAAG、CAAC、CAAU、CAGA、CAGG、CAGC、CAGU、CACA、CACG、CACC、CACU、CAUA、CAUG、CAUC、CGAG、CGAC、CGAU、CGGA、CGGG、CGGC、CGGU、CGCA、CGUA、CGUG、CCAG、CCAC、CCAU、CCGA、CCGG、CCGC、CCGU、CCCA、AGAA、AGAG、AGAC、AGAU、CCCG、CCCU、AGGG、AGGC、AGGU、AGCA、CCUA、CCUG、CCUC、CCUU、CUAA、CUAG、CUAC、CUAU、AGCG、AGUA、CUGA、CUGG、CUGC、CUGU、CUCA、CUUG、CUUC、AGCC、AGCU。
6.根据权利要求1或2的生物活性核苷酸分子,其选自GUCUAUCAGCACAAUtt(SEQ ID NO:1)、GCUUAACUGUAUCUGGAGCtt(SEQ ID NO:2)、UUAACUGUAUCUGGAGCtt(SEQ ID NO:3)、AACUGUAUCUGGAGCtt(SEQ ID NO:4)、GCUCACCAAUGGAGAtt(SEQID NO:5)、GGCUGAACAAAGGAGAtt(SEQ ID NO:6)和UGGCUGGCUGGCUGGCtt(SEQ ID NO:7)。
7.药物,其包含根据权利要求1至6之一的生物活性核苷酸分子。
8.根据权利要求1至6之一的生物活性核苷酸分子,其用于肿瘤疾病或病毒引起的疾病的治疗和/或预防。
9.根据权利要求1至6的生物活性核苷酸分子用于选择性地杀伤真核细胞特别是动物细胞、植物细胞或真菌细胞的用途。
10.根据权利要求1至6的生物活性核苷酸分子用于选择性地杀伤病毒感染的细胞的用途。
11.根据权利要求1至6的生物活性核苷酸分子用于选择性地杀伤原核细胞的用途。
12.根据权利要求1至6的生物活性核苷酸分子的用途,其特征在于,所述生物活性核苷酸分子与蛋白酶抑制剂组合使用。
13.用于使用和施用根据权利要求1至5的生物活性核苷酸分子的应用试剂盒,其至少由下列所组成:
-至少一个安剖瓶(安剖瓶A),其包含所述生物活性分子并且还可以包含:
-至少一个其他的具有转染系统例如细胞渗透性肽、纳米粒子、聚乙烯亚胺或脂质的安剖瓶(安剖瓶B),
-至少一个其他的安剖瓶(安剖瓶C),其包含用于与所述生物活性分子或转染系统结合的其它成分,
-用于安剖瓶A、B的内容物的稀释缓冲液和反应缓冲液,
-一个或多个探针或注射器,其具有针头和其它所需材料,用于将安剖瓶内容物的混合物注射至包含目标细胞的培养基中,以及
-用于使用和施用的说明书。
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CN (1) | CN103597075A (zh) |
DE (1) | DE102011009470A1 (zh) |
WO (1) | WO2012098234A1 (zh) |
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DE102013003869B4 (de) | 2013-02-27 | 2016-11-24 | Friedrich-Schiller-Universität Jena | Verfahren zur gezielten Abtötung von Zellen durch zur mRNA-Anbindung ausgerichtete Nukleotid-Moleküle sowie Nukleotid-Moleküle und Applikationskit für solche Verwendung |
WO2020023268A1 (en) | 2018-07-24 | 2020-01-30 | Amgen Inc. | Combination of lilrb1/2 pathway inhibitors and pd-1 pathway inhibitors |
DE102019000490A1 (de) | 2019-01-23 | 2020-07-23 | HAEMES Verwaltungsgesellschaft mbH | Verwendung von Oligonukleotiden für die Behandlung von Tumoren |
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WO2009083790A2 (en) * | 2007-12-28 | 2009-07-09 | Qiagen Sciences, Inc | 'apoptosis inducing positive control for expression modulating experiments' |
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US5898031A (en) | 1996-06-06 | 1999-04-27 | Isis Pharmaceuticals, Inc. | Oligoribonucleotides for cleaving RNA |
DK2813582T3 (en) | 2000-12-01 | 2017-07-31 | Max-Planck-Gesellschaft Zur Förderung Der Wss E V | Small RNA molecules that mediate RNA interference |
US7790867B2 (en) * | 2002-12-05 | 2010-09-07 | Rosetta Genomics Inc. | Vaccinia virus-related nucleic acids and microRNA |
JP2006525811A (ja) * | 2003-05-16 | 2006-11-16 | ロゼッタ インファーマティクス エルエルシー | Rna干渉の方法と組成物 |
AU2005212433B2 (en) * | 2003-05-23 | 2010-12-16 | Sirna Therapeutics, Inc. | RNA interference mediated inhibition of gene expression using multifunctional short interfering nucleic acid (multifunctional sINA) |
US7404969B2 (en) * | 2005-02-14 | 2008-07-29 | Sirna Therapeutics, Inc. | Lipid nanoparticle based compositions and methods for the delivery of biologically active molecules |
KR100793505B1 (ko) * | 2006-05-30 | 2008-01-14 | 울산대학교 산학협력단 | 복수의 표적 mRNA에 적용 가능한 siRNA염기서열을 추출하는 방법 |
WO2008094516A2 (en) * | 2007-01-29 | 2008-08-07 | City Of Hope | Multi-targeting short interfering rnas |
DE102007008596B4 (de) | 2007-02-15 | 2010-09-02 | Friedrich-Schiller-Universität Jena | Biologisch wirksame Moleküle auf Grundlage von PNA und siRNA, Verfahren zu deren zellspezifischen Aktivierung sowie Applikationskit zur Verabreichung |
WO2009020344A2 (en) * | 2007-08-06 | 2009-02-12 | Postech Acad Ind Found | Small interfering rnas (sirnas) controlling multiple target genes and method for preparing the same |
EP2190994A2 (en) * | 2007-09-17 | 2010-06-02 | Intradigm Corporation | Compositions comprising stat3 sirna and methods of use thereof |
DE102009043743B4 (de) * | 2009-03-13 | 2016-10-13 | Friedrich-Schiller-Universität Jena | Zellspezifisch wirksame Moleküle auf Grundlage von siRNA sowie Applikationskits zu deren Herstellung und Verwendung |
GB2468477A (en) * | 2009-03-02 | 2010-09-15 | Mina Therapeutics Ltd | Double stranded RNA molecule comprising siRNA and miRNA precursors |
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2012
- 2012-01-20 JP JP2013549831A patent/JP2014511173A/ja active Pending
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- 2012-01-20 EP EP12704718.1A patent/EP2665816A1/de not_active Withdrawn
- 2012-01-20 CN CN201280006049.7A patent/CN103597075A/zh active Pending
- 2012-01-20 WO PCT/EP2012/050879 patent/WO2012098234A1/de active Application Filing
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WO2009083790A2 (en) * | 2007-12-28 | 2009-07-09 | Qiagen Sciences, Inc | 'apoptosis inducing positive control for expression modulating experiments' |
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DARJUS TSCHAHARGANEH 等: "Non-specific Effects of siRNAs on Tumor Cells with Implications on Therapeutic Applicability Using RNA Interference", 《PATHOLOGY ONCOLOGY RESEARCH》, vol. 13, no. 2, 31 December 2007 (2007-12-31) * |
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JP2014511173A (ja) | 2014-05-15 |
DE102011009470A1 (de) | 2012-08-09 |
EP2665816A1 (de) | 2013-11-27 |
US20170233760A1 (en) | 2017-08-17 |
WO2012098234A1 (de) | 2012-07-26 |
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