CN110177877A - 反义寡核苷酸 - Google Patents
反义寡核苷酸 Download PDFInfo
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- CN110177877A CN110177877A CN201780081337.1A CN201780081337A CN110177877A CN 110177877 A CN110177877 A CN 110177877A CN 201780081337 A CN201780081337 A CN 201780081337A CN 110177877 A CN110177877 A CN 110177877A
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- antisense oligonucleotides
- cancer
- gldc
- exon
- cell
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Abstract
本发明涉及用于调节甘氨酸脱羧酶活性(GLDC)的反义寡核苷酸。具体地,本发明涉及能够诱导RNA外显子跳跃的反义寡核苷酸。还要求保护使用所述反义寡核苷酸治疗癌症和诱导外显子跳跃的药物组合物、试剂盒和方法。此外,提供一种基于评估源自患者的样品中GLDC核酸、蛋白质或活性的水平,用于帮助分类或确定癌症的预后或者为癌症患者选择治疗策略的方法。
Description
技术领域
本发明涉及反义寡核苷酸。具体地,本发明涉及能够诱导RNA外显子跳跃的反义寡核苷酸。
背景技术
更具体地,本申请涉及癌症领域,具体涉及以下领域:过量表达甘氨酸脱羧酶(GLDC)的癌症,诸如非小细胞肺癌(NSCLC),以及其他癌症,诸如淋巴瘤(例如,滤泡性淋巴瘤、伯基特淋巴瘤、弥漫性大B细胞淋巴瘤)、胶质母细胞瘤、乳腺癌、前列腺癌、肺癌和结肠癌等。本文中示出了直接和选择性靶向GLDC蛋白丰度(例如,通过反义寡核苷酸(ASO)介导的外显子跳跃)导致细胞周期停滞和/或癌细胞凋亡。还提供了证据表明正常组织中GLDC的部分消耗(例如,通过反义寡核苷酸(ASO)介导的外显子跳跃)没有副作用。
GLDC是属于氧化还原酶家族的酶,特别是那些以二硫化物作为受体作用于供体的CH-NH2基团的酶。该酶参与甘氨酸、丝氨酸和苏氨酸代谢。它采用磷酸吡哆醛作为辅助因子。GLDC是来自所有真核生物中催化甘氨酸的降解的甘氨酸裂解系统的四种蛋白质中的一种。对于甘氨酸脑病,人体中高水平的甘氨酸或甘氨酸积聚是已知的。
在本说明书中列出或讨论明显在先公开的文件不应被视为承认该文件是现有技术的一部分或是公知常识。
本文中所提及的任何文件均通过引用整体并入本文。
发明内容
在本发明的第一方面中,提供了一种用于调节GLDC活性的分离的反义寡核苷酸。
优选地,术语“调节”是指激活、抑制、延迟、阻遏或干扰以下各项中的一项或多项;GLDC的活性;对GLDC的RNA剪接或翻译后加工;GLDC的磷酸化;GLDC的表达水平,其包括mRNA和/或前mRNA表达和蛋白质表达在内的GLDC表达的水平;或GLDC的亚细胞定位。优选地,在本发明中,寡核苷酸调节以下各项中的一项或多项的活性或水平:前mRNA、成熟mRNA和蛋白质表达。在各种实施例中,寡核苷酸抑制GLDC的表达或修饰其表达产物。在特定实施例中,寡核苷酸通过诱导GLDC前mRNA或成熟mRNA的外显子的跳跃来抑制GLDC的表达以诱导细胞周期停滞和/或凋亡。更具体地,寡核苷酸抑制GLDC在表达GLDC的细胞中的表达。
优选地,寡核苷酸与GLDC前mRNA或成熟mRNA的靶区域特异性杂交。如本文中所使用的,“杂交”意指根据Watson-Crick DNA互补性、Hoogstein结合(Hoogstein binding)或本领域已知的其他序列特异性结合的规则在两条或三条核酸链之间通过氢键产生的相互作用。杂交可以在本领域已知的不同严格条件下执行。
更优选地,本发明的反义寡核苷酸对一种或多种RNA结合剪接调节剂发挥空间位阻效应。例如,在实施例中,寡核苷酸通过空间位阻阻止mRNA的翻译。
优选地,寡核苷酸与GLDC RNA的外显子、内含子或外显子-内含子边界靶区域特异性杂交,该靶区域是选自包括以下各项的组中的任一项:外显子2、外显子3、外显子6、外显子7、外显子8、外显子9、外显子10、外显子12、外显子13、外显子15、外显子16、外显子19、外显子20、外显子21、内含子6、内含子7、以及内含子8。在下表中示出这些序列。
表:靶外显子和内含子的基因组序列
优选地,反义寡核苷酸包括选自SEQ ID NO:1至94中任一个的序列。在表1中示出SEQ ID NO:1至12。除表1中所示的那些序列之外,表2还示出了shAON的备选序列(SEQ IDNO:13至94),该shAON也能够诱导GLDC转录物中的外显子跳跃。
表1
表2
在本发明的各种实施例中,表1或表2中列出的每个序列可以靶向一个或多个外显子/内含子。
“寡核苷酸”意指任何多核苷酸。“多核苷酸”是由核苷酸组成的寡聚体。多核苷酸可以包括DNA、其RNA修饰形式、或其组合。如本文中所使用的术语“核苷酸”或其复数可与本文中所讨论的和本领域已知的修饰形式互换。在某些实例中,本领域使用术语“核碱基”,其涵盖天然存在的核苷酸以及可以聚合的核苷酸的修饰。因此,核苷酸或核碱基意指天然存在的核碱基:腺嘌呤(A)、鸟嘌呤(G)、胞嘧啶(C)、胸腺嘧啶(T)和尿嘧啶(U);以及非天然存在的核碱基,诸如黄嘌呤、二氨基嘌呤、8-氧代-N6-甲基腺嘌呤、7-脱氮黄嘌呤、7-脱氮鸟嘌呤、N4,N4-乙酸胞嘧啶、N',N'-乙醇-2,6-二氨基嘌呤、5-甲基胞嘧啶(mC)、5-(C[3]-C6)-炔基-胞嘧啶、5-氟尿嘧啶、5-溴尿嘧啶、假异胞嘧啶、2-羟基-5-甲基-4-三唑并吡啶、异胞嘧啶、异鸟嘌呤、肌苷;以及在Benner等人的美国专利号5,432,272以及Susan M.Freier和Karl-Heinz Altmann,1997,Nucleic Acids Research,第25卷:第4429-4443页中描述的“非天然存在的”核碱基。术语“核碱基”不仅包括已知的嘌呤和嘧啶杂环,而且还包括其杂环类似物和互变异构体。其他天然和非天然存在的核碱基包括在美国专利号3,687,808(Merigan等人)中,Sanghvi在第15章中,Antisense Research and Applications,第289-302页,Ed.S.T.Crooke,S.T.和B.Lebleu主编,CRC出版社,1993年中,在Englisch等人,1991,Angewandte Chemie,国际版本,30:613-722(尤其参见第622和623页),以及在Concise Encyclopedia of Polymer Science and Engineering,J.I.Kroschwitz Ed.,John Wiley&Sons,1990,第858-859页,Cook,Anti-Cancer Drug Design 1991,6,585-607中所描述的那些核碱基,其中每篇通过引用整体并入本文。在各个方面中,多核苷酸还包括一个或多个“核苷碱基”或“碱基单元”,其包括可以像核碱基一样起作用的化合物(诸如杂环化合物),这些核碱基包括某些“通用碱基”,这些通用碱基不是传统意义上的核苷碱基但是用作核苷碱基。通用碱基包括3-硝基吡咯、可选取代的吲哚(例如,5-硝基吲哚)、以及可选取代的次黄嘌呤。其他所需的通用碱基包括吡咯以及二唑或三唑衍生物,其包括本领域已知的那些通用碱基。
多核苷酸还可以包括修饰核碱基。“修饰碱基”在本领域中被理解为可以与天然碱基(例如,腺嘌呤、鸟嘌呤、胞嘧啶、尿嘧啶和/或胸腺嘧啶)配对和/或可以与非天然存在的碱基配对的碱基。在EP 1 072 679和WO 97/12896中描述了示例性修饰碱基,其公开内容通过引用并入本文。修饰核碱基包括但不限于5-甲基胞嘧啶(5-me-C)、5-羟甲基胞嘧啶、黄嘌呤、次黄嘌呤、2-氨基腺嘌呤、腺嘌呤和鸟嘌呤的6-甲基和其他烷基衍生物、腺嘌呤和鸟嘌呤的2-丙基和其他烷基衍生物、2-硫尿嘧啶、2-硫代胸腺嘧啶和2-硫胞嘧啶、5-卤代尿嘧啶和胞嘧啶、5-丙炔基尿嘧啶和胞嘧啶以及嘧啶碱基的其他炔基衍生物、6-偶氮尿嘧啶、胞嘧啶和胸腺嘧啶、5-尿嘧啶(假尿嘧啶)、4-硫尿嘧啶、8-卤基腺嘌呤和鸟嘌呤、8-氨基腺嘌呤和鸟嘌呤、8-硫羟基腺嘌呤和鸟嘌呤、8-硫烷基腺嘌呤和鸟嘌呤、8-羟基腺嘌呤和鸟嘌呤以及其他8-取代腺嘌呤和鸟嘌呤、5-卤基尿嘧啶和胞嘧啶、特别是5-溴基尿嘧啶和胞嘧啶、5-三氟甲基尿嘧啶和胞嘧啶以及其他5-取代尿嘧啶和胞嘧啶、7-甲基鸟嘌呤和7-甲基腺嘌呤、2-F-腺嘌呤、2-氨基-腺嘌呤、8-氮杂鸟嘌呤和8-氮杂腺嘌呤、7-脱氮鸟嘌呤和7-脱氮腺嘌呤和3-脱氮鸟嘌呤和3-脱氮腺嘌呤。其他修饰碱基包括三环嘧啶(诸如吩恶嗪胞苷(1H-嘧啶并[5,4-b][1,4]苯并恶嗪-2(3H)-酮)、吩噻嗪胞苷(1H-嘧啶并[5,4-b][1,4-苯并噻嗪-2(3H)-酮)),G-夹子(诸如取代吩恶嗪胞苷(例如,9-(2-氨基乙氧基)-H-嘧啶并[5,4-b][1,4]苯并恶嗪-2(3H)-酮)、咔唑胞苷(2H-嘧啶并[4,5-b]吲哚-2-酮)、吡啶并吲哚胞苷(H-吡啶并[3',2':4,5]吡咯[2,3-d]嘧啶-2-酮))。修饰碱基还可以包括其中嘌呤或嘧啶碱基被其他杂环(例如,7-脱氮腺嘌呤、7-脱氮鸟苷、2-氨基吡啶和2-吡啶酮)取代的那些碱基。附加核碱基包括美国专利号3,687,808中公开的那些核碱基;The Concise EncyclopediaOf Polymer Science And Engineering,第858-859页,Kroschwitz,J.I.,ed.John Wiley&Sons,1990,Angewandte Chemie中公开的那些核碱基;Englisch等人,1991,AngewandteChemie,国际版本,30:613所公开的那些核碱基;以及Sanghvi,Y.S.,第15章,AntisenseResearch and Applications,第289-302页,Crooke,S.T.和Lebleu,B.,ed.,CRC出版社,1993所公开的那些核碱基。这些碱基中的某些碱基可以用于增加多核苷酸的结合亲和力,并且包括5-取代嘧啶、6-氮杂嘧啶、以及N-2、N-6和O-6取代嘌呤,其包括2-氨基丙基腺嘌呤、5-丙炔基尿嘧啶、以及5-丙炔基胞嘧啶。已经显示5-甲基胞嘧啶取代使核酸双链体稳定性增加0.6-1.2℃,并且在某些方面中,与2'-O-甲氧基乙基糖修饰组合。参见美国专利号3,687,808、美国专利号4,845,205;5,130,302;5,134,066;5,175,273;5,367,066;5,432,272;5,457,187;5,459,255;5,484,908;5,502,177;5,525,711;5,552,540;5,587,469;5,594,121;5,596,091;5,614,617;5,645,985;5,830,653;5,763,588;6,005,096;5,750,692;以及5,681,941,其公开内容通过引入并入本文。
根据本发明,本领域技术人员可以容易地设计反义多核苷酸。例如,本领域的一般教导包括但不限于Wee和Pramono等人,PLoS One 3:e1844(2008);Pramono ZA等人,WO/2011/078,797;Pramono和Wee等人,Hum Gene Ther 23:781-790(2012);Aartsma-Rus等人,Methods Mol Biol.867:117-29(2012);Aartsma-Rus等人,Methods Mol Biol.867:97-116(2012);van Roon-Mom等人,Methods Mol Biol.867:79-96(2012),其中每个都通过引用并入本文。通用指南还包括试图避免3个连续的G或C核苷酸,选择不利于形成自身结构的长度和序列(避免发夹结构(hairpinning)),并且避免可能形成引物二聚体的那些序列。在一些实施例中,本公开的反义多核苷酸是被设计为与外显子或内含子或内含子-外显子边界特异性杂交的反义多核苷酸,使得反义多核苷酸与完全在PRDM15核酸的外显子内的序列特异性杂交,或当反义多核苷酸与PRDM15核酸特异性杂交时,反义多核苷酸的约一个核苷酸跨越所述内含子-外显子边界。在其中反义多核苷酸与完全在外显子内的序列特异性杂交的一些实施例中,应当设想反义多核苷酸的末端为来自外显子末端的约1、2、3、4、5、6、7、8、9、10或者更多个核苷酸。
在其他实施例中,本公开的反义多核苷酸是被设计为与GLDC核酸的内含子-外显子边界特异性杂交的反义多核苷酸,使得反义多核苷酸的约2、3、4、5、6、7、8、9、10或更多个核苷酸跨越所述内含子-外显子边界。应当理解,核苷酸可以在外显子侧或内含子侧上“跨越内含子-外显子边界”。因此,主要与内含子序列特异性杂交并且仅与相邻外显子的一个核苷酸杂交的反义多核苷酸可能“跨越内含子-外显子边界”一个核苷酸。同样,与外显子序列特异性杂交并且仅与相邻内含子的一个核苷酸杂交的反义多核苷酸可能“跨越内含子-外显子边界”一个核苷酸。在任一上述实施例中,反义多核苷酸的长度为至少约10个核苷酸且至多约15、20、25、30、35、40、45、50或更多个核苷酸。
优选地,反义寡核苷酸可以包括修饰的多核苷酸主链。该修饰的多核苷酸主链可以包括取代至少一个多核苷酸的糖的修饰的部分。
设想使用修饰的多核苷酸,其中多核苷酸中核苷酸单元的一个或多个糖和/或一个或多个核苷酸间键分别被“非天然存在的”糖(即,除核糖或脱氧核糖以外的糖)或核苷酸间键取代。在一个方面中,该实施例设想了肽核酸(PNA)。在PNA化合物中,多核苷酸的糖主链被含酰胺(例如,N-(2-氨基乙基)-甘氨酸单元之间的肽键)主链取代。例如,参见美国专利号5,539,082;5,714,331;和5,719,262,以及Nielsen等人,Science,1991,254,1497-1500,其公开内容通过引用并入本文。修饰的多核苷酸还可以含有一个或多个取代的糖基团。在一个方面中,糖的修饰包括锁定核酸(LNA),其中2'-羟基基团与糖环的3'或4'碳原子连接,从而形成双环糖基团。在某些方面中,键是桥连2'氧原子和4'碳原子的亚甲基(-CH[2]-)[n]基团,其中n为1或2。在WO 98/39352和WO 99/14226中描述了LNA及其制备,其公开内容通过引入并入本文。在本发明中,优选地,反义寡核苷酸包括修饰的多核苷酸主链。该修饰的多核苷酸主链可以包括取代至少一个多核苷酸的糖的修饰的部分。该修饰的部分可以选自包括磷酰二酰胺吗啉代寡聚物(PMO)、肽缀合的磷酰二酰胺吗啉代寡聚物(PPMO)、以及非肽树枝状聚合物八胍部分标记的吗啉代寡聚物的组。
在各种实施例中,修饰的多核苷酸主链包括至少一个修饰的核苷酸间键。该修饰的核苷酸间键包括修饰的磷酸酯。更优选地,该修饰的磷酸酯是选自包括取代硫原子的非桥氧原子、膦酸酯、硫代磷酸酯、磷酸二酯、磷酸莫来酸酯、磷哌嗪酸酯和氨基磷酸酯的组的任一个。
在本发明的各种实施例中,反义寡核苷酸包含选自包括以下各项的组的主链:核糖核酸、脱氧核糖核酸、DNA硫代磷酸酯、RNA硫代磷酸酯、2'-O-甲基-寡核糖核苷酸和2'-O-甲基-寡脱氧核糖核苷酸、2'-O-烃基核糖核酸、2'-O-烃基DNA、2'-O-烃基RNA硫代磷酸酯、2'-O-烃基DNA硫代磷酸酯、2'-F-硫代磷酸酯、2'-F-磷酸二酯、2'-甲氧基乙基硫代磷酸酯、2-甲氧基磷酸二酯、脱氧亚甲基(甲基亚氨基)(脱氧MMI)、2'-O-羟基MMI、脱氧甲基膦酸酯、2'-O-烃基膦酸甲酯、吗啉代、4'-硫代DNA、4'-硫代RNA、肽核酸、3'-酰胺化物、脱氧3'-酰胺化物、2'-O-烃基3'-酰胺化物、锁核酸、环己烷核酸、三环-DNA、2'-氟-阿拉伯核酸、N3'-P5'氨基磷酸酯、氨基甲酸酯连接、磷酸三酯连接、尼龙主链修饰、以及前述主链的混合物。
优选地,寡核苷酸与一种或多种缀合物化学连接,该缀合物增强反义寡核苷酸的活性、细胞分布或细胞摄取。
本公开的化合物还可以用作预防剂或治疗剂,其可以用于治疗遗传疾病。在实施例中,反义寡核苷酸可以用于治疗表达GLDC的癌症患者。可以对患者施用另外的抗癌剂或治疗剂。癌症是选自包括以下各项的组的任一个:非小细胞肺癌(NSCLC)、乳腺癌、卵巢癌、血液系统恶性肿瘤、肺癌、前列腺癌、胃癌、睾丸癌、喉癌、肝癌、子宫癌、结肠直肠癌、黑色素瘤、胶质母细胞瘤、肉瘤、视网膜母细胞瘤、甲状腺癌和胶质瘤、类癌、以及叶状肿瘤。血液系统恶性肿瘤是淋巴瘤或白血病。特别地,淋巴瘤是B细胞淋巴瘤。更具体地,B细胞淋巴瘤是滤泡性淋巴瘤或弥漫性大B细胞淋巴瘤。
在本发明的另一方面中,提供了一种药物组合物,其包括根据本发明第一方面的反义寡核苷酸以及药学上可接受的载体。该组合物适于对患者进行肠胃外施用,要么以裸露形式要么与递送剂复合。该载体选自由以下各项组成的组:纳米颗粒,诸如聚合物纳米颗粒;脂质体,诸如pH敏感性脂质体、抗体缀合脂质体;病毒载体、阳离子脂质、聚合物、UsnRNA(诸如U7snRNA)、以及细胞穿透肽。反义寡核苷酸的施用方式包括口服、或直肠、或透粘膜、或肠内、或肌内、或皮下、或髓内、或鞘内、或直接心室内、或静脉内、或玻璃体内、或腹膜内、或鼻内或眼内。
药学上可接受的载体通常是指适合对受试者施用的材料,其中载体在生物学上无害,或不会产生不良作用。这些载体通常是药物的惰性成分。通常,载体与活性成分一起施用于受试者,而不会引起任何不良生物学作用或以有害方式与包含它的药物组合物的任何其他组分相互作用。在Martin,Remington's Pharmaceutical Sciences,18th Ed.,MackPublishing Co.,Easton,Pa.,(1990)中描述了合适的药物载体,其全部内容通过引用并入本文。
在本公开的更具体形式中,提供了药物组合物,其包括治疗有效量的反义多核苷酸以及药学上可接受的稀释剂、防腐剂、增溶剂、乳化剂、佐剂和/或载体。这些组合物包括各种缓冲液含量的稀释剂(例如,磷酸盐、Tris-HCl、乙酸盐)、pH和离子强度的稀释剂,以及添加剂,诸如去污剂和增溶剂(例如,吐温80、聚山梨醇酯80)、抗氧化剂(例如,抗坏血酸、偏亚硫酸氢钠)、防腐剂(例如,硫柳汞、苯甲醇)和膨胀物质(例如,乳糖、甘露醇)。该材料可以掺入聚合化合物的颗粒制剂中,诸如例如但不限于聚乳酸或聚乙醇酸,或掺入脂质体中。还可以使用月桂酸。这些组合物可能影响所公开的组合物的物理状态、稳定性、体内释放速率、以及体内清除速率。组合物可以以液体形式制备,或者可以呈干燥粉末,诸如冻干形式。
应当领会,根据本公开提供的药物组合物可以通过本领域已知的任何方式施用。优选地,用于施用的药物组合物通过注射、口服或通过肺部或鼻内途径施用。在各种实施例中,反义多核苷酸通过静脉内、动脉内、腹膜内、肌内或皮下施用途径递送。
本发明的反义分子涵盖任何药学上可接受的盐、酯、或这些酯的盐、或任何其他化合物,其在对包括人在内的动物施用时能够(直接或间接)提供生物活性代谢物或其残留物。因而,例如,本公开还涉及本发明的化合物的前药和药学上可接受的盐、这些前药的药学上可接受的盐、以及其他生物等效物。
术语“药学上可接受的盐”是指本发明的化合物的生理学上和药学上可接受的盐:即,保留母体化合物的所需生物活性并且不会产生不期望的毒理学作用的盐。
对于多核苷酸,药学上可接受的盐的优选示例包括但不限于(a)与诸如钠、钾、铵、镁、钙之类的阳离子和诸如精胺和亚精胺之类的多胺形成的盐;(b)与无机酸(例如,盐酸、氢溴酸、硫酸、磷酸、硝酸)形成的酸加成盐;(c)与有机酸(诸如例如,乙酸、草酸、酒石酸、琥珀酸、马来酸、富马酸、葡萄糖酸、柠檬酸、苹果酸、抗坏血酸、苯甲酸、单宁酸、棕榈酸、海藻酸、聚谷氨酸、萘磺酸、甲磺酸、对甲苯磺酸、萘二磺酸、聚半乳糖醛酸)形成的盐;以及(d)由元素阴离子(诸如氯、溴和碘)形成的盐。本发明的药物组合物可以若干种方式施用,这取决于是否需要局部或全身治疗以及取决于待治疗区域。施用可以是局部的(包括眼内以及包括直肠递送在内的粘膜)、肺部的(例如,通过吸入粉末或气溶胶)(包括通过喷雾器、气管内、鼻内、表皮和透皮)、口服或肠胃外。肠胃外施用包括静脉内、动脉内、皮下、腹膜内或肌内注射或输注;或颅内,例如,鞘内或心室内施用。具有至少一个2'-O-甲氧基乙基修饰的多核苷酸被认为是特别适用于口服施用。
可以根据制药工业中熟知的常规技术制备本公开的药物制剂,其可以方便地以单位剂型呈现。这些技术包括使活性成分与药物载体或赋形剂缔合的步骤。一般而言,通过将活性成分与液体载体或细碎的固体载体或两者均匀缔合来制备制剂,然后,如果有必要,则使产品成形。
本公开还设想了与附加治疗剂的组合疗法。可以与本公开的组合物同时递送的治疗剂的示例包括但不限于糖皮质激素类固醇(例如但不限于强的松和地夫可特)、血管紧张素转化酶抑制剂、β肾上腺素能受体阻断剂、抗-纤维化剂及其组合。
在一些实施例中,本发明可以用于基因疗法,诸如例如,使用包括本发明的多核苷酸的载体(例如,表达载体)来指导多核苷酸在合适的宿主细胞中的表达。这些载体用于例如扩增宿主细胞中的多核苷酸以产生其有用量,以及用于使用重组技术表达蛋白质。在一些实施例中,载体是表达载体,其中本发明的多核苷酸与包括表达控制序列的多核苷酸可操作地连接。
如此,本发明的另一方面提供一种治疗患者癌症的方法,该方法包括:施用根据本发明第一方面的反义寡核苷酸、或包括反义寡核苷酸的药物有效量的组合物。载体选自由以下各项组成的组:纳米颗粒,诸如聚合物纳米颗粒;脂质体,诸如pH敏感性脂质体、抗体缀合脂质体;病毒载体、阳离子脂质、聚合物、UsnRNA(诸如U7snRNA)、以及细胞穿透肽。
反义寡核苷酸或组合物的施用方式可以包括口服、或直肠、或透粘膜、或肠内、或肌内、或皮下、或髓内、或鞘内、或直接心室内、或静脉内、或玻璃体内、或腹膜内、或鼻内或眼内。话虽如此,但是经证实的全身施用选项包括静脉内、腹膜内、鼻内和鞘内。ASO与递送载体(诸如纳米颗粒、基于聚合物或脂质体的载体)的复合可以进一步增强ASO对特定组织的递送效率。
癌症可以是选自包括以下各项的组的任一种:血液系统恶性肿瘤、肺癌、乳腺癌、前列腺癌、胃癌、睾丸癌、喉癌、肝癌、子宫癌、结肠直肠癌、黑素瘤、胶质母细胞瘤、肉瘤、以及视网膜母细胞瘤。
在实施例中,癌症是非小细胞肺癌(NSCLC)。
在本发明的另一方面中,提供了反义寡核苷酸在制造用于治疗癌症的药物中的用途,该反义寡核苷酸调节GLDC的活性。寡核苷酸调节以下各项中的一项或多项的活性或水平:前mRNA、成熟mRNA和蛋白质表达。在实施例中,癌症是非小细胞肺癌(NSCLC)。
在本发明的另一方面中,提供了一种用于帮助分类或确定患有癌症的患者的预后或者为癌症患者选择治疗策略的方法,该方法包括:评估样本中的GLDC核酸、蛋白质或活性的水平。该方法包括以下步骤:利用关于样本中GLDC核酸、蛋白质或活性的水平的信息选择治疗方案。在各种实施例中,样本来自于患者,并且样本是怀疑患有癌症或已经发现癌症的组织样本,或包含来自所述组织的细胞。如果样本中GLDC核酸、蛋白质或活性的水平是升高的水平,则所选择的治疗方案包括用GLDC活性抑制剂或GLDC的前mRNA、mRNA和蛋白质表达水平的调节剂来治疗患者。在各种实施例中,抑制剂或调节剂包括根据本发明第一方面的反义寡核苷酸。
在本发明的另一方面中,提供了一种诱导GLDC前mRNA的外显子跳跃的方法,该方法包括:向细胞递送根据本发明第一方面的反义多核苷酸或包括该反义寡核苷酸的药物组合物,从而诱导GLDC前mRNA的外显子跳跃。
优选地,外显子2、外显子3、外显子6、外显子7、外显子8、外显子9、外显子10、外显子12、外显子13、外显子15、外显子16、外显子19、外显子20或外显子21中的任一个被跳跃。
在各种实施例中,细胞可以是人体细胞。人体细胞是癌细胞,癌症是选自包括以下各项的组的任一种:NSCLC、血液系统恶性肿瘤、肺癌、乳腺癌、前列腺癌、胃癌、睾丸癌、喉癌、肝癌、子宫癌、结肠直肠癌、黑色素瘤、胶质母细胞瘤、肉瘤、以及视网膜母细胞瘤。
在本发明的另一方面中,提供了一种试剂盒,其包括根据本发明第一方面的反义寡核苷酸(可选地在容器中),以及包装插入件、包装标签、说明书或其他标签。
有利地,本发明提供一种诱导GLDC mRNA外显子跳跃的反义寡核苷酸、以及所述反义寡核苷酸用于治疗表达GLDC的癌症的用途。
本领域普通技术人员应当领会,上述方法的应用广泛应用于鉴定适用于治疗许多其他疾病的反义分子。
附图说明
为了使本发明可以被完全理解并易于实施,现在将仅通过非限制性示例对本发明的优选实施例进行描述,该描述参考所附说明性附图。
在附图中:
图1是选择导致GLDC靶外显子的有效和特异性跳跃的shAON。使用Lipofectamin2000将100nM的靶向GLDC的shAON转染到A549细胞中。转染后16-24小时收获细胞,提取总RNA,并且逆转录成cDNA。通过PCR扩增覆盖靶外显子的区域,然后对PCR产物进行光密度分析,确定shAON诱导特异性靶外显子跳跃的效率。shAON效率代表相对于总扩增子的具有外显子跳跃的扩增子的百分比。转染后5小时,向培养基中加入100μg/ml的环己酰亚胺,以防止蛋白质合成和RNA降解。a)PCR产物的琼脂糖凝胶电泳图像,其证明特异性外显子跳跃。b)所计算的由每个shAON诱导的外显子跳跃效率。c)条带的DNA测序对应于从琼脂糖凝胶切下的所跳跃的转录物[如(a)中所示]以确认特异性靶外显子的跳跃。d)通过shAON转染在与上述相同的条件下但在不存在环己酰亚胺的情况下估计跳跃的转录物被NMD降解的程度。在两个实验条件下跳跃效率之间的比较表明,环己酰亚胺保护所跳跃的转录物免于NMD影响,因为在经环己酰亚胺处理的样本中所跳跃的转录物的比例更高。
图2是3种所选择的shAON诱导靶外显子跳跃并且在低剂量下抑制GLDC蛋白表达。a)剂量响应曲线。b)时间过程实验显示在长达144小时的不同时间点的跳跃效率。c)用β-肌动蛋白作为上样对照的GLDC蛋白的Western印迹。
图3是shAON抑制A549细胞生长/增殖和肿瘤发生。a)转染10nM的shAON的A549细胞的生长曲线;NC-乱序shAON,lipo only-仅限脂质体2000。b)转染后120小时各种浓度的shAON对A549细胞活力的影响。c)在用10nM的shAON 7C、7D或8D转染后120小时,A549、MRC5和HLF细胞之间细胞活力的比较。d)转染shAON的A549细胞的软琼脂测定。转染后7天对菌落数进行计数,并且相对于NC进行归一化。‘*’指示结果与乱序AON对照显著差异(p<0.01,学生t检验)。
图4是shAON诱导的肺肿瘤球体细胞中的GLDC敲低(GLDC knockdown)抑制细胞生长/增殖和肿瘤发生。a)肿瘤球体细胞中shAON诱导的外显子跳跃效率的剂量响应曲线(通过光密度分析测量)。转染后24小时收获细胞。在shAON转染后5小时加入100μg/ml的环己酰亚胺以抑制所跳跃的转录物经历NMD。b)用β-肌动蛋白作为上样对照,肿瘤球体细胞(转染后72小时)中GLDC蛋白(200nM)的Western印迹。c)转染后72小时使用细胞活力测定测量shAON剂量响应。d)转染150nM shAON的肿瘤球体细胞的生长曲线。e)转染shAON的肿瘤球体细胞的软琼脂测定。转染后7天对菌落数进行计数,并且相对于NC样本进行归一化。‘*’指示结果与乱序AON对照显著差异(p<0.01,学生的t检验)。
图5是GLDC shAON可以有效诱导GLDC在其他类型的癌症中的下调。a)不同细胞系中GLDC表达的实时PCR测量。b)GLDC shAON(100nM)诱导MLL细胞系中的外显子跳跃效率(在shAON转染后5小时加入25μg/ml环己酰亚胺)。
图6是GLDC shAON诱导外显子跳跃并抑制免疫缺陷小鼠皮下生长的人体肿瘤异种移植物的肿瘤生长。a)比较来自用shAON 7D或NC(乱序)(7-8只小鼠/治疗组)治疗的小鼠的肿瘤质量的点图。指示具有显示第25百分点和第75百分点的误差条的中值。来自7D治疗的小鼠的肿瘤的质量中值比来自NC治疗的小鼠的质量中值小60%;P值=0.000676(非配对的t样本检验)和0.00391(未配对的Wilcoxon秩和检验)。b)凝胶电泳分析显示shAON 7D诱导的外显子7跳跃与从小鼠提取的20个肿瘤样本(12个来自乱序对照组,8个来自shAON 7D组)中的NC相比较。凝胶照片中不包括由于RNA降解而没有可检测的PCR产物的样本。
图7是shAON 7D能够在不存在转染剂(脂质体)的情况下诱导外显子跳跃、GLDC下调和TS32细胞生长抑制。在转染后24小时,shAON 7D(100nM)在有或无脂质体的情况下诱导特异性外显子跳跃(a)和GLDC转录物下调(b)的比较。(c)转染后72小时,shAON 7D在不存在脂质体的情况下对TS32细胞生长的影响。在不存在转染剂(脂质体)的情况下,浓度为100nM的shAON 7D能够诱导外显子7跳跃、GLDC下调和TS32细胞生长抑制,其效率分别为55%,37%和18%。当shAON 7D浓度增加至200nM时,细胞生长抑制增加至32%。
具体实施方式
本发明涉及靶向甘氨酸脱羧酶(GLDC)表达的反义寡核苷酸(shAON)的空间位阻。特别地,这种shAON可以用作非小细胞肺癌(NSCLC)和其他癌症的候选药物。shAON用于诱导无义介导的GLDC mRNA衰变的应用尚未在先前的工作中说明。因此,对于NSCLC和其他癌症,本发明可以用于开发针对GLDC的一组高效(IC50<10nM)候选药物的新化学式。
示例1
这里,我们已经鉴定了12种新型shAON(空间位阻反义寡核苷酸),它们各自降解GLDC(甘氨酸脱羧酶)mRNA作为NSCLC(非小细胞肺癌)的治疗策略(附图中的图1)。GLDC在来自NSCLC的肿瘤引发细胞中高度表达并且促进细胞转化和肿瘤发生(Zhang等人,Cell2012;Jain等人,Science 2012);催化失活的GLDC不能促进细胞转化。shAON是合成的单链化学修饰的RNA分子,其中每个碱基用硫代磷酸酯主链所连接的2'-O-甲基修饰。每个shAON被设计为在特定位点互补地结合新生GLDC mRNA,并且对RNA结合剪接调节剂发挥空间位阻效应,这对外显子识别及其剪接至关重要。从机理上讲,当shAON诱导排除特异性框外外显子(specific out-of-frame exon)时,其中特异性框外外显子在成熟RNA中生成大量下游提前终止密码子,所得mRNA经由无义介导的衰变过程被靶向降解。
我们选择三种最有效的shAON用于进一步体外验证人类癌细胞系和患者的原发癌细胞。在癌细胞系中,每个shAON诱导GLDC mRNA中的特异性外显子跳跃以及随后用IC50<10nM抑制GLDC蛋白表达(图2),并且抑制>70%的癌细胞生长(图3)。在原发性癌细胞中,shAON抑制肺肿瘤球体细胞的细胞生长/增殖和肿瘤发生(图4)。另外,shAON在下调急性白血病细胞系中的GLDC时有效(图5)。我们进一步选择一种shAON用于植入来自NSCLC患者的肺肿瘤球体细胞的小鼠的体内研究。当经由腹膜内注射向移植小鼠全身施用shAON时,其抑制60%的人体肿瘤生长(图6)。
下文对本发明进行更详细的描述。
材料与方法
细胞系培养
将人肺腺癌上皮细胞系A549维持在补充有10%FBS、2mM L-谷氨酰胺和1%青霉素-链霉素的DMEM培养基中。将正常人胎肺成纤维细胞MRC-5细胞和人成体肺成纤维细胞HLF细胞维持在补充有10%FBS和1%青霉素-链霉素的DMEM培养基中。
肿瘤球体培养
如前所述(Zhang,WC;等人,2012)制备肿瘤球体TS32细胞,并且维持在DMEM/F12中,该DMEM/F12含有ITS(Sigma)并且在未处理的有盖培养皿中补充有0.4%BSA(Sigma)、20ng/ml EGF、4ng/ml碱性成纤维细胞生长因子(bFGF)(Invitrogen)、1%青霉素-链霉素(Gibco)。每3天补充一次新鲜培养基。用Accutase(EMD-Millipore)分裂细胞以获得单细胞悬浮液。
shAON设计和合成
shAON使用我们获得专利的计算方法进行设计,由Sigma-Aldrich Pte Ltd(新加坡)合成,用硫代磷酸酯主链的2'-O-甲基(2OMePS)修饰。在所有实验中都包括具有乱序序列的shAON。
用shAON细胞转染
对于所有细胞/细胞系,在细胞转染期间,不含抗生素(即,青霉素-链霉素)的生长培养基用作转染培养基。
A549细胞以1.0-1.5×105个细胞/孔接种于含有1mL转染培养基的6孔板中,并且孵育过夜。细胞达到约40%汇合,培养基在转染前降至900μL。在Opti-MEM培养基(Invitrogen)中制备各种浓度的shAON(100pmol)与脂质体2000(单位为μL)的固定比例为1:2的10×转染混合物至总体积100μL,在室温下孵育20分钟,并且加入到细胞培养物中。
TS32细胞以1.0-1.5×105个细胞/ml的密度接种于含有900μL转染培养基的24孔板中。在Opti-MEM培养基中制备具有不同量的shAON和固定量的脂质体2000(5μL)的10×转染混合物至总体积100μL,在室温下孵育20分钟,并且加入到细胞培养物中。为了确保重复性,通常重复转染两次或三次。
为了测量shAON诱导的外显子跳跃效率,在转染后5小时在细胞培养基(100μg/ml)中加入环己酰亚胺(Sigma)以停止蛋白质合成和RNA降解。
量化shAON诱导的外显子跳跃效率和mRNA下调
在使用shAON转染后24小时收获细胞,并且使用Qiagen Rneasy小量试剂盒(Qiagen RNeasy Mini Kit)提取总RNA,用DNase(AmbionTurbo DNA-free试剂盒)处理以除去污染的DNA,并且根据制造商的说明使用具有随机六聚体的SuperScriptTM IIIReverse转录酶(Invitrogen)随机转录成cDNA。
为了测量shAON诱导的外显子跳跃效率,在两侧上使用扩增覆盖靶外显子和至少1个相邻外显子的区域的引物执行PCR反应。然后,通过琼脂糖凝胶电泳和DNA测序验证PCR产物以确认特异性靶外显子的跳跃。通过计算所靶向的外显子跳跃产物相对于总产物的量,通过凝胶图像的光密度分析来估计外显子跳跃效率。使用ImageJ软件(Rasband,WS,ImageJ,U.S.National Institutes of Health,Bethesda,MD,USA)执行密度测定分析。
使用引物在所靶向的外显子中和在所靶向的外显子外部执行定量实时PCR(qPCR),以使用GADPH作为对应样本中的内源参照并且相对于使用具有乱序序列的相同浓度的shAON转染的细胞来测量全长GLDC转录物的量,用于计算跳跃效率。
细胞活力测定
使用噻唑基蓝四唑溴化物(MTT)测定(Sigma)或CellTiter-Blue细胞活力测定(Promega)在96孔板(每孔100μL培养基中存在3.5×103个细胞)中执行细胞活力和增殖的测量。使用shAON和脂质体2000的混合物在96孔板中转染细胞。对于MTT测定(用于贴壁细胞),使用具有MTT(0.5mg/ml)的DMEM培养基(100μL/孔)代替培养基,在37℃下孵育4小时,然后更换为100μL的异丙醇。在波长570nm处使用微量培养板读数器(Molecular Devices)测量吸光度,其中在630nm处减去背景,并且使用吸光度对活细胞数的校准曲线将其转换为活细胞数。对于CellTiter-Blue细胞活力测定(对于悬浮细胞),加入Cell-Titer Blue试剂(11μL/孔),在37℃下孵育4小时。使用微量培养板读数器(Molecular Devices)测量荧光,其中激发波长为570nm,而发射波长为600nm。为了确保再现性,执行测定至少两次,并且在每次实验中重复三次转染。
软琼脂试验
shAON转染后24小时,收获细胞,在DMEM培养基中用0.4%的noble琼脂重悬,并涂布到含有固化底层(DMEM培养基中的0.6%的noble琼脂)的96孔板(5×103细胞/孔)上。使用100μL的生长培养基覆盖每个孔,并且在标准培养条件下孵育1周。使用CellTiter Blue染色细胞,并且使用荧光微量培养板读数器执行菌落计数。
Western印迹(需要仔细检查浓度和公司)
通过在4度下温和摇动10分钟,使用含有Halt蛋白酶抑制剂混合物的M-PER裂解缓冲液从细胞中提取蛋白质。通过在4度下以最高速度(12000g?)离心20分钟,除去细胞碎片,并根据制造商的说明使用BioRad蛋白质测定测量上清液的蛋白质浓度。在根据制造商的说明制备的8%的Bis-Tris SDS-PAGE凝胶(Bio-rad)中上样来自每个样本的16μg蛋白质,然后转移到Bio-rad硝酸纤维素膜上。使用PBST中5%牛奶封闭1小时后,将膜在3%牛奶/PBST中与多克隆兔抗人GLDC抗体(1:2000)在4度下一起孵育过夜,然后在3%牛奶/PBST中与辣根过氧化物酶缀合的抗兔IgG(1:4000)在室温下一起孵育3小时。β-肌动蛋白用作Western印迹的上样对照,而小鼠抗人β-肌动蛋白抗体(1:1000)和辣根过氧化物酶缀合的抗小鼠IgG(1:4000)分别用作第一和第二抗体。使用Supersignal West Pico化学发光底物(Thermo Scientific)通过ECL系统实现可视化。
小鼠异种移植物实验
在无血清培养基和Matrigel(BD)(1:1)中,向4-6周龄的NOD.Cg-PrkdcscidIl2rgtm1Wj1/SzJ小鼠皮下移植(500,000细胞/小鼠)肿瘤球体的单细胞悬浮液。腹膜内注射50mg/kg剂量的shAON,每周3次。注射6周后,处死小鼠,提取肿瘤,并且称重。在使用MicroSmash MS-100(3×45s,4500rpm)中的0.5mg玻璃珠(Sigma-Aldrich)破碎细胞后,分别使用Qiagen RNeasy小量试剂盒和M-PER裂解缓冲液从肿瘤样本中提取总RNA和蛋白质。
结果
选择导致GLDC靶外显子的有效和特异性跳跃的shAON
在GLDC转录物中的25个外显子中,选择了5个框外外显子(外显子7、8、13、15和16)作为我们设计shAON的靶。当它们中的每个被单独地跳跃时,所得转录物将在所靶向的外显子下游具有移码的阅读框,并且在所跳跃的转录物中生成多个PTC。结果,靶基因将经由无义介导降解(NMD)途径的组合下调,其中转录物被主动降解或导致截短的和无功能的基因产物的翻译。我们使用我们先前开发和验证的运算方法设计了20个shAON(4个shAON/外显子)。每次实验中包括具有乱序序列的一个shAON作为阴性对照。这些shAON使用硫代磷酸酯主链和2'-O-甲基核糖修饰(2OMePS)合成,以提高稳定性和序列特异性。
我们检验了所设计的shAON通过转染A549人肺腺癌上皮细胞来诱导GLDC转录物的特异性外显子跳跃的能力(图1),并且基于所计算的跳跃效率来选择在使用100nM的shAON转染后24小时诱导GLDC靶外显子在A549细胞中跳跃方面最佳的shAON。在所设计的20个shAON中,12个能够诱导靶外显子的跳跃;表1列出了12个有效shAON的式。总体而言,靶向外显子7和8的shAON在诱导靶外显子跳跃方面更有效。选择三个shAON(7D、7C和8D),因为它们能够诱导靶外显子在超过80%的GLDC转录物中跳跃。
表1
GLDC shAON诱导靶外显子跳跃并且抑制低剂量的GLDC蛋白表达
通过PCR,然后通过光密度分析,评估所选择的3个shAON对诱导A549细胞中特异性外显子跳跃的时间和剂量响应影响。如图2a所示,所有3个shAON均可以诱导低剂量的特异性外显子跳跃,其中IC50范围为3.5至7nM。基于使用10nM shAON进行的时程实验(图2b),它们对诱导外显子跳跃的影响随时间降低,但仍可以在转染后72小时诱导>40%的GLDC转录物跳跃。
我们进行了定量实时PCR(qPCR)以测量所选择的3个shAON对下调A549细胞中GLDCmRNA转录物的影响。与使用乱序shAON转染的细胞相比,qPCR分析表明在使用10nM的GLDCshAON转染后24小时,GLDC全长转录物下调>70%。转染后72小时,3个shAON中的每个仍然可以诱导>40%的下调,这与光密度测定的结果一致。
执行Western印迹分析以检查所选择的shAON对蛋白质水平的GLDC表达的影响(图2c)。在使用10或20nM的所选择的shAON转染后72小时,蛋白质水平降低至几乎完全耗尽。
GLDC shAON抑制A549细胞生长/增殖和肿瘤发生
已经发现肺癌细胞对高水平的GLDC成瘾(Zhang WC等人,Cell.2012年1月20日;148(1-2):259-72)。由于shAON可以在转染细胞时诱导有效敲低GLDC,因此我们检验了它们对癌细胞生长和增殖的影响。在使用不同浓度的所选择的shAON转染后0、24、48、72和120小时,使用MTT测定测量A549细胞活力,并且与几种阴性对照进行比较,这些阴性对照包括未处理的细胞、使用脂质体2000转染的细胞、或乱序shAON。shAON诱导的GLDC敲低似乎在转染后长达120小时内抑制细胞增殖(图3a和图3b)。细胞生长的抑制从使用5nM的shAON的35%抑制增加到使用30nM的shAON的几乎100%抑制。如图3c所示,当使用10nM的shAON转染时,两种非癌细胞(HLF和MRC-5)的生长似乎没有被抑制,而A549细胞中约70%的细胞生长被抑制。
然后,检查shAON转染的A549细胞在软琼脂中形成菌落的能力,其是一种通常与致瘤性相关的不依赖锚定生长(anchorage-independent growth)的量度。转染后7天,与用乱序shAON处理的细胞相比,使用三个GLDC shAON处理的细胞在软琼脂中产生少于70-92%的菌落,表明shAON诱导的GLDC敲低极大地抑制了癌细胞致瘤性。
肺肿瘤球体细胞中的shAON诱导的GLDC敲低抑制细胞生长/增殖和肿瘤发生
癌症干细胞(CSC)或肿瘤引发细胞(TIC)具有驱动并维持肿瘤生长的能力,并且当在体外培养中生长时,可以通过球体形成分离和富集。因此,我们检验了GLDC shAON在影响肿瘤球体细胞生长的作用,这些肿瘤球体细胞高度富含肿瘤引发细胞。如图4a所示,GLDCshAON可以有效诱导特异性靶外显子在GLDC转录物中的跳跃,其中跳跃效率范围为51-93%,而IC50范围为65nM-220nM(图4a)。在使用Western印迹测量的蛋白质水平下,还有效抑制了GLDC蛋白质表达(图4b)。需要更大量的shAON来诱导肿瘤球体细胞而非A549细胞中的有效外显子跳跃和GLDC敲低。
与使用乱序shAON处理的肿瘤球体细胞相比,如使用CellTiter Blue测定测量的,GLDC shAON处理的细胞的生长/增殖被抑制了57-75%。软琼脂测定还表明由经GLDC shAON处理的细胞形成的菌落数比由经乱序shAON处理的细胞形成的菌落数减少了61-73%。
GLDC shAON可以有效诱导GLDC在其他类型的癌症中的下调
不仅在非小细胞肺癌中发现了GLDC的过表达,而且在其他类型的癌症中也有报道(Zhang WC,2012)。我们使用qPCR评估了两种MLL白血病细胞系中的GLDC表达,并确认GLDC在它们两者中均被过表达,而非癌细胞表达比GLDC低600倍(图5a)。因此,我们检验了GLDCshAON是否在这两种MLL细胞系中均会有效。如图5b所示,GLDC shAON在两种MLL细胞系中诱导特异性外显子在超过50%的GLDC转录物中的跳跃,表明我们设计的shAON可能对其他类型的癌症也有效。
GLDC shAON抑制移植肿瘤的生长
为了检验shAON 7D在体内实验中的作用,我们将肿瘤球体细胞移植到免疫缺陷小鼠中,然后腹膜内注射shAON 7D。如图6所示,来自经7D和NC处理的小鼠的肿瘤分别使用中值0.325g和0.820g进行加权,即,抑制60%的肿瘤生长(来自未配对的t样本检验,P值=0.000676或来自未配对的Wilcoxon秩和检验,P值=0.00391)。20个肿瘤样本(分别来自对照组12个以及来自shAON 7D组8个)的RNA分析表明7D处理导致所有处理的肿瘤样本中GLDC外显子7或外显子7和8(次要)跳跃,而在NC处理的肿瘤样本中几乎没有观察到GLDC外显子跳跃。
尽管在前面的描述中已经描述了本发明的优选实施例,但是本领域技术人员应当理解,在不背离本发明的情况下,可以对设计或构造的细节进行许多变化或修改。
Claims (42)
1.一种用于调节GLDC活性的分离的反义寡核苷酸。
2.根据权利要求1所述的反义寡核苷酸,其中,寡核苷酸调节以下各项中的一项或多项的活性或水平:前mRNA、成熟mRNA和蛋白质表达。
3.根据权利要求2所述的反义寡核苷酸,其中,寡核苷酸抑制GLDC的表达或修饰其表达产物。
4.根据权利要求3所述的反义寡核苷酸,其中,寡核苷酸通过诱导GLDC前mRNA或成熟mRNA的外显子跳跃来抑制GLDC的表达以诱导细胞周期停滞或凋亡。
5.根据前述权利要求中任一项所述的反义寡核苷酸,其中,寡核苷酸与GLDC前mRNA或成熟mRNA的靶区域特异性杂交,并且反义寡核苷酸对一个或多个RNA结合剪接调节剂发挥空间位阻效应。
6.根据权利要求5所述的反义寡核苷酸,其中,寡核苷酸通过空间位阻阻止mRNA的翻译。
7.根据前述权利要求中任一项所述的反义寡核苷酸,其中,寡核苷酸与GLDCRNA的外显子、内含子或外显子-内含子边界靶区域特异性杂交,靶区域是选自包括以下各项的组的任一个:外显子2、外显子3、外显子6、外显子7、外显子8、外显子9、外显子10、外显子12、外显子13、外显子15、外显子16、外显子19、外显子20、外显子21、内含子6、内含子7、以及内含子8。
8.根据前述权利要求中任一项所述的反义寡核苷酸,其中,反义寡核苷酸包括选自SEQID NO:1至94中任一个的序列。
9.根据前述权利要求中任一项所述的反义寡核苷酸,其中,反义寡核苷酸包括修饰的多核苷酸主链。
10.根据权利要求9所述的反义寡核苷酸,其中,修饰的多核苷酸主链包括取代至少一个所述多核苷酸的糖的修饰的部分。
11.根据权利要求10所述的反义寡核苷酸,其中,修饰的部分选自由以下各项组成的组:磷酰二酰胺吗啉代寡聚物(PMO)、肽缀合的磷酰二酰胺吗啉代寡聚物(PPMO)、以及非肽树枝状聚合物八胍部分标记的吗啉代寡聚物。
12.根据权利要求9-11中任一项所述的反义寡核苷酸,其中,修饰的多核苷酸主链包括至少一个修饰的核苷酸间键。
13.根据权利要求12所述的反义寡核苷酸,其中,修饰的核苷酸间键包括修饰的磷酸酯。
14.根据权利要求13所述的反义寡核苷酸,其中,修饰的磷酸酯选自由以下各项组成的组:取代硫原子的非桥氧原子、膦酸酯、硫代磷酸酯、磷酸二酯、磷酸莫来酸酯、磷哌嗪酸酯、以及氨基磷酸酯。
15.根据前述权利要求中任一项所述的反义寡核苷酸,其中,反义寡核苷酸包括主链,该主链选自包括以下各项的组:核糖核酸、脱氧核糖核酸、硫代磷酸DNA、硫代磷酸RNA、2'-O-甲基-寡核糖核苷酸和2'-O-甲基-寡脱氧核糖核苷酸、2'-O-烃基核糖核酸、2'-O-烃基DNA、2'-O-烃基RNA硫代磷酸酯、2'-O-烃基DNA硫代磷酸酯、2'-F-硫代磷酸酯、2'-F-磷酸二酯、2'-甲氧基乙基硫代磷酸酯、2-甲氧乙基磷酸二酯、脱氧亚甲基(甲基亚氨基)(脱氧MMI)、2'-O-烃基MMI、脱氧-甲基膦酸酯、2'-O-烃基甲基膦酸酯、吗啉代、4'-硫代DNA、4'-硫代RNA、肽核酸、3'-酰胺化物、脱氧3'-酰胺化物、2'-O-烃基3'-酰胺化物、锁核酸、环己烷核酸、三环-DNA、2'-氟-阿拉伯糖核酸、N3'-P5'氨基磷酸酯、氨基甲酸酯连接、磷酸三酯连接、尼龙主链修饰、以及上述主链的混合物。
16.根据前述权利要求中任一项所述的反义寡核苷酸,其中,寡核苷酸与一种或多种缀合物化学连接,该缀合物增强反义寡核苷酸的活性、细胞分布或细胞摄取。
17.根据前述权利要求中任一项所述的反义寡核苷酸,其中,寡核苷酸调节以下各项中的一项或多项的活性或水平:前mRNA、成熟mRNA和蛋白质表达。
18.根据权利要求1-17中任一项所述的反义寡核苷酸,其用于治疗表达GLDC的癌症患者。
19.根据权利要求18所述的反义寡核苷酸,其中,对患者施用另外的抗癌剂或治疗剂。
20.根据权利要求18或19中任一项所述的反义寡核苷酸,其中,癌症是选自包括以下各项的组的任一种:非小细胞肺癌(NSCLC)、乳腺癌、卵巢癌、结肠癌、黑素瘤、睾丸癌、甲状腺癌、胶质瘤、类癌、以及叶状肿瘤。
21.一种药物组合物,其包括根据权利要求1至20中任一项所述的反义寡核苷酸以及药学上可接受的载体。
22.根据权利要求21所述的组合物,其中,组合物适于对患者进行肠胃外施用,要么以裸露方式要么与递送剂复合。
23.根据权利要求22所述的组合物,其中,载体选自包括以下各项组成的组:纳米颗粒,诸如聚合物纳米颗粒;脂质体,诸如pH敏感性脂质体、抗体缀合脂质体;病毒载体、阳离子脂质、聚合物、UsnRNA(诸如U7snRNA)、以及细胞穿透肽。
24.根据权利要求21-23中任一项所述的组合物,其中,反义寡核苷酸的施用方式包括口服、或直肠、或透粘膜、或肠内、或肌内,或皮下、或髓内、或鞘内、或直接心室内、或静脉内、或玻璃体内、或腹膜内、或鼻内、或眼内。
25.一种治疗患者癌症的方法,该方法包括:施用根据权利要求1至20中任一项所述的反义寡核苷酸、或施用药学有效量的根据权利要求21至24中任一项所述的组合物。
26.根据权利要求25所述的方法,其中,载体选自由以下各项组成的组:纳米颗粒,诸如聚合物纳米颗粒;脂质体,诸如pH敏感性脂质体、抗体缀合脂质体;病毒载体、阳离子脂质、聚合物、UsnRNA(诸如U7snRNA)、以及细胞穿透肽。
27.根据权利要求25或26中任一项所述的方法,其中,反义寡核苷酸或组合物的施用方式包括口服、或直肠、或透粘膜、或肠内、或肌内,或皮下、或髓内、或鞘内、或直接心室内、或静脉内、或玻璃体内、或腹膜内、或鼻内、或眼内。
28.根据权利要求25至27中任一项所述的方法,其中,癌症是非小细胞肺癌(NSCLC)。
29.反义寡核苷酸在制造用于治疗癌症的药物中的用途,该反义寡核苷酸调节GLDC活性。
30.根据权利要求29所述的用途,其中,寡核苷酸调节以下各项中的一项或多项的活性或水平:前mRNA、成熟mRNA和蛋白质表达。
31.根据权利要求30所述的用途,其中,寡核苷酸抑制GLDC的表达或修饰其表达产物。
32.根据权利要求29-31中任一项所述的用途,其中,癌症是非小细胞肺癌(NSCLC)。
33.一种用于帮助分类或确定患有癌症的患者的预后或为患有癌症的患者选择治疗策略的方法,所述方法包括:评估样本中GLDC核酸、蛋白质或活性的水平。
34.根据权利要求33所述的方法,还包括以下步骤:利用关于样本中GLDC核酸、蛋白质或活性的水平的信息选择治疗方案。
35.根据权利要求33或34中任一项所述的方法,其中,样本从所述患者获得,并且样本是怀疑患有癌症或已经发现癌症的组织样本,或者含有来自所述组织的细胞。
36.根据权利要求33至35中任一项所述的方法,其中,如果样本中GLDC核酸、蛋白质或活性的水平是升高的水平,则所选择的治疗方案包括用GLDC活性的抑制剂或GLDC的前mRNA、mRNA和蛋白表达水平的调节剂来治疗患者。
37.根据权利要求36所述的方法,其中,抑制剂或调节剂包括根据权利要求1至20中任一项所述的反义寡核苷酸。
38.一种诱导GLDC前mRNA的外显子跳跃的方法,该方法包括:向细胞递送根据权利要求1至20中任一项所述的反义寡核苷酸或根据权利要求21至24中任一项所述的组合物,从而诱导GLDC前mRNA的外显子跳跃。
39.根据权利要求38所述的方法,其中,外显子2、外显子3、外显子6、外显子7、外显子8、外显子9、外显子10、外显子12、外显子13、外显子15、外显子16、外显子19、外显子20、或外显子21中的任一个被跳跃。
40.根据权利要求38或39中任一项所述的方法,其中,细胞是人体细胞。
41.根据权利要求40所述的方法,其中,人体细胞是癌细胞,癌症是选自包括以下各项的组的任一种:血液系统恶性肿瘤、肺癌、乳腺癌、前列腺癌、胃癌、睾丸癌、喉癌、肝癌、子宫癌、结肠直肠癌、黑色素瘤、胶质母细胞瘤、肉瘤、以及视网膜母细胞瘤。
42.一种试剂盒,其包含权利要求1至20中任一项所述的反义寡核苷酸,可选地在容器中;以及包含包装插入件、包装标签、说明书或其他标签。
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