CN111836623A - 诱导三重基序包含蛋白16(trim16)信号传导的组合物和方法 - Google Patents
诱导三重基序包含蛋白16(trim16)信号传导的组合物和方法 Download PDFInfo
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Abstract
本公开提供了在细胞中诱导三重基序包含蛋白16(TRIM16)活性的组合物和方法。这种活性可以表现在癌细胞中的程序性细胞死亡中。该组合物包含含有病原体相关分子模式(PAMP)的核酸分子。PAMP包含分子可以包含含有末端三磷酸酯的5'臂区域,包含至少8个连续尿嘧啶残基的聚尿嘧啶核心,和包含至少8个核酸残基的3'臂区域,其中3'臂区域的最5’端核酸残基不是尿嘧啶并且其中3'臂区域至少30%是尿嘧啶残基。该组合物可以与其他癌症治疗剂组合,或与其他癌症治疗剂组合使用,包括与其他免疫治疗剂组合。
Description
相关申请的交叉引用
本申请要求2018年2月2日提交的美国临时申请号62/625623的权益,其全部内容通过引用并入本文。
关于序列表的声明
与本申请相关的序列表以文本格式代替纸质副本提供,并且在此通过引用并入说明书中。包含序列表的文本文件的名称是68142_Seq_final_2019-01-31.txt。文本文件为29KB;创建于2019年1月31日;并经由EFS-Web与说明书的递交一起提交。
政府许可权的声明
本发明是在国立卫生研究院(NIH)授予的拨款号R01 AI118916的政府支持下完成的。政府拥有本发明的某些权利。
发明背景
癌症的特征是细胞失去控制细胞周期的能力,导致异常和不受控制的细胞生长。转化的细胞经常失去调节其分裂和生长以及对诱导健康细胞中程序性细胞死亡(PCD)的典型信号作出应答的能力。治疗癌症的传统策略包括手术以去除癌性组织(例如肿瘤)和施用主要影响体内癌细胞的毒性组合物。利用受试者免疫系统的组合物和/或信号传导通路来攻击癌细胞的免疫疗法的发展已经取得了长足的进步。这样的治疗方法包括,例如,施用含有毒性有效载荷的癌症特异性抗体,施用防止癌细胞阻断或阻止宿主针对癌细胞的应答的检查点抑制剂,以及过继性细胞疗法,包括使用改变的免疫细胞以特异性启动并靶向对癌细胞的宿主免疫应答的CAR T方法。
然而,尽管抗癌疗法取得了许多进步,但是仍然存在与毒性副作用、癌细胞的抗性、疾病的复发以及许多治疗的难以负担和不可行有关的问题。因此,仍然需要以最小的异地毒性促进细胞死亡的癌症治疗。此外,为了确保医保的可达性和减少随之而来的费用,仍然需要可以广泛应用于多种类型的癌症并且不需要根据具体情况个性化的治疗。本公开解决了这些和相关需求。
发明概述
提供本概述以简化形式介绍一些概念,这些概念将在下面的详细描述中进一步描述。本概述不旨在标识所要求保护的主题的关键特征,也不旨在用于帮助确定所要求保护的主题的范围。
在一方面,本公开提供了诱导细胞中三重基序包含蛋白16(TRIM16)活性的方法。该方法包括使细胞与有效量的含有病原体相关分子模式(PAMP)的核酸分子接触。含有PAMP的核酸分子包含:包含末端三磷酸根的5'臂区域,包含至少8个连续尿嘧啶残基的聚尿嘧啶核心,和包含至少8个核酸残基的3'臂区域。3'臂区域的最5’端核酸残基不是尿嘧啶并且其中3'臂区域至少30%是尿嘧啶残基。
在一些实施方案中,聚尿嘧啶核心由8至30个尿嘧啶残基组成。在一些实施方案中,3'臂区域的最5’端核酸残基是胞嘧啶残基或鸟嘌呤残基。在一些实施方案中,3'臂区域至少40%、50%、60%、70%、80%或90%是尿嘧啶残基。在一些实施方案中,3'臂区域包含至少7个连续尿嘧啶残基。在一些实施方案中,5'臂区域还包含位于末端三磷酸根和聚尿嘧啶核心之间的一个或多个核酸残基。在一些实施方案中,5'臂区域由末端三磷酸根组成,并且其中末端三磷酸根直接连接至聚尿嘧啶核心的5'端。在一些实施方案中,末端三磷酸根、5'臂区的一个或多个核酸残基和聚尿嘧啶核心在丙型肝炎病毒中不天然地一起出现。
在一些实施方案中,含有PAMP的核酸分子在细胞中诱导视黄酸诱导型基因I(RIG-I)样受体(RLR)与TRIM16的相互作用。在一些实施方案中,RLR与TRIM16的相互作用诱导细胞中的细胞死亡信号传导。在一些实施方案中,RLR是RIG-I。
在一些实施方案中,该方法还包括使细胞与干扰素(IFN)接触。在一些实施方案中,该方法还包括使细胞与包含可操作地连接至启动子的编码TRIM16的序列的核酸接触,其中启动子序列能够在细胞中诱导所编码的TRIM16的表达。在一些实施方案中,所编码的TRIM16具有与SEQ ID NO:1所示的氨基酸序列至少90%同一性的氨基酸序列。
在一些实施方案中,TRIM16活性包括诱导程序性细胞死亡。
在一些实施方案中,细胞是癌细胞。在一些实施方案中,癌细胞是实体瘤癌细胞。在一些实施方案中,癌细胞是选自胰腺癌、膀胱癌、结肠直肠癌、乳腺癌、前列腺癌、肾癌、肝细胞癌、肺癌、卵巢癌、子宫颈癌、胃癌、食道癌、头颈癌、黑素瘤、神经内分泌癌、CNS癌、脑瘤、骨癌和软组织肉瘤的实体瘤癌细胞。在一些实施方案中,癌细胞是血液学癌细胞。
在一些实施方案中,细胞在体外与有效量的含有PAMP的核酸分子接触。在一些实施方案中,在哺乳动物受试者体内使细胞与有效量的含有PAMP的核酸分子接触。在一些实施方案中,该方法还包括向受试者施用用于癌症的免疫疗法。在一些实施方案中,免疫疗法包括癌症特异性抗体或其功能片段、免疫检查点抑制剂和过继性细胞疗法,包括CAR T细胞疗法。在一些实施方案中,受试者是人。
在另一方面,本公开提供了在有此需要的受试者中治疗癌症的方法。该方法包括向受试者施用治疗有效量的含有病原体相关分子模式(PAMP)的核酸分子。含有PAMP的核酸分子包含:包含末端三磷酸根的5'臂区域,包含至少8个连续尿嘧啶残基的聚尿嘧啶核心,和包含至少8个核酸残基的3'臂区域。3'臂区域的最5’端核酸残基不是尿嘧啶并且其中3'臂区域至少30%是尿嘧啶残基。
在一些实施方案中,聚尿嘧啶核心由8至30个尿嘧啶残基组成。在一些实施方案中,3'臂区域的最5’端核酸残基是胞嘧啶残基或鸟嘌呤残基。在一些实施方案中,3'臂区域至少40%,50%,60%,70%,80%或90%是尿嘧啶残基。在一些实施方案中,3'臂区域包含至少7个连续尿嘧啶残基。在一些实施方案中,5'臂区域还包含位于末端三磷酸根和聚尿嘧啶核心之间的一个或多个核酸残基。在一些实施方案中,5'臂区域由末端三磷酸根组成,并且其中末端三磷酸根直接连接至聚尿嘧啶核心的5'端。在一些实施方案中,末端三磷酸根、5'臂区域的一个或多个核酸残基和聚尿嘧啶核心在丙型肝炎病毒中不天然地一起出现。
在一些实施方案中,含有PAMP的核酸分子在受试者的癌细胞中诱导视黄酸诱导型基因I(RIG-I)样受体(RLR)与TRIM16的相互作用。在一些实施方案中,RLR与TRIM16的相互作用诱导癌细胞中的细胞死亡信号传导。在一些实施方案中,RLR是RIG I。
在一些实施方案中,该方法还包括向受试者施用有效量的干扰素(IFN)。在一些实施方案中,该方法还包括向受试者施用包含可操作地连接至启动子的编码TRIM16的序列的核酸,其中启动子序列能够在受试者内的癌细胞中诱导所编码的TRIM16的表达。
在一些实施方案中,癌症是选自胰腺癌、膀胱癌、结肠直肠癌、乳腺癌、前列腺癌、肾癌、肝细胞癌、肺癌、卵巢癌、子宫颈癌、胃癌、食道癌、头颈癌、黑素瘤、神经内分泌癌、CNS癌、脑瘤、骨癌和软组织肉瘤的实体瘤癌。在一些实施方案中,癌症是血液学癌症。
在一些实施方案中,该方法还包括向受试者施用用于癌症的免疫疗法。在一些实施方案中,免疫疗法包括癌症特异性抗体或其功能片段、免疫检查点抑制剂和过继性细胞疗法,包括CAR T细胞疗法。在一些实施方案中,受试者是人。
在另一方面,本公开提供了治疗组合物,其包含本文所述的含有病原体相关分子模式(PAMP)的核酸分子和干扰素(IFN)的组合。在一些实施方案中,IFN是1型IFN。在一些实施方案中,IFN是聚乙二醇化的IFN。在一些实施方案中,IFN是IFNλ。在一些实施方案中,组合物还包含可操作地连接至启动子的编码TRIM16的核酸。在一些实施方案中,将组合物配制在脂质体中。
在另一方面,本公开提供了治疗组合物,其包含本文所述的含有病原体相关分子模式(PAMP)的核酸分子和可操作地连接至启动子的编码TRIM16的核酸的组合。在一些实施方案中,该组合物还包含干扰素(IFN)。在一些实施方案中,所述IFN是1型IFN。在一些实施方案中,IFN是聚乙二醇化的IFN。在一些实施方案中,将组合物配制在脂质体中。
附图说明
当结合附图时,通过参考以下详细描述,本发明的前述方面和许多附带的优点将变得更加容易理解同时更好地理解,其中:
图1A-1D示例说明了PAMP RNA以剂量依赖的方式,但是在不同的剂量范围诱导先天性免疫信号传导和胱天蛋白酶活性。图1A是western印迹,显示了在所有剂量的HCV PAMPRNA在人肝细胞癌(Huh7)细胞中对先天性免疫信号传导的剂量依赖性激活,以及对细胞凋亡的剂量依赖性诱导。图1B以图形方式示例说明了细胞死亡分析,揭示了与胱天蛋白酶活性的激活相对应的对细胞死亡的诱导。条形图显示20小时后细胞死亡的百分比。图1C是western印迹,揭示了PAMP RNA诱导的对先天性免疫信号传导的激活和胱天蛋白酶激活的不同阈值。图1D以图形方式示例说明了细胞死亡分析,揭示了直至200ng阈值的细胞死亡诱导的剂量依赖性增加。条形图显示20小时后细胞死亡的百分比。
图2A-2C示例说明了HCV PAMP RNA对细胞凋亡的诱导依赖于RIG-I并通过经典的胱天蛋白酶依赖性通路被激活。图2A以图形方式示例说明了敲除RIG-I挽救了HCV PAMPRNA对细胞凋亡的诱导(图2A-1)。RIG-I依赖性胱天蛋白酶激活诱导了细胞凋亡并且用泛胱天蛋白酶抑制化合物zVAD的处理消除了细胞凋亡(图2A-2)。与模拟相比,*P<0.05和****P<0.0001。图2B是Western印迹,示例说明了用zVAD的处理挽救了HCV PAMP RNA诱导的细胞凋亡,但不改变其对先天性免疫应答的激活。图2C以图形方式示例说明了RIG-I的缺失消除了响应HCV PAMP RNA的p53依赖性促凋亡基因NOXA和PUMA的转录激活。
图3A-3C示例说明了I型IFN通路在RIG-I介导的对细胞凋亡的诱导中起作用。图3A以图形方式示例说明了敲低I型IFN受体链IFNAR1部分地消除了RIG-I介导的对细胞凋亡的诱导。图3B是western印迹,显示了在IFNAR和RIG-I KO细胞中先天性免疫应答的诱导均被削弱。图3C示例说明了用B18R(纯化的牛痘病毒编码的IFNAR信号传导的蛋白抑制剂)阻断I型IFN信号传导导致RIG-I介导的对细胞凋亡的诱导受损(左图)。Western印迹揭示了B18R对I型IFN信号传导的阻断(右)。与Huh7+PAMP相比,*P<0.05。
图4A-4E示例说明了TRIM16是RIG-I的新的相互作用物。图4A示意性地说明了在通过仙台病毒激活后鉴定结合RIG-I的蛋白质的工作流程。图4B显示了Western印迹,示例说明了RIG-I的N-末端对于结合TRIM16是必需的。图4C显示了Western印迹,示例说明了RIG-I的CARD结构域对于结合TRIM16是必需且足够的。图4D显示了Western印迹,示例说明了TRIM16的N-末端B-Box结构域对于结合RIG-I是必需且足够的。*表示非特异性条带。图4B是描绘RIG-I与TRIM16之间的相互作用的概述的卡通图。
图5A-5D示例说明了TRIM16不参与宿主先天性免疫应答。图5A显示了Western印迹,示例说明了敲除TRIM16对PAMP介导的先天性免疫应答没有影响。图5B是Western印迹,示例说明了相比之下,敲除TRIM25消除了PAMP介导的先天性免疫应答;左图显示了western印迹,其中对照细胞(EV)对PAMP应答,而TRM25 KO细胞对PAMP没有应答。图5B,右图显示了描绘TRIM25 KO细胞中PAMP信号传导丧失的图。图5C是western印迹,示例说明了TRIM16在由组成型活性N-RIG诱导的RIG-I介导的先天性免疫应答中没有作用。图5D以图形方式示例说明了TRIM16在仙台病毒诱导的先天性免疫应答中没有作用。
图6A和6B示例说明了TRIM16而不是TRIM25参与RIG-I介导的对细胞凋亡的诱导。图6A示例说明了敲除TRIM16损害了HCV PAMP RNA对细胞凋亡的诱导。与EV+PAMP相比,*P<0.05。相反,图6B示例说明了敲除TRIM25对细胞凋亡的诱导没有显著影响。
图7A和7B示例说明了PAMP RNA处理引起HepG2(图7A)和Huh7(图7B)肝细胞癌细胞的溶瘤破坏,但对原代肝细胞没有影响(图7B)。用指定量的PAMP RNA或xRNA(阴性对照)处理细胞,并监测标记可渗透/死亡中或死的细胞的Sytox绿色染料的摄取。将数据绘制为20小时时间过程中死细胞的数量。PAMP处理特异性诱导肿瘤细胞的溶瘤破坏(HepG2和Huh7细胞是人的癌衍生的细胞),而不诱导正常的非癌细胞(原代人肝细胞)。
发明详述
本公开基于研究揭示了诱导RIG-I样受体(RLR)信号传导不仅可以导致触发先天性免疫应答,而且可以选择性地诱导癌细胞中的细胞凋亡。这项工作提出了对抗癌症的新治疗策略。
先天性免疫力是身体抵抗感染的第一道防线。先天性免疫应答对于编程有效的适应性免疫和免疫记忆以产生对消除致病性病原体关键性的强而持久的免疫应答也是必要的。针对病毒感染的先天性免疫应答的关键组分是干扰素调节因子(IRF)3的激活和1型干扰素(IFN)的诱导。IRF3诱导抗病毒基因的表达并且也诱导IFN。抗病毒基因可以抑制被感染细胞中的病毒复制,而IFN则通过表达数百种具有抗病毒和免疫调节活性的干扰素刺激基因(ISG)来指导对被感染细胞和邻近旁观者细胞中的病毒复制的抑制。除IFN抑制病毒感染外,病毒感染细胞的程序性死亡还可以防止病毒传播。因此,IRF3作用、IFN作用和细胞死亡信号传导的组合通过尚未确定的机制赋予了病毒控制的协同程序。本公开基于确定IFN与细胞死亡信号传导之间相互作用的研究。这些努力已经确定可以利用IFN与细胞死亡应答之间的联系用于控制肿瘤生长的治疗策略。
对IRF激活和IFN的诱导在病毒感染开始时通过细胞模式识别受体(PRR)识别病原体相关分子模式(PAMP)而被触发,PPR包括Toll样受体(TLR)、RIG-I样受体(RLR)、Nod样受体(NLR)或环状GMP-AMP合酶(cGAS)/干扰素基因刺激物(STING)。PRR检测病毒PAMP或PAMP产物以触发细胞内信号传导级联,最终通过干扰素调节因子(IRF)3/7和核因子Kappa B(NF-κB)激活转录机制以指导先天性免疫激活。特别是,RNA病毒在很大程度上是通过RLR的作用来感知的,RLR是识别并结合PAMP RNA基序的胞质RNA解旋酶。RIG-I是RLR家族的创始成员,该家族还包括MDA5和LGP2。RIG-I识别包含5'三磷酸根(5'ppp)和短dsRNA结构基序和/或聚尿苷基序(这将RNA分子标记为非己方)的RNA。RIG-I蛋白结构具有位于中心的DExH盒解旋酶结构域、位于N末端的两个胱天蛋白酶募集结构域(CARD)、和C末端阻遏子结构域,该结构域调节信号传导,通过将分子保持在封闭的信号传导关闭状态直至配体结合导致信号传导开启状态的其构象改变,从而驱动抗病毒防御。PAMP基序先前已针对RIG-I识别和结合在丙型肝炎病毒(HCV)RNA中进行了表征,其以聚尿苷和单链RNA结构为特征。当配制用于细胞递送时,包含5'ppp的该基序(称为PAMP-RNA)的合成修饰是有力且特异性的RIG-I激动剂,其被RIG-I结合而诱导RIG-I信号传导。PAMP RNA指导RIG-I介导的先天性免疫作用,提供抗病毒和免疫增强活性来控制病毒感染。
一旦识别并结合PAMP-RNA,RIG-I发生促进其与信号传导辅因子的相互作用的构象变化,这使RIG-I结合线粒体抗病毒信号传导(MAVS)蛋白,从而下游激活潜在的转录因子包括IRF3、IRF7和NF-κB转录因子,导致它们易位至细胞核并诱导转录程序。RIG-I信号传导通路受多种RIG-I或MAVS相互作用伙伴,包括TRAF3、TRAF2、TRAF6、TANK、SIKE、NEMO/IKKγ、FADD、RIP1、HDAC6、TRADD、胱天蛋白酶8/10和STING/MITA/MPYS,以及负信号传导调节剂,例如RNF125、ISG15、DAK、Atg12-Atg5、NLRX1、胱天蛋白酶-1,以及几种去泛素化酶、乙酰化酶、蛋白激酶和蛋白磷酸酶的调控。研究还表明,RIG-I信号传导也通过RIG-I泛素化或与游离泛素的相互作用以及通过可逆的磷酸化和乙酰化而受控制。
蛋白质的三重基序(TRIM)家族由具有E3泛素连接酶活性和多种生物学功能的超过70种家族成员组成,所述生物学功能包括在抗病毒免疫、自身免疫和癌症中的作用。TRIM家族成员具有共享的保守序列,包括识别E2缀合酶的基因(RING)结构域、在一些成员中至少一个介导自缔合的B-Box结构域和对于TRIM二聚化必不可少的卷曲螺旋(CC)结构域。成员具有可变的C末端结构域,其可以介导特定的底物相互作用。已知许多TRIM家族成员调节TLR和RLR信号传导通路。当暴露的RIG-I CARD结构域被TRIM25的C末端SPRY结构域结合时,家族成员TRIM25通过RIG-I的K63多泛素化调节RIG-I信号传导。RIG-I的多聚泛素化促进了其与MAVS的相互作用以及随后的下游信号传导,因此将TRIM25指定为RIG-I先天性免疫激活的关键辅因子。RIG-I与其他TRIM家族成员的相互作用尚未确定。
先前的研究表明RIG-I激活可以通过多种机制诱导细胞死亡。RLR信号传导可以通过诱导Puma和Noxa(Bcl-2家族成员,是必需的细胞凋亡调节剂)来触发程序性细胞死亡。另一系列研究描述了RLR诱导的IRF3介导的细胞凋亡通路(RIPA),其需要通过RLR信号传导通路激活IRF3,IRF3结合促凋亡蛋白BAX并诱导其易位至线粒体并触发细胞色素C的释放和随后的胱天蛋白酶激活。有趣的是,在通过TRAF2和TRAF6依赖性机制募集到泛素化复合物LUBAC之后,RIPA独立于IRF3转录活性并依赖于IRF3泛素化。RIG-I激活还可以触发炎症小体诱导的细胞焦亡,其通过RIG-I CARD结构域与炎症小体组分相互作用来激活胱天蛋白酶-1并释放促炎性细胞因子IL-1β和IL-18。因此,尽管很明显RIG-I的PAMP识别可以触发程序性细胞死亡,但是目前尚不清楚确定RIG-I激活导致先天性免疫信号传导还是细胞死亡的机制。
如下文更详细描述,该工作在RIG-I细胞死亡信号传导的系统研究中探讨了PAMP-RNA诱导细胞死亡的能力,以确定负责RIG-I诱导的细胞死亡的分子决定因素。PAMP-RNA剂量滴定研究确定了触发RIG-I诱导的细胞死亡对比先天性免疫激活的阈值剂量。RNA-PAMP通过RIG-I和胱天蛋白酶依赖性信号传导(通过IFN处理增强)诱导细胞死亡。使用基于蛋白质组学的筛选,鉴定了新的RIG-I结合伙伴TRIM16,它对PAMP-RNA和RIG-I诱导的细胞死亡是必要的。另外,显示TRIM16与RIG-I CARD结合以指导响应PAMP-RNA的细胞死亡信号传导。因此,证明了TRIM16是RIG-I信号传导的辅因子,其确定了新的PAMP敏感的细胞死亡通路。利用这点证明RLR信号传导是特异性地诱导癌细胞中的细胞死亡的新治疗靶标。
根据前述内容,在一方面,本公开提供了诱导细胞中三重基序包含蛋白16(TRIM16)活性的方法。在一些实施方案中,通过使细胞与有效量的视黄酸诱导型基因I(RIG-I)样受体激活剂接触来诱导TRIM16活性。在一些实施方案中,RLR激活剂是含有病原体相关分子模式(PAMP)的核酸分子。因此,在这样的实施方案中,该方法包括使细胞与有效量的含有PAMP的核酸分子接触。含有PAMP的核酸分子在本文中通常也可以称为核酸PAMP。如本文所使用的,PAMP是病原体相关的分子模式,即,核酸分子的结构/序列中的模式,其被RLR识别,从而导致细胞中RLR的激活。在一些实施方案中,RLR是RIG-I。本公开涵盖的示例性PAMP和含有PAMP的核酸分子在美国公开号2015/0017207和2018/0104325中公开,其涉及先天性免疫应答信号传导的PAMP诱导,并且通过引用将其全部并入本文。本文涉及本公开涵盖的含PAMP的核酸的元件和示例性实施方案。
首先,本文所用的术语“核酸”是指单体单元或“残基”的聚合物。核酸的单体亚基或残基各自包含氮碱基(即核碱基)、五碳糖和磷酸基团。每个残基的身份通常在本文中参考每个残基的核碱基(或含氮碱基)结构的身份来指示。典型的核碱基包括腺嘌呤(A)、鸟嘌呤(G)、胸腺嘧啶(T)、尿嘧啶(U)(在RNA中代替胸腺嘧啶(T)残基)和胞嘧啶(C)。然而,如本领域所公知的,本公开的核酸可以包括任何修饰的核碱基、核碱基类似物和/或非典型核碱基。核酸单体或残基的修饰涵盖导致非典型亚基结构的核酸单体或残基的结构中的任何化学变化。这样的化学变化可以由例如表观遗传修饰(例如对基因组DNA或RNA)或者辐射、化学或其他手段导致的损伤引起。可以由修饰产生的非典型亚基的示例说明性和非限制性实例包括尿嘧啶(对于DNA)、5-甲基胞嘧啶、5-羟甲基胞嘧啶、5-formethyl胞嘧啶、5-羧基胞嘧啶b-葡萄糖基-5-羟基-甲基胞嘧啶、8-氧鸟嘌呤、2-氨基-腺苷、2-氨基-脱氧腺苷、2-硫代胸苷、吡咯并嘧啶、2-硫代胞苷或无碱基病变。无碱基病变是沿着脱氧核糖主链的位置,但是缺乏碱基。天然核苷酸的已知类似物以类似于天然存在的核苷酸的方式与核酸杂交,例如肽核酸(PNA)和硫代磷酸DNA。
附着有核碱基的五碳糖可以根据核酸的类型而变化。例如,糖在DNA中是脱氧核糖,而在RNA中是核糖。在本文的某些情况下,核酸残基也可就核苷结构来提述,例如腺苷、鸟苷、5-甲基尿苷、尿苷和胞苷。此外,核苷的替代命名法还包括在核碱基前指示“核糖”或“脱氧核糖”前缀以指示五碳糖的类型。例如,本文中偶尔使用的“核糖胞嘧啶”等同于胞苷残基,因为它指示RNA分子在该残基处存在核糖。核酸聚合物可以是或包含脱氧核糖核苷酸(DNA)聚合物,核糖核苷酸(RNA)聚合物(包括mRNA)。核酸也可以是或包含PNA聚合物,或者本文所述的任何聚合物类型的组合(例如,包含具有不同糖的残基)。
在一些实施方案中,含有PAMP的核酸是合成的。在本文中,术语“合成的”是指核酸的非天然特征。可以使用标准合成技术从头合成此类核酸。备选地,可以使用重组技术从天然存在的病原体序列生成或衍生核酸PAMP,这些重组技术在本领域中是众所周知的。在一些实施方案中,合成的核酸PAMP构建体的序列不是天然存在的。
在一些实施方案中,含有PAMP的核酸是RNA构建体。在这些实施方案的一些中,含有PAMP的核酸衍生自或反映HCV聚U/UC区域的序列,并且在这种情况下,可以通常指聚U/UCPAMP RNA构建体。在一些实施方案中,聚U/UC PAMP RNA构建体是合成的。
本公开的含有PAMP的核酸通常包含(a)包含末端三磷酸根的5'臂区域;(b)聚尿嘧啶核心(也称为聚U核心);和(c)3'臂区域。在一个实施方案中,三个区域(a、b和c)在单个核酸聚合物大分子中共价连接。共价键可以是直接的(没有夹杂的一个或多个接头序列)或间接的(有夹杂的一个或多个接头序列)。在一个实施方案中,5'臂区域共价连接至聚U核心的5'端。在一个实施方案中,3'臂区域共价连接至聚U核心的3'臂区域。聚合物可以是单链或双链的,或者可以以单链和双链部分的组合出现。
在一个实施方案中,聚U核心包含至少8个连续尿嘧啶残基。在进一步的实施方案中,包含8至30个连续尿嘧啶残基,例如8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续尿嘧啶残基。在一实施方案中,聚U核心包含超过8个连续尿嘧啶残基。在一个实施方案中,聚U核心包含12个或更多个连续尿嘧啶残基。在一些实施方案中,聚U核心由多个连续尿嘧啶残基组成,例如8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个连续尿嘧啶残基。
在一个实施方案中,3'臂区域包含不是尿嘧啶残基的最5’端核酸残基。相反,3'臂区域的最5’端核酸残基可以是腺嘌呤、鸟嘌呤或胞嘧啶残基,或任何非典型残基。在一个实施方案中,3'臂区域的最5’端核酸残基是胞嘧啶残基或鸟嘌呤残基。
在一个实施方案中,3'臂区域的核苷酸组成是至少约40%为尿嘧啶残基。在一些实施方案中,3'臂区域是至少约45%,是至少约50%,是至少约60%,是至少约70%,是至少约80%或是至少约90%为尿嘧啶残基。在一个实施方案中,3'臂区域包含多个短区段(例如,长度在约2至约15个核苷酸)的连续尿嘧啶残基,其间夹杂有一个或多个胞嘧啶残基。在一个实施方案中,3'臂区域包含一段连续尿嘧啶残基,其不超过合成的含有PAMP的核酸分子的聚U核心的长度。在一个实施方案中,3'臂区域不包含一段连续尿嘧啶残基,其长度超过合成的含有PAMP的核酸分子的聚U核心的长度。在一些实施方案中,3'臂区域包含至少7个连续尿嘧啶残基,例如7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28或29个连续尿嘧啶残基。
最少,5'臂区域由末端三磷酸根(ppp)部分组成。在这样的实施方案中,三磷酸根在合成的含有PAMP的核酸分子的5'末端并且可以表示为“5'-ppp”。在进一步的一个实施方案中,末端三磷酸根直接连接至聚U核心序列的5'端。在备选的实施方案中,5'臂区域包含5'端末端三磷酸根和一个或多个另外的核酸残基,其序列终止于3'端。该实施方案的5'臂区域中的一个或多个另外的核酸残基位于末端三磷酸根与聚U核心的最5’端尿嘧啶残基之间。本领域普通技术人员将容易理解在5'臂区域中的一个或多个另外的核酸残基可以是任何数量的核酸残基并且可以呈现任何序列而没有限制。5'臂区域中的一个或多个另外的核酸残基的序列不影响含有PAMP的核酸分子的功能。例如,如美国公开号2015/0017207和2018/0104325中所述,添加聚U核心区域到包含5'-三磷酸根的非刺激性核酸(例如HCV X区域)赋予了对先天性免疫系统信号传导的刺激性特性。在一个实施方案中,在5'臂区域中的一个或多个另外的核酸残基的序列不由天然存在的HCV基因组序列的天然存在于该HCV株的聚U/UC区域的“上游”或5'端的整个5'端部分组成。换句话说,在该实施方案中,整个合成的含有PAMP的核酸分子不是天然存在的HCV基因组,完整具有5'三磷酸根、整个编码区和非翻译的3’聚U/UC区。因此,在该实施方案中,5'臂区域、5'臂区域的一个或多个核酸残基和聚尿嘧啶核心不在HCV基因组中天然地一起出现。然而,在该实施方案中,5'臂区域的一个或多个核酸残基可以包含存在于5'臂区域和聚尿嘧啶核心之间的整个天然存在的序列的亚片段或由其组成。备选地,在该实施方案中,5'臂区域的一个或多个核酸残基可以包含除了存在于5'末端与聚尿嘧啶核心之间的一部分或整个天然存在的HCV基因组序列之外的序列。
在一些实施方案中,含有PAMP的核酸分子还包含编码功能性基因产物,例如多肽或干扰RNA构建体的另外的核酸结构域。所述另外的核酸结构域可以是3'臂结构域的部分,作为其中一个或多个另外的核酸残基的全部或部分。在另一个实施方案中,所述另外的核酸结构域可以位于包含末端三磷酸根的5'臂区域、聚尿嘧啶核心和3'臂区域中的任何之间。
在美国专利申请公开号2015/0017207和2018/0104325(通过引用并入本文)中披露了所公开的含有PAMP的核酸所涵盖的PAMP的核酸序列的非限制性实例,并在本文中以SEQ ID NO:2-92列出。公开的含有PAMP的核酸可以包含本文列出的任何序列。应当理解,这种示例性的含有PAMP的核酸将符合本文所述的含有PAMP的分子的一般结构参数,包括具有末端三磷酸根(ppp)部分。在一个实施方案中,含有PAMP的分子包含序列:GGCCAUCCUGUUUUUUUCCCUUUUUUUUUUUCUCCUUUUUUUUUCCUCUUUUUUUCCUUUUCUUUCCUUU(SEQ ID NO:)。在另一个实施方案中,含有PAMP的核酸包含序列:GGCCAUUUUCUUUUUUUUUUCUCUUUUUUUUUUUUUUUUUUAUUUUCUUUAAU(SEQ ID NO:)。再次,应当理解具有所示序列的示例性含有PAMP的核酸也将具有上述特征,包括5'末端三磷酸根(ppp)部分。
如美国公布号2015/0017207和2018/0104325中所述,具有HCV衍生的RNA PAMP(具有聚尿嘧啶核心序列)的核酸可以触发视黄酸诱导型基因I(RIG-I)样受体(RLR)信号传导。因此,在一些实施方案中,含有PAMP的核酸分子能够诱导视黄酸诱导型基因I(RIG-I)样受体(RLR)激活。在一个实施方案中,RLR是RIG-I。本领域普通技术人员将能够容易地确定RLR的激活,例如通过测定已知的下游RLR调节的基因的转录,如下文更详细地描述。例如,在一些实施方案中,可以通过IFN-β或ISG54表达的增加来建立RLR激活。在另一个实施方案中,可以通过IRF-3磷酸化的增加来建立RLR激活。
如以下更详细描述的,蛋白质组学分析将TRIM16鉴定为特异性导致细胞死亡信号传导的关键RLR结合伙伴。因此,PAMP诱导的RLR(例如,RIG-I)信号传导包括RLR-TRIM16相互作用,其可以导致程序性细胞死亡。因此,该方法涵盖其中含有PAMP的核酸分子在细胞中诱导视黄酸诱导型基因I(RIG-I)样受体(RLR)与TRIM16的相互作用的实施方案。示例性TRIM16具有SEQ ID NO:1所示的氨基酸序列,或与其具有至少约80%、约85%、约90%、约95%、约98%或约99%序列同一性的功能性变体。功能性变体指示尽管蛋白质中有任何序列变异,但该蛋白质保留了TRIM16的活性。例如,TRIM16(也称为EBBP)先前已被表征鉴定为雌激素应答性基因,参与许多生物学过程,包括在几种癌症中抑制肿瘤进展。本文公开的研究证明RLR(例如RIG-I)/TRIM16相互作用诱导程序性细胞死亡信号传导。因此,被诱导的TRIM16活性可以最终导致程序性细胞死亡。在一些实施方案中,该方法包括诱导细胞中的程序性细胞死亡。在一些实施方案中,使含有PAMP的核酸分子以约80ng/mL或更高的浓度与细胞接触以产生对细胞中程序性细胞死亡的诱导。
因此,在一些实施方案中,该方法包括使细胞与浓度为至少约80ng/mL至约500ng/mL,例如100ng/mL至250ng/mL的含有PAMP的核酸分子接触。在一些实施方案中,该方法包括使细胞与浓度为至少约80ng/mL、约90ng/mL、约100ng/mL、约110ng/mL、约120ng/mL、约130ng/mL、约140ng/mL、约150ng/mL、约160ng/mL、约170ng/mL、约180ng/mL、约190ng/mL、约200ng/mL、约210ng/mL、约220ng/mL、约230ng/mL、约240ng/mL、约250ng/mL、约260ng/mL、约270ng/mL、约280ng/mL、约290ng/mL、约300ng/mL、约310ng/mL、约320ng/mL、约330ng/mL、约340ng/mL、约350ng/mL、约360ng/mL、约370ng/mL、约380ng/mL、约390ng/mL、约400ng/mL、约410ng/mL、约420ng/mL、约430ng/mL/mL、约440ng/mL、约450ng/mL、约460ng/mL、约470ng/mL、约480ng/mL、约490ng/mL和约500ng/mL的含有PAMP的核酸分子接触。
在一些实施方案中,该方法还包括使细胞与干扰素(IFN)接触。在一些实施方案中,IFN是1型IFN。在一些实施方案中,IFN是聚乙二醇化的IFN。在一些实施方案中,IFN是IFNλ。
在一些实施方案中,使细胞与约5至约1500个单位IFN每ml,例如约100至约1000个单位IFN每ml,例如约100至约500个单位IFN每ml,例如约200至约1000个单位IFN每ml,例如约300至约700个单位IFN每ml,和例如约500至约1000个单位IFN每ml接触。IFN的示例性剂量包括约100个单位IFN每ml,约200个单位IFN每ml,约300个单位IFN每ml,约400个单位IFN每ml,约500个单位IFN每ml,约600个单位IFN每ml,约700个单位IFN每ml,约800个单位IFN每ml,约900个单位IFN每ml,约1000个单位IFN每ml,约1200个单位IFN每ml和约1500个单位IFN每ml。本文使用的术语“单位”是指国际单位。
通过还向细胞提供编码TRIM16的核酸可以促进或进一步增强TRIM16活性。如上所述,示例性TRIM16蛋白序列是SEQ ID NO:1所示或与其具有至少约80%、约85%、约90%、约95%、约98%或约99%序列同一性的功能性变体。因此,所述核酸可以是编码如SEQ ID NO:1所示的氨基酸序列或与其具有至少约80%、约85%、约90%、约95%、约98%或约99%序列同一性的功能性变体的任何核酸。核酸可以可操作地连接至启动子序列,其中启动子序列能够诱导所编码的TRIM16在细胞中的表达。术语“启动子”是指能够激活基因和/或其剪接变体同等型的转录(表达)的调节性核苷酸序列。启动子通常位于基因的上游,但是可以位于基因附近的其他区域,甚至是基因内。启动子通常包含RNA聚合酶和一个或多个转录因子的结合位点,其参与转录复合物的组装。如本文所用,术语“可操作地连接”是指启动子和编码核酸以使得启动子可以通过细胞的转录机制激活编码核酸的转录的方式相对于彼此配置和定位。启动子可以是组成型或诱导型的。组成型启动子可以基于靶细胞的特征和胞浆中可用的具体转录因子来确定。本领域普通技术人员可以根据预期的人选择合适的启动子,因为各种启动子是本领域已知和常用的。
细胞可以是需要TRIM16活性的任何细胞。在一些实施方案中,细胞是癌细胞。癌细胞可以来自实体瘤或血液学癌症。
代表性实体瘤是胰腺癌、膀胱癌、结肠直肠癌、乳腺癌、前列腺癌、肾(肾脏)癌、肝细胞癌、肺癌、卵巢癌、宫颈癌、胃癌、食道癌、头颈癌、黑素瘤、神经内分泌癌、CNS癌、脑瘤、骨癌、软组织肉瘤。代表性血液学癌症包括白血病、淋巴瘤和骨髓瘤。
在一些实施方案中,可以执行方法以使得细胞在体外与有效量的含有PAMP的核酸分子接触。通常,将一种或多种细胞保持在合适的培养基中。将含有PAMP的核酸分子的量以适合于细胞连续培养的运载体中的所需浓度施用给培养基中的细胞。
在其他实施方案中,可以在体内执行方法,例如在患有将得益于对TRIM16活性的诱导的疾病或病况的人或非人哺乳动物受试者(例如另一灵长类动物、马、狗、猫、小鼠、大鼠、豚鼠、兔子等)中。考虑到经由RLR(例如,RIG-I)的足够的PAMP信号传导对TRIM16的激活导致细胞凋亡,该方法有利地在受试者中的疾病或病况是癌症(例如实体瘤癌症或血液学癌症)的情况下执行。本公开涵盖的示例性癌症包括上述那些,包括胰腺癌、膀胱癌、结肠直肠癌、乳腺癌、前列腺癌、肾(肾脏)癌、肝细胞癌、肺癌、卵巢癌、子宫颈癌、胃癌、食道癌、头颈癌、黑素瘤、神经内分泌癌、CNS癌、脑肿瘤、骨癌、软组织肉瘤和骨髓瘤。在这些体内实施方案中,以适合于特定疾病(例如癌症)的方式将有效量的含有PAMP的核酸施用给受试者。在一些实施方案中,该方法是组合疗法的一个方面并因此还包括向受试者施用免疫疗法,例如癌症特异性抗体或其功能片段、免疫检查点抑制剂和过继性细胞疗法,包括CAR T细胞疗法。
因此,在另一方面,本公开提供了在有此需要的受试者中治疗癌症的方法。该方法包括向受试者施用治疗有效量的含有病原体相关分子模式(PAMP)的核酸分子。
如本文所用,术语“治疗”是指受试者(例如人或非人哺乳动物,例如另一灵长类动物、马、狗、小鼠、大鼠、豚鼠、兔子等)的疾病、病症或病况(例如如上所述的癌症)的医学管理。治疗可以涵盖在治疗或缓解疾病或病况(例如癌症)中成功的任何标志,包括任何参数,例如症状的减少、减轻、削弱或使疾病或病况对受试者更宽容,减慢退行或衰退的速度,或使退行更加减弱。特别地,在癌症的情况下,术语治疗可以涵盖与不进行治疗相比,减慢或抑制癌症生长的速度,或降低复发的可能性。在一些实施方案中,治疗涵盖导致患者中一些可检测程度的癌细胞死亡。症状的治疗或缓解可以基于客观或主观参数,包括医师检查的结果。因此,术语“治疗”包括施用本公开的组合物以减轻或者阻滞或抑制与疾病或病况(例如,癌症)相关的症状或病况的发展。术语“治疗效果”是指受试者中疾病或病况、疾病或病况的症状或者疾病或病况的副作用的缓解、减少或消除。术语“治疗有效的”是指产生治疗效果的组合物的量,该量可以容易地确定。
在一些实施方案中,含有PAMP的核酸分子包含包括末端三磷酸根的5'臂区域,包含至少8个连续尿嘧啶残基的聚尿嘧啶核心,和包含至少8个核酸残基的3'臂区域,其中3'臂区域的最5’端核酸残基不是尿嘧啶并且其中3'臂区域至少30%是尿嘧啶残基。为了简洁起见,不再重复在上文中提供的对含有PAMP的核酸分子的更详细描述。如所描述的,含有PAMP的分子具有在受试者的癌细胞中诱导视黄酸诱导型基因I(RIG-I)样受体(RLR)与TRIM16的相互作用的功能。在一些实施方案中,RLR是RIG-I。RLR与TRIM16的相互作用导致在受试者的癌细胞中对细胞死亡信号传导的诱导。
已经证明干扰素(IFN)可以与程序性细胞死亡信号传导协同相互作用,从而导致对细胞甚至肿瘤的增强破坏。实际上,以下描述的研究确定了PAMP诱导的细胞死亡(通过刺激RLR/TRIM16相互作用)诱导了胱天蛋白酶依赖性细胞死亡,其通过添加IFN处理而得到进一步增强。因此,在一些实施方案中,治疗的方法还包括向受试者施用对受试者有效量的干扰素(IFN)。在一些实施方案中,IFN是1型IFN。在一些实施方案中,IFN是聚乙二醇化的IFN。在一些实施方案中,IFN是IFNλ。
IFN可以以本领域普通技术人员确定的任何治疗有效量给药。示例性剂量范围包括约5至约1500个单位IFN每ml,例如约100至约1000个单位IFN每ml、例如约100至约500个单位IFN每ml、例如约200至约1000个单位IFN每ml、例如约300至约700个单位IFN每ml和例如约500至约1000个单位IFN每ml。IFN的示例性剂量包括约100个单位IFN每ml、约150个单位IFN每ml、约200个单位IFN每ml、约250个单位IFN每ml、约300个单位IFN每ml、约350个单位IFN每ml、约400个单位IFN每ml、约450个单位IFN每ml、约500个单位IFN每ml、约550个单位IFN每ml、约600个单位IFN每ml、约650个单位IFN每ml、约700个单位IFN每ml、约750个单位IFN每ml、约800个单位IFN每ml、约850个单位IFN每ml、约900个单位IFN每ml、约950个单位IFN每ml、约1000个单位IFN每ml、约1200个单位IFN每ml和约1500个单位IFN每ml。本文使用的术语“单位”是指国际单位。
在一些实施方案中,该方法还包括向受试者施用包含可操作地连接至启动子序列的编码TRIM16的序列的核酸。如上所述,该核酸可以编码包含SEQ ID NO:1所示的氨基酸序列的TRIM16蛋白,或者与其具有至少约80%、约85%、约90%、约95%、约98%或约99%的序列同一性的功能性变体。可以选择启动子,以使其可操作于诱导癌细胞中TRIM的表达。根据本领域的常规知识,可以将这样的启动子配置为在预期的靶癌细胞类型中诱导型或组成型表达。可以将核酸(和启动子)掺入合适的载体中以促进编码核酸在癌细胞中的递送和表达。载体可以是病毒载体、环状核酸构建体(例如质粒)或纳米颗粒。多种病毒载体是本领域已知的并且被本公开所涵盖。参见,例如,Machida,C.A.(ed.),Viral Vectors for GeneTherapy:Methods and Protocols,Humana Press,Totowa,New Jersey(2003);Muzyczka,N.,(ed.),Current Topics in Microbiology and Immunology:Viral ExpressionVectors,Springer-Verlag,Berlin,Germany(2012),各自通过引用整体并入本文。在一些实施方案中,病毒载体是腺相关病毒(AAV)载体、腺病毒载体、逆转录病毒载体或慢病毒载体。AAV载体的一个具体实施方案包括AAV2.5血清型。
该方法可用于治疗任何形式的癌症(例如实体瘤或血液学癌症),只要癌细胞保持有必需的TRIM16信号传导通路即可。上文描述了该方面所涵盖的示例性癌症,并且包括但不限于胰腺癌、膀胱癌、结肠直肠癌、乳腺癌、前列腺癌、肾(肾脏)癌、肝细胞癌、肺癌、卵巢癌、宫颈癌、胃癌、食道癌、头颈癌、黑素瘤、神经内分泌癌、CNS癌、脑肿瘤、骨癌、软组织肉瘤和骨髓瘤。
考虑到含有PAMP的核酸的功能作用是诱导细胞中的程序性细胞死亡信号传导,该方面的方法特别适用于与其他抗癌疗法的组合。因此,在一些实施方案中,该方法还包括向受试者施用抗癌治疗剂。该抗癌治疗剂可以是对受试者的癌细胞具有可证明的治疗作用的任何毒素、小分子、大分子治疗剂。在一些实施方案中,该方法还包括向受试者施用用于癌症的免疫疗法,例如癌症特异性抗体或其功能片段、免疫检查点抑制剂和过继性细胞疗法,包括CAR T细胞疗法。所公开的含有PAMP的核酸可以与另外的抗癌治疗剂(包括IFN和/或TRIM16编码核酸)同时或与之分开施用。
在另一方面,本公开提供了治疗组合物,该组合物包含含有病原体相关分子模式(PAMP)的核酸分子和另外的治疗剂。在一些实施方案中,含有PAMP的核酸分子包含包括末端三磷酸根的5'臂区域,包含至少8个连续尿嘧啶残基的聚尿嘧啶核心,和包含至少8个核酸残基的3'臂区域,其中3'臂区域的最5’端核酸残基不是尿嘧啶并且其中3'臂区域至少30%是尿嘧啶残基。为了简洁起见,不再重复以上提供的对含有PAMP的核酸分子的更详细描述。如所描述的,含有PAMP的分子具有在受试者的癌细胞中诱导视黄酸诱导型基因I(RIG-I)样受体(RLR)与TRIM16的相互作用的功能。在一些实施方案中,RLR是RIG-I。RLR与TRIM16的相互作用产生在受试者的癌细胞中对细胞死亡信号传导的诱导。
在一个实施方案中,另外的治疗剂可以是干扰素(IFN),如上文更详细描述的。在一些实施方案中,IFN是1型IFN。在一些实施方案中,IFN是聚乙二醇化的IFN。在一些实施方案中,IFN是IFNλ。在一个实施方案中,另外的治疗剂是编码包含可操作地连接至启动子序列的TRIM16编码序列的核酸。如上所述,核酸可以编码包含SEQ ID NO:1所示的氨基酸序列的TRIM16蛋白,或者与其具有至少约80%、约85%、约90%、约95%、约98%或约99%序列同一性的功能性变体。可以选择启动子,以使其可操作以诱导癌细胞中TRIM的表达。在另一个实施方案中,治疗组合物包含含有PAMP的核酸、IFN和可操作地连接至启动子序列的编码TRIM16的核酸。
本公开涵盖治疗组合物的制剂,其以适合于在受试者(例如患有癌症的哺乳动物受试者)的体内治疗环境中应用的施用方法的方式配置。施用可以通过本领域已知的任何方法进行,包括但不限于口服、肠胃外、脊柱内、脑池内、硬膜下、直肠、真皮内、透皮、肌内或局部施用。在具体的实施方案中,施用是肿瘤内。为了促进递送,可以将治疗性组合物与药学上可接受的运载体、赋形剂或稀释剂配制成多种组合物。“药学上可接受的”表示选择的运载体、赋形剂或稀释剂不会不利地影响含有PAMP的核酸分子和任何另外的治疗剂或组合物的接受者的生物学活性。
根据本领域普通技术和知识,公开的含有PAMP的核酸和另外的治疗剂,包括IFN,编码核酸和/或包含核酸的载体,毒素,免疫治疗化合物等可以以任何适当的治疗递送系统或形式配制。示例性的非限制性递送系统可以包括颗粒制剂,例如乳剂、微粒和纳米颗粒,其可以是例如颗粒和/或基质,以及脂质体等,这对于抗原的递送是有利的。在一个实施方案中,将公开的含有PAMP的核酸和另外的治疗剂配制成脂质体递送形式。脂质体制剂的示例性应用描述于Yallapu,U.等人,Liposomal Formulations in Clinical Use:AnUpdated Review,Pharmaceutics 9(2):12(2017),通过引用将其全部并入本文。
一般定义
除非本文具体定义,否则本文使用的所有术语具有对于本公开技术领域的技术人员相同的含义。对于本领域的定义和术语,从业者尤其参见Ausubel,F.M.,等人(eds.),Current Protocols in Molecular Biology,John Wiley&Sons,New York(2010),Coligan,J.E.,等人(eds.),Current Protocols in Immunology,John Wiley&Sons,NewYork(2010),Mirzaei,H.和Carrasco,M.(eds.),Modern Proteomics–SamplePreparation,Analysis and Practical Applications in Advances in ExperimentalMedicine and Biology,Springer International Publishing,2016,以及Comai,L,等人(eds.),Proteomic:Methods and Protocols in Methods in Molecular Biology,Springer International Publishing,2017。
为了方便起见,在此提供在说明书、实例和所附权利要求中本文所采用的某些术语。提供这些定义以帮助描述特定的实施方案并且无意于限制所要求保护的发明,因为本发明的范围仅由权利要求书限制。
除非明确指出仅指代替代方案或替代方案是互斥的,否则在权利要求中使用术语“或”来表示“和/或”,尽管本公开支持仅指代替代方案和“和/或”的定义。
除非明确指出,否则在与权利要求书或说明书中的词语“包括”结合使用时,词语“一”和“一个”表示一个或多个。
除非上下文另有明确要求,否则在整个说明书和权利要求书中,词语“包括”、“包含”等应理解为包括性含义,而不是排他性或穷举性含义,其表示以“包括但不限于”的意义。使用单数或复数的词语也分别包括复数和单数。词语“约”指示在所述参考数字之上或之下的微小变化范围内的数字。例如,“约”可以指高于和/或低于所指示的参考数字的10%、9%、8%、7%、6%、5%、4%、3%、2%或1%的范围内的数字。
如本文所用,术语“多肽”或“蛋白质”是指聚合物,其中单体是通过酰胺键连接在一起的氨基酸残基。当氨基酸是α-氨基酸时,可以使用L-旋光异构体或D-旋光异构体,L-异构体是优选的。本文所用的术语多肽或蛋白质涵盖任何氨基酸序列,并且包括修饰的序列,例如糖蛋白。术语多肽专门用于涵盖天然存在的蛋白质以及重组或合成产生的蛋白质。
技术人员将认识到,在序列中改变、添加或删除单个氨基酸或一定百分比的氨基酸的对肽、多肽或蛋白质序列的单个取代、缺失或添加是“保守修饰的变体”,其中改变导致氨基酸被化学上相似的氨基酸取代。提供功能上相似的氨基酸的保守氨基酸取表是本领域普通技术人员众所周知的。以下六组是被认为是彼此的保守替代的氨基酸的实例:
(1)丙氨酸(A)、丝氨酸(S)、苏氨酸(T)、
(2)天冬氨酸(D)、谷氨酸(E)、
(3)天冬酰胺(N)、谷氨酰胺(Q)、
(4)精氨酸(R)、赖氨酸(K)、
(5)异亮氨酸(I)、亮氨酸(L)、甲硫氨酸(M)、缬氨酸(V),和
(6)苯丙氨酸(F)、酪氨酸(Y)、色氨酸(W)。
对序列同一性的提述是指两个聚合序列例如蛋白质序列的相似度。序列同一性的确定可以由本领域普通技术人员使用公认的算法和/或技术容易地完成。通常通过在比较窗口中比较两个最佳比对的序列来确定序列同一性,其中与参考序列(不包含添加或缺失)相比,比较窗口中肽或多核苷酸序列的部分可以包含添加或缺失(即缺口)以优化两个序列的比对。百分比如下计算:通过确定两个序列中出现相同氨基酸残基或核酸碱基的位置的数量以得到匹配位置的数量,用匹配位置的数量除以在比较窗口中的位置总数并将结果乘以100来得到序列同一性的百分比。很容易获得多种软件驱动算法,例如BLAST N或BLAST P来执行这种比较。
公开了可用于所公开的方法和组合物,可与所公开的方法和组合物结合使用,可用于制备所公开的方法和组合物,或是所公开的方法和组合物的产物的材料、组合物和组分。应当理解,当公开了这些材料的组合、子集、相互作用、组等时,尽管可以不明确地公开特别提及这些化合物的每个单独的组合和排列,但是具体地涵盖了各种单独的和集体的组合中的每一个。该概念适用于本公开的所有方面,包括但不限于所描述的方法中的步骤。因此,任何前述实施方案的特定要素可以被组合或替代为其他实施方案中的要素。例如,如果有多个另外的步骤可以执行,则应理解,这些另外的步骤中的每一个都可以用所公开方法的任何特定方法步骤或方法步骤的组合来执行,并且特定地涵盖每个这样的组合或组合的子集并且应该认为其被公开。另外,应当理解,本文描述的实施方案可以使用任何合适的材料例如本文其他地方描述的或本领域已知的材料实施。
本文所引用的出版物及其所引用的主题特别地通过引用将其全部并入本文。
以下描述了证明使用PAMP-RNA诱导RLR信号传导可以诱导程序性细胞死亡并且可以应用于癌症特定背景的研究。
介绍
视黄酸诱导型基因I(RIG-I)通过结合病毒RNA中的病原体相关分子模式(PAMP)充当胞质病原体识别受体发挥作用。已经将RIG-I识别病毒RNA的分子签名鉴定为含5'-三磷酸根(5'ppp)的特定基序,包括富含聚尿苷(聚U)RNA基序和短双链(ds)RNA基序,其发现于RNA病毒复制期间在被感染细胞的细胞质中积聚的病毒RNA产物中。本发明人先前基于丙型肝炎病毒(HCV)RNA(PAMP-RNA)的聚U基序开发了合成的5'ppp RNA RIG-I配体,其被RIG-I特异识别并结合。RIG-I的PAMP-RNA激活触发细胞内信号传导级联反应,其介导RIG-I效应子功能以诱导先天性免疫并提供抗病毒治疗和免疫佐剂作用。
这项研究证明了惊人的发现,即RIG-I的PAMP-RNA激活可以诱导细胞凋亡型细胞死亡。对PAMP-RNA作用的剂量范围研究揭示了低剂量的PAMP-RNA水平触发通过RIG-I的先天性免疫激活,而增加的PAMP-RNA水平介导细胞死亡的RIG-I信号传导。这揭示了PAMP-RNA敏感性和作用的新的细胞死亡通路。蛋白质组学分析鉴定了TRIM16是关键的RIG-I结合伙伴,它特异性指导PAMP-RNA经由RIG-I信号传导诱导的细胞死亡。TRIM16对于细胞死亡信号传导是必需的,但不是通过RIG-I的先天性免疫激活必需的。另外,I型干扰素(IFN)进一步增强了TRIM16介导的细胞死亡。这些研究证明TRIM16是RIG-I结合伙伴,指导应答PAMP-RNA处理的细胞死亡。因此,证明PAMP-RNA信号传导和TRIM16/RIG-I/IFN轴是用于癌症疗法的细胞死亡的新肿瘤抑制程序。
结果
HCV PAMP RNA诱导的细胞凋亡是剂量依赖性的
研究发现,RIG-I激动剂可以诱导先天性免疫信号传导激活和细胞凋亡。为了用PAMP-RNA证实这一点,执行了剂量滴定研究以评估在Huh7细胞中诱导每个生物学事件所需的PAMP-RNA的量(图1A-1D)。有趣的是,先天性免疫信号传导以10ng/mL或更高的阈值被诱导,对IRF3诱导的ISG56和ISG15表达以及IFN依赖性的IFITM1的诱导证明了这一点,而PAMP-RNA诱导的细胞死亡的分析发生在100ng/mL的最小浓度。这些滴定研究指示PAMP-RNA在200ng/mL诱导了最高水平的细胞死亡(图1C和1D)。因此,在其余研究中,将200ng/mL用作诱导RIG-I介导的细胞凋亡和PAMP-RNA诱导细胞死亡的最佳浓度。
PAMP-RNA诱导的细胞凋亡是RIG-I和胱天蛋白酶依赖性的
为了确认PAMP-RNA驱动RIG-I依赖性信号传导,并评估胱天蛋白酶激活在应答由PAMP RNA诱导的RIG-I信号传导_ENREF_27的细胞死亡应答中的作用,在有或没有用泛胱天蛋白酶抑制剂zVAD处理的RIG-I敲低细胞和空载体对照细胞中对HCV PAMP诱导的细胞死亡程度和胱天蛋白酶活性进行评估。实际上,RIG-I敲除的细胞中RIG-I的丧失消除了PAMP-RNA诱导的细胞死亡,而在PAMP-RNA处理之前对空载体对照细胞的zVAD处理则降低了诱导的细胞死亡的频率(图2A-1、-2)。对先天性免疫信号传导蛋白表达的免疫印迹分析显示zVAD处理不影响对ISG56的PAMP-RNA诱导,从而指示在这些细胞中对胱天蛋白酶的抑制不抑制由PAMP-RNA触发的先天性免疫应答(图2B)。此外,细胞中RIG-I的丧失降低了p53依赖性促细胞凋亡基因NOXA和PUMA的表达(图3C),已知它们在RIG-I依赖性细胞凋亡期间是被诱导的。因此,PAMP-RNA可以通过RIG-I和胱天蛋白酶依赖性过程驱动细胞死亡。
I型IFN通路参与PAMP-RNA介导的细胞死亡
为了确定IFN对细胞死亡的PAMP-RNA信号传导的影响,在Huh7对照细胞和缺乏IFNAR或RIG-I的细胞中评估了细胞死亡。显示出IFNAR的丧失部分地消除了由PAMP-RNA触发的RIG-I介导的对细胞死亡的诱导(图3A)。在用I型IFN信号传导抑制剂B18R预处理的Huh7细胞中,RIG-I介导的对细胞凋亡的诱导的部分受损进一步证实了这一发现(图3B)。
TRIM16是新的RIG-I结合因子
为了确定参与通过PAMP-RNA的细胞先天性免疫或细胞死亡信号传导的RIG-I的可能辅因子,在由仙台病毒(SeV)激活RIG-I后,执行对P85CH8细胞的蛋白质组学筛选来筛选RIG-I结合蛋白(图4A)。SeV是有力的RIG-I激活剂并因此在这些研究中用作PAMP-RNA替代物,以在细胞培养物中提供对RIG-I的强而均匀的激活。用带FLAG标签的RIG-I构建体或FLAG构建体对照转染细胞,并模拟感染或用SeV感染。16小时后,通过FLAG亲和纯化分离RIG-I和RIG-I相关蛋白,并通过LC/MS-MS鉴定。该筛选鉴定了先天性免疫的已知RIG-I相互作用子,例如OASL。重要的是,分析鉴定出与FLAG-RIG-I关联但与单独的FLAG珠不关联的对应于TRIM16(一种E3泛素连接酶)的5个独特的肽序列。TRIM16与RIG-I相互作用是之前不知道的。在表达RIG-I-FLAG和TRIM16 V5构建体的HEK293细胞中,使用共免疫沉淀确认了TRIM16和RIG-I的状态为结合伙伴(图4B)。对RIG-I和TRIM16相互作用必要的结构域进一步图谱定位,其通过对来自每种蛋白质的共表达的截短突变体进行共免疫沉淀分析。这些分析揭示了RIG-I CARD结构域(aa1-176)对于RIG-I与TRIM16缔合是必要且足够的(图4C)。同样,与TRIM16截短物的相互共免疫沉淀揭示了TRIM16的B-Box结构域对于TRIM16与RIG-I缔合是必要且足够的(图4D)。
TRIM16的丧失不影响RIG-I对先天性免疫反应的诱导
为了阐明TRIM16在先天性免疫信号传导中的可能作用,评估了TRIM16丧失对PAMP-RNA诱导的先天性免疫激活的影响。使用TRIM16的CRISPR/cas9靶向敲除在Huh7细胞中耗尽TRIM16表达。与工程化以敲除RIG-I或TRIM25(RIG-I信号传导的正调控因子)的表达的Huh7细胞系相反,来自两个不同CRISPR/cas9靶向序列的Huh7 TRIM16 KO细胞系均表现出对通过PAMP-RNA的RIG-I先天性免疫信号传导的强力诱导,如RIG-I应答性基因ISG56、ISG15、IFNβ和TNFα的诱导所证明的(图5A和5B)。此外,在存在主要活性形式的RIG-I(N-RIG)时或在SeV感染后,TRIM16表达的丧失不影响RIG-I下游先天性信号传导程序(图5C和5D)。总之,这些实验证明TRIM16不参与RIG-I诱导的宿主先天性免疫信号传导过程。
TRIM16而不是TRIM25参与RIG-I介导的细胞凋亡
为了确定TRIM16是否特异性地参与由PAMP-RNA诱导的RIG-I介导的细胞死亡,评估了TRIM16丧失对PAMP-RNA诱导的细胞死亡的影响。将TRIM16 KO和TRIM25 KO Huh7细胞分别用PAMP-RNA、胱天蛋白酶抑制剂(ZVAD)或两者处理,并通过实时成像细胞死亡测定法进行评估。与对照相比,TRIM16的丧失或ZVAD处理抑制了PAMP-RNA诱导的细胞死亡(图6A)。相反,TRIM25表达的丧失不影响PAMP-RNA诱导的细胞死亡的程度(图6B)。这些研究揭示了PAMP-RNA通过新的RIG-I结合伙伴TRIM16特异性信号传导RIG-I介导的凋亡性细胞死亡。
PAMP-RNA介导的细胞死亡是癌细胞特有的
为了确定RLR(例如RIG-I)介导的程序性细胞死亡信号传导对健康细胞和癌细胞是否具有同等影响,将PAMP RNA暴露于肝癌细胞系和正常原代人肝细胞。具体而言,将HEPG2和Huh7肝细胞癌细胞和原代肝细胞用增加量的PAMP RNA或xRNA(用作阴性对照)处理,并在至少24小时的过程中监测指示可渗透/死亡中或死细胞的Sytox绿色染料的摄取。观察到PAMP处理特定地诱导肿瘤细胞(即,HepG2和Huh7细胞,它们是人的癌来源的细胞)的溶瘤破坏,而正常的非癌原代人肝细胞不是这样。具体地,图7A和7B示例说明了PAMP RNA处理引起HepG2(图7A)和Huh7(图7B)肝细胞癌细胞的溶瘤破坏,但对原代肝细胞没有影响(图7B)。
这证明健康细胞对于通过RLR(例如RIG-I)的PAMP诱导的程序性细胞死亡信号传导是不应性的。因此,可以特定地靶向破坏癌细胞,同时最小化或防止健康细胞中的异地毒性。
讨论
这项研究证明TRIM16是PAMP-RNA/RIG-I通路中细胞死亡信号传导的效应子。这些数据支持TRIM16作用的模型,其中PAMP-RNA和其他RIG-I配体激活RIG-I以赋予与TRIM16的结合。结果,TRIM16指导胱天蛋白酶的下游激活以介导细胞凋亡性细胞死亡。RIG-I介导的细胞死亡的诱导发生在比先天性免疫激活的RIG-I信号传导更高剂量的PAMP-RNA,这可能强调了先天性免疫保护细胞免受感染相对于细胞凋亡清除被感染的细胞来保护组织和生物体的不同作用。因此,细胞和组织的更高剂量的PAMP-RNA处理为定向细胞死亡结果的策略提供了一种方法,其中TRIM16介导PAMP-RNA/RIG-I信号传导的细胞死亡轴。
TRIM16(也称为EBBP)更早时候被鉴定为雌激素应答性基因,已参与许多生物学过程。TRIM16已被图谱定位为调节应激诱导的蛋白质聚集物和调节内膜损伤内稳态中的自噬。TRIM16与角质形成细胞分化和类视黄醇代谢有关。一项研究发现类风湿关节炎中TRIM16表达与滑膜增生之间存在相关性。另一项研究发现TRIM16与炎性小体组分胱天蛋白酶1和NLRP1相互作用以增强IL-1b分泌,这表明TRIM16在炎性小体调节中起作用。然而,最值得注意的是,TRIM16与癌症中的肿瘤抑制显著相关。已经阐明了TRIM16介导的肿瘤抑制的几种机制。在肺癌、卵巢癌和乳腺癌细胞模型系统中的研究发现TRIM16通过下调肿瘤促进性声波刺猬(sonic hedgehog)通路来抑制肿瘤进展期间的上皮向间质转化(EMT)。同样,肝细胞癌和前列腺癌的细胞模型已经显示TRIM16通过抑制调节细胞-细胞粘附分子E-钙粘蛋白表达的转录抑制剂来抑制EMT。对黑色素瘤中TRIM16的研究发现TRIM16通过调节IFN-β1产生抑制增殖和迁移。研究还发现TRIM16调节肿瘤中中间纤维家族成员波形蛋白的稳定性,减少皮肤癌细胞的迁移,并已显示通过抑制细胞周期来抑制神经母细胞瘤生长。最后,在乳腺癌细胞系中的研究发现TRIM16的过表达通过胱天蛋白酶-2的激活诱导细胞凋亡。
值得注意的是,FishTRIM16L基因是从橙色斑点石斑鱼中克隆出来的并与智人有29%的同一性,并且显示出拮抗鱼细胞中的先天性免疫信号传导。相反,该研究发现人TRIM16的丧失不影响先天性免疫激活的PAMP-RNA/RIG诱导。鱼和人TRIM16在先天性免疫应答中功能上的这种差异可能是由于缺乏序列同一性,而鱼TRIM16L蛋白缺乏B-box结构域并预计不与RIG-I结合,但可能对鱼先天性免疫产生独特的作用。
有趣的是,本研究揭示了HCV PAMP诱导的细胞死亡部分地依赖于IFN,这与先前的研究表明RIPA独立于IFN形成直接对比。不受任何特定理论的束缚,这种明显的差异很可能是由于在两项研究中使用了不同的细胞系,但是该结果表明IFN可以增强细胞死亡信号传导,与IFN促细胞凋亡作用相一致。由于RIG-I本身是ISG,因此IFN可以提高RIG-I的水平以增强PAMPR-RNA信号传导强度用于TRIM16的下游衔接。备选地,其他ISG可以与PMPA-RNA信号传导合作以增强TRIM16细胞死亡表型。
总而言之,这项研究将TRIM16鉴定为介导由PAMP-RNA诱导的细胞死亡信号传导的新的RIG-I结合伙伴。观察到该作用对于癌细胞是特有的,并且不负面影响相同起源组织的健康细胞。因此,用PAMP-RNA靶向这种新的RIG-I/TRIM16通路来指导细胞死亡结果是用于指导癌细胞中细胞死亡的治疗方法的可行策略。
为了示例说明而非限制本公开,提供以下实施例。
实施例1
示例性方法和材料
细胞培养、抑制剂和病毒
将人Huh 7肝细胞癌、原始永生化的人PH5CH8肝细胞和人胚胎肾脏HEK293细胞生长在“完全DMEM”(补充有10%胎牛血清(FBS)、10mM L-谷氨酰胺、5mM丙酮酸钠、抗生素-抗真菌溶液和5x非必需氨基酸的Dulbecco's改良Eagle培养基(DMEM))中。根据公认的方案维持来自仙台病毒(SenV)的Cantell株的细胞。对于测定,对细胞进行模拟处理,用脂质体中配制的PAMP或XRNA转染,用组成型活性RIG-I(N-RIG)转染或用100HAU/ml的SenV感染指定的时间,然后分析细胞死亡(通过incucyte分析)、胱天蛋白酶3/7活性或收获细胞裂解物用于免疫印迹和qRT-PCR分析(如Kentsis,A.&Borden,K.L.Construction ofmacromolecular assemblages in eukaryotic processes and their role in humandisease:linking RINGs together.Curr Protein Pept Sci 1,49-73(2000),Diaz-Griffero,F.,等人,A B-box 2 surface patch important for TRIM5alpha self-association,capsid binding avidity,and retrovirus restriction.J Virol 83,10737-10751(2009)所描述的,其每一个均通过引用整体并入本文)。抑制剂使用以下浓度:zVAD(SM-Biochemicals)以1:1000使用,TNFa(Peprotech)[0.5μL TNF/孔和400μL总培养基/孔],以及牛痘病毒B18R Carrier-Free Recombinant Protein(eBioscience)[1μg/ml]。在PAMP或X RNA转染前三小时添加抑制剂。
免疫印迹分析
用于免疫印迹分析的蛋白提取物是通过在冰上在补充有1μM冈田酸、1μM磷酸酶抑制剂混合物II(Calbiochem)和10μM蛋白酶抑制剂(Sigma)的MCLB裂解缓冲液(50mM TrisHCl[pH 7.5]、150mM NaCl、0.5%NP-40)中裂解细胞,然后在16,000xg于4℃离心10分钟以澄清裂解液而制备的。使用SDS-聚丙烯酰胺凝胶电泳分离相等的蛋白质的量,然后进行免疫印迹。以下一抗用于免疫印迹分析:PARP(Cell Signaling Technologies)、RIG-I Alme-1(Adipogen)、ISG56(在UT Southwestern Antibody Core设施制造的rb-a-IFIT1#971)、IFITM1(Protein Tech)、ISG15(Cell Signaling Technologies)、肌动蛋白(Millipore)、pSTAT1 Y701(Cell Signaling Technologies)、FLAG(Sigma),V5和(Life Technologies)。使用Alexa Fluor 680/790驴抗兔和驴抗小鼠(Jackson Immunoresearch)作为二抗。
免疫沉淀
在PH5CH8或293细胞中外源表达FLAG-RIG-I和TRIM16-V5全长或突变体构建体后,使用α-FLAG琼脂糖珠(Sigma)和α-V5缀合的琼脂糖珠(Sigma)于4℃免疫沉淀细胞提取物过夜。免疫沉淀后,在洗脱之前,使用MCLB裂解缓冲液将样品洗涤3次。将洗脱液煮沸5分钟,并使用SDS凝胶电泳分离并通过免疫印迹进行分析。α-V5(Life Technologies)和α-FLAG(Sigma)用作一抗,并孵育过夜。使用Alexa Fluor 680/790驴抗兔和驴抗小鼠(JacksonImmunoresearch)作为二抗。
细胞死亡分析
将细胞以100k个细胞每孔接种到24孔培养皿中。用100nM Sytox(Sytox)(#S7020,Thermo Fischer)处理细胞以测定细胞死亡和/或通透性,或在单独的孔中用Syto-Green(S7559,Thermo Fischer)处理以测定总细胞数。用IncuCyte成像平台(Essen Bioscience)对细胞进行成像。在每个时间点,每孔拍摄九张图像。每种处理一式两份或一式三份运行。通过在接近运行开始的两个小时内平均Syto-Green计数来计算细胞死亡百分比。通过重复对这些进行平均并用于标准化Sytox计数以计算细胞死亡百分比。
胱天蛋白酶3/7测定法
使用在IncuCyte成像平台(如上所述)上的Caspase 3/7Green Apoptosis AssayReagent(Essen)和3/7Assay System(Promega)测量胱天蛋白酶3/7活性。根据制造商的说明进行使用和分析。
构建体和细胞转染
RIG-I截短突变体构建体先前已经生产和描述(PMID:16558524,PMID:17190814)。使用基于PCR的克隆策略产生了TRIM16截短突变体。用于TRIM16 cDNA生产的寡核苷酸序列可在要求时获得。用如描述的2μg/mL PAMP-RNA10(PMID:18548002),对照XRNA(衍生自丙型肝炎病毒基因组的含5'ppp的RNA基序,与PAMP-RNA临近且长度相等但不结合RIG-I或诱导RIG-I信号传导10),编码TRIM16(全长或截短的突变体)或RIG-I(全长或截短的突变体)的cDNA构建体转染细胞,其使用转染试剂盒(Mirus Bio LLC)。
RT-qPCR
在RLT缓冲液(Qiagen)中收获细胞,并使用RNeasy试剂盒(Qiagen)纯化总细胞RNA。使用iScript cDNA合成试剂盒(Bio-rad)通过随机引物和寡聚(dT)引物从纯化的RNA合成cDNA。使用7300或Viia 7 RT-PCR仪(Applied Biosystems)通过SYBR Green相对定量方法测量RNA水平。通过减去管家基因RPLPO或POLRA的各自CT值对样品进行标准化,并相对于未经处理的对照计算的特定基因的诱导倍数。引物序列列于表1。
敲低细胞系的生成
先前已经描述了表达CRISPR和非靶向性指导RNA(对照)或用于敲除RIG-I或IFN-受体链(IFNAR)的指导RNA的Huh7细胞(PMID:27841874)。使用以下组的指导RNA选择表达CRISPR和特异于TRIM16或TRIM25的指导RNA的Huh7细胞,以产生每个特定的敲低敲除细胞群体:
TRIM16指导RNA 5'至3';每个靶向外显子6
GTCGGTGTCAGAGGTCAAAG(SEQ ID NO:)
GGTGTCAGAGGTCAAAGCGG(SEQ ID NO:)
GTCGGTGTCAGAGGTCAAAG(SEQ ID NO:)
TRIM25指导RNA 5'至3';每个靶向外显子3
GATGACTGCAAACAGAAAGG(SEQ ID NO:)
TCAGATGACTGCAAACAGAA(SEQ ID NO:)
GCAGCTACCAACAAGAATACA(SEQ ID NO:)
通常,每个基因选择3个独立的gRNA。通过InFusion克隆(TaKaRa)将gRNA序列亚克隆到pRRL-空-gRNA-Cas9-T2A-Puro载体32中。通过旋转转染将构建体转导到Huh7细胞中,并通过嘌呤霉素选择来分离多克隆群体。然后通过RNA和免疫印迹分析来筛选嘌呤霉素抗性细胞,以验证目标基因是否被充分敲除。
蛋白质组学分析
如上所述,用全长FLAG-RIG-I转染PH5CH8细胞,并模拟感染或用仙台病毒感染以激活RIG-I(100HAU/mL)达16小时。然后收获细胞以产生蛋白质提取物。将每种蛋白提取物200ug与抗FLAG亲和珠(Sigma)混合,并于4℃孵育2小时。然后将珠子在含有150mM NaCl的Tris缓冲盐水中洗涤3次,其后在含有250mM NaCl的Tris缓冲盐水中再洗涤3次。然后通过将珠子与过量的FLAG肽一起孵育来洗脱结合的蛋白质。然后对洗脱液进行串联液相色谱质谱法(LC-MS/MS)分析,以鉴定RIG-I和RIG-I-蛋白质复合物。鉴定了TRIM16的5个独特的肽。
统计学分析
使用GraphPad Prism 6软件(GraphPad,La Jolla,CA)执行统计学分析。根据每个实验中变量和时间点的数量,通过学生t检验或多元方差分析(ANOVA),然后进行Bonferroni事后分析,对组之间的均值差异进行统计学分析。通过对数秩检验分析Kaplan-Meier生存分析。通过未配对的t检验分析平均生存率和临床症状的比较。具体的统计学检验、P值和样本大小显示在图例中。
尽管已经示例说明和描述了示例说明性实施方式,但是应当理解,可以在不脱离本发明的精神和范围的情况下进行各种改变。
序列表
<110> University of Washington
Gale, M.J.
<120> 诱导三重基序包含蛋白16(TRIM16)信号传导的组合物和方法
<130> UWOTL-1-68142
<150> US 62/625,623
<151> 2018-02-02
<160> 91
<170> PatentIn version 3.5
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<212> PRT
<213> 人
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Met Ala Glu Leu Asp Leu Met Ala Pro Gly Pro Leu Pro Arg Ala Thr
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Glu Lys Leu Gly Arg Glu Thr Glu Glu Gln Asp Ser Asp Ser Ala Glu
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Gln Gly Asp Pro Ala Gly Glu Gly Lys Glu Val Leu Cys Asp Phe Cys
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Leu Asp Asp Thr Arg Arg Val Lys Ala Val Lys Ser Cys Leu Thr Cys
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Met Val Asn Tyr Cys Glu Glu His Leu Gln Pro His Gln Val Asn Ile
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Lys Leu Gln Ser His Leu Leu Thr Glu Pro Val Lys Asp His Asn Trp
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Arg Tyr Cys Pro Ala His His Ser Pro Leu Ser Ala Phe Cys Cys Pro
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Asp Gln Gln Cys Ile Cys Gln Asp Cys Cys Gln Glu His Ser Gly His
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Thr Ile Val Ser Leu Asp Ala Ala Arg Arg Asp Lys Glu Ala Glu Leu
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Gln Cys Thr Gln Leu Asp Leu Glu Arg Lys Leu Lys Leu Asn Glu Asn
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Ala Ile Ser Arg Leu Gln Ala Asn Gln Lys Ser Val Leu Val Ser Val
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Ser Glu Val Lys Ala Val Ala Glu Met Gln Phe Gly Glu Leu Leu Ala
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Ala Val Arg Lys Ala Gln Ala Asn Val Met Pro Phe Leu Glu Glu Lys
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Glu Gln Ala Ala Leu Asn Gln Ala Asn Gly Ile Lys Ala His Leu Glu
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Tyr Arg Ser Ala Glu Met Glu Lys Ser Lys Gln Glu Leu Glu Arg Met
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Ala Ala Ile Ser Asn Thr Val Gln Phe Leu Glu Glu Tyr Cys Lys Phe
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Lys Asn Thr Glu Asp Ile Thr Phe Pro Ser Val Tyr Val Gly Leu Lys
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Asp Lys Leu Ser Gly Ile Arg Lys Val Ile Thr Glu Ser Thr Val His
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Leu Ile Gln Leu Leu Glu Asn Tyr Lys Lys Lys Leu Gln Glu Phe Ser
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Lys Glu Glu Glu Tyr Asp Ile Arg Thr Gln Val Ser Ala Val Val Gln
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Arg Lys Tyr Trp Thr Ser Lys Pro Glu Pro Ser Thr Arg Glu Gln Phe
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Leu Gln Tyr Ala Tyr Asp Ile Thr Phe Asp Pro Asp Thr Ala His Lys
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Tyr Leu Arg Leu Gln Glu Glu Asn Arg Lys Val Thr Asn Thr Thr Pro
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Trp Glu His Pro Tyr Pro Asp Leu Pro Ser Arg Phe Leu His Trp Arg
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Gln Val Leu Ser Gln Gln Ser Leu Tyr Leu His Arg Tyr Tyr Phe Glu
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Val Glu Ile Phe Gly Ala Gly Thr Tyr Val Gly Leu Thr Cys Lys Gly
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ggccauuucc uguuuuuuuu uuuuuuuggu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60
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ggccauuucc uguuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuucuuu uccuucuuuu 60
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ggccaaccug uuuuuuuuuu uuuuuuuuuu uuuuuuccuu uuuuuuuuuu uuuuuuuuuu 60
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ggccauccug uuuuuuuccc uuuuuuuuuu ucuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60
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ggccauuucc uguuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60
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<400> 48
ggccauuccu guuuuuuuuu uuuuuuucuu uuguuuuuuu uguuuuuuuu uuuuuuuuuc 60
cuuucuuuuu uuuuuuuuuu ccuuucuucu uuaau 95
<210> 49
<211> 72
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 49
ggccauuucc uguuuuuuuu uuuuuuuuuu uucuuuccuu cuuuuuuccu uucuuuuccu 60
uccuucuuua au 72
<210> 50
<211> 87
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 50
ggccauuucc uguuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuucuuu uccuucuuuu 60
ucccuuuuuc uuucuuccuu cuuuaau 87
<210> 51
<211> 143
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 51
ggccauuucc uguuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60
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<210> 52
<211> 108
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 52
ggccauuucc uguuuuuuuu uuuuuucccu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60
uuuuuucuuu ccuucuuuuu uuuccuuucu uuuccuuccu ucuuuaau 108
<210> 53
<211> 82
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 53
ggcauccugu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60
uuuuuuuuuu uuucuuuucu uu 82
<210> 54
<211> 130
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 54
acacuccauu ucuuuuuuug uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60
uuuuuuuuuu uuuuuuuuuu uuuuuuucuu uuucuuuccu uucuuuucug acuucuaauu 120
uuccuucuua 130
<210> 55
<211> 129
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 55
guccuucugu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60
uuuuuuuuuu uuuuuuuuuu uuuuuuuccu uacccuuucc uucuuuucuu ccuuuuuuuu 120
ccuuacuuu 129
<210> 56
<211> 57
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 56
ggguccccuu guuuuuuuuu uuucuuuccu ucuuuccuuu ccuaaucuuu cuuucuu 57
<210> 57
<211> 96
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 57
agccauuucc uguuuuuuuu uuuuuuuuuu uuuuuuuuuu cuuuuuuuuu uucuuuccuu 60
uccuucuuuu uuuccuuucu uuuucccuuc uuuaau 96
<210> 58
<211> 115
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 58
ggccauuucc uguuuuuuuu uuuuuuuguu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60
uuuuuuuuuu uuuuuuuuuu uuuccuuucc uuuuuuuuuu uuuuucccuu uuuau 115
<210> 59
<211> 107
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 59
ggccauccug uuuuuuuuuu uuuuuuuuuu uucuuuuuuu uuuuuuuuuu cuuuuuuuuu 60
cuucuuuuuc uuuccuuuuu uuuuuuuuuu uuuuuuuuuc uucuuuc 107
<210> 60
<211> 96
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 60
ggccauuucc uguuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuucu 60
uuuucccucu uuuucuucuc uuuuuccuuc uuuaau 96
<210> 61
<211> 111
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 61
gcuaacuguu ccuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuucuuuu 60
uuuuuuuuuu cccucuuucu ucccuucuca ucuuauucua cuuucuuucu u 111
<210> 62
<211> 107
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 62
gcuaacuguu ccuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu cuuuuuuuuu 60
uuuuuucccu cuuucuuccc uucucaucuu auucuacuuu cuuucuu 107
<210> 63
<211> 92
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 63
gcuaacuguu ccuuuuuuuu uuucuuuuuu uuuuuuuuuu uuuuuuuuuu uccuucuuuc 60
uuucuuucuu accuuacuuu acuuucuuuu cu 92
<210> 64
<211> 111
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 64
gcuaacaguu ucucuuuuuu uuuuuuuccu uuuuuauuuu uauuuuuuuu uuuuuuuuuu 60
uuuuuuuauu uucuuuuccu uucuuucuca ccuuacauua cuuucuuucu u 111
<210> 65
<211> 77
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 65
gcuaauuucc uuauuguuuu uuuuuuuuuu uuuuucuuuc cauuuccuuc cuucuuacuu 60
cacuuuaccu ucuuucu 77
<210> 66
<211> 124
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 66
gcuaacuguu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60
uuuuuuuuuu uuuuuuuuuu uuuuuccuuu ccuuucuuuc uuaccuuacu uuacauucuu 120
uucu 124
<210> 67
<211> 119
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 67
gcuaacuguu ccuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60
uuuuuuuuuu uuuuuuuuuu uucuuuccuu ccuuucucac cuucuuuuac uucuuuccu 119
<210> 68
<211> 93
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 68
gcuaacuguu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuucuuuucu 60
uuccuuuccu ucuuacucua cuuuacuuuu ucu 93
<210> 69
<211> 129
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 69
gcuaacuguu cuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60
uuuuuuuuuu uuuuuuuuuu uuuuuuuuuc uuuuccuucu ucuuucuuac cuuauuuucc 120
uucuuucuu 129
<210> 70
<211> 86
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 70
gcuaacuguu uuuuuuuuuu uuuuuuuuuu uuuuuuuucu uuuuuuuucu uuucuuuccu 60
ucuuaccuua cuuuacuuuc uuuucu 86
<210> 71
<211> 133
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 71
gcuaacuguu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60
uuuuuuuuuu uuuuuuuuuu uuuuuuuuuc cuuuuuccuu uuccuucucu uuuuaccuua 120
cuuuacuuuu cuu 133
<210> 72
<211> 144
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 72
gcuaacuguc ccuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60
uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuucuuu uuuucucuuu uccuucuuuc 120
uuaccuuauu uuacuuucuu uccu 144
<210> 73
<211> 119
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 73
gcuaacuguc ccuuuuuuuu uguuuuuuuu uuuuuuuuuu uuuuuuuuuu uucuuuuuuu 60
uuuuuuuuuu uguuucuuuu ccuucucauu uccuucuuau cuuaauuacu uccuuuccu 119
<210> 74
<211> 88
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 74
gcuaacuguu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuccu ucuuccuuuc 60
cuucuuaccu uacuuuauuu ucuuuccu 88
<210> 75
<211> 101
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 75
gcuaacuguu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60
uucuuucuuu ucuuuucuca ccuuacuuua cuuccuuucu u 101
<210> 76
<211> 86
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 76
gcuaguuuuc uuuuuuuuuu uuuuuuuuuu uuuuguuuuu uuuuuuuuuc cucuuuuucc 60
guauuuuuuu uuuuuccucu uuucuu 86
<210> 77
<211> 105
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 77
ggccauccug uuuuuuuccc uuuuuuuuuu ucuuuuuuuu uuuuuuuuuu uuuuuuuuuu 60
uuuuuucucc uuuuuuuuuc cucuuuuuuu ccuuuucuuu ccuuu 105
<210> 78
<211> 79
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 78
ccauuuuucu uuuuuuuuuu uuguuuguuu uuuuuuuuuu uuucuuuccu ucuuuccuga 60
cuuuuaauuu uccuucuua 79
<210> 79
<211> 116
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 79
ccauuuuucu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuugu 60
uuguuuuuuu uuuuuuuuuu cuuuccuucu uuccugacuu uuaauuuucc uucuua 116
<210> 80
<211> 84
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 80
ggccauuucc uguuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuacccu uuuuucucuu 60
uuuuuuuuuu ccuucuucuu uaau 84
<210> 81
<211> 74
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 81
ggccauuucc uguuuuuuuu uuuuuuuuuu acccuuuuuu cucuuuuuuu uuuuuuuuuu 60
ccuucuucuu uaau 74
<210> 82
<211> 38
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 82
ggccauuuuc uguuuuuuuu uuuuuuauuu ucuuuaau 38
<210> 83
<211> 53
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 83
ggccauuuuc uuuuuuuuuu cucuuuuuuu uuuuuuuuuu uauuuucuuu aau 53
<210> 84
<211> 82
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 84
ggccauuuuc uguuuuuuuu uuuuuuuuuu uuccuuuuuu uuuuuuccuc uuuuuuuuuu 60
uuuuuuuuuu auuuucuuua au 82
<210> 85
<211> 82
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 85
ggccauuuuc uguuuuuuuu uuuucuuuuu uuuuuuuuuu uuccuuuuuu uuucucuuuu 60
uuuuuuuuuu auuuucuuua au 82
<210> 86
<211> 85
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 86
ggccauccug uuuuuuuuuu uuuuuuuuuu uuuuguuuuu uuuuuuuuuu uucuuuuucc 60
uuuuuuuuuu uuuauuuucu ucuuu 85
<210> 87
<211> 59
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 87
gguccuaagu uuuuuuuuuu uucuuccuuc cuucuuuccu uuucuaauuu uccuucuuu 59
<210> 88
<211> 62
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 88
gguccuaagu uguuuuuuuu uuuuuuuccu uccuucuuuc ccuuuucuaa uuuuccuucu 60
uu 62
<210> 89
<211> 74
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 89
gguccuaagu uguuuuuuuu uuuuuuuuuu uuuuuccuuu ccuuccuucu uuccuuuucu 60
aauuuuccuu cuuu 74
<210> 90
<211> 95
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 90
ggccauuucu guuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuuuuuuuuu uuguuuccuu 60
cuuuuuccuu uucuuuuuuu uuuucucccu uuaau 95
<210> 91
<211> 70
<212> RNA
<213> 人工序列
<220>
<223> 合成RNA
<400> 91
ggccauuucu guuuuuuuuu uuuuuguuuc cuucuuuuuc cuuuucuuuu uuuuuuuuuc 60
ucccuuuaau 70
Claims (48)
1.在细胞中诱导三重基序包含蛋白16(TRIM16)活性的方法,该方法包括使细胞与有效量的含有病原体相关分子模式(PAMP)的核酸分子接触,其中含有PAMP的核酸分子包含:
包含末端三磷酸根的5'臂区域,
包含至少8个连续尿嘧啶残基的聚尿嘧啶核心,和
包含至少8个核酸残基的3'臂区域,其中3'臂区域的最5’端核酸残基不是尿嘧啶并且其中3'臂区域至少30%是尿嘧啶残基。
2.权利要求1的方法,其中聚尿嘧啶核心由8至30个尿嘧啶残基组成。
3.权利要求1的方法,其中3'臂区的最5’端核酸残基是胞嘧啶残基或鸟嘌呤残基。
4.权利要求1的方法,其中3'臂区至少40%、50%、60%、70%、80%或90%是尿嘧啶残基。
5.权利要求1的方法,其中3'臂区域包含至少7个连续尿嘧啶残基。
6.权利要求1的方法,其中5'臂区域还包含位于末端三磷酸根和聚尿嘧啶核心之间的一个或多个核酸残基。
7.权利要求1的方法,其中5'臂区域由末端三磷酸根组成,并且其中末端三磷酸根直接连接至聚尿嘧啶核心的5'端。
8.权利要求1的方法,其中末端三磷酸根、5'臂区域的一个或多个核酸残基和聚尿嘧啶核心在丙型肝炎病毒中不天然地一起出现。
9.权利要求1的方法,其中含有PAMP的核酸分子在细胞中诱导视黄酸诱导型基因I(RIG-I)样受体(RLR)与TRIM16的相互作用。
10.权利要求9的方法,其中RLR与TRIM16的相互作用诱导细胞中的细胞死亡信号传导。
11.权利要求9的方法,其中RLR是RIG-I。
12.权利要求1的方法,还包括使细胞与干扰素(IFN)接触。
13.权利要求1的方法,还包括使细胞与包含可操作地连接至启动子序列的编码TRIM16的序列的核酸接触,其中启动子序列能够在细胞中诱导所编码的TRIM16的表达。
14.权利要求13的方法,其中所编码的TRIM16具有与SEQ ID NO:1所示氨基酸序列有至少90%同一性的氨基酸序列。
15.权利要求1的方法,其中TRIM16活性包括诱导程序性细胞死亡。
16.权利要求1的方法,其中细胞是癌细胞。
17.权利要求16的方法,其中癌细胞是实体瘤癌细胞。
18.权利要求16的方法,其中癌细胞是选自胰腺癌、膀胱癌、结肠直肠癌、乳腺癌、前列腺癌、肾癌、肝细胞癌、肺癌、卵巢癌、宫颈癌、胃癌、食道癌、头颈癌、黑素瘤、神经内分泌癌、CNS癌、脑肿瘤、骨癌和软组织肉瘤的实体瘤癌细胞。
19.权利要求16的方法,其中癌细胞是血液学癌细胞。
20.前述权利要求中任一项的方法,其中在体外使细胞与有效量的含有PAMP的核酸分子接触。
21.权利要求1-19中任一项的方法,其中在哺乳动物受试者体内使细胞与有效量的含有PAMP的核酸分子接触。
22.权利要求21的方法,还包括向受试者施用用于癌症的免疫疗法。
23.权利要求22的方法,其中免疫疗法包括癌症特异性抗体或其功能片段、免疫检查点抑制剂和过继性细胞疗法,包括CAR T细胞疗法。
24.权利要求21的方法,其中受试者是人。
25.在有此需要的受试者中治疗癌症的方法,该方法包括向该受试者施用治疗有效量的含有病原体相关分子模式(PAMP)的核酸分子,其中含有PAMP的核酸分子包含:
包含末端三磷酸根的5'臂区域,
包含至少8个连续尿嘧啶残基的聚尿嘧啶核心,和
包含至少8个核酸残基的3'臂区域,其中3'臂区域的最5’端核酸残基不是尿嘧啶并且其中3'臂区域至少30%是尿嘧啶残基。
26.权利要求25的方法,其中聚尿嘧啶核心由8至30个尿嘧啶残基组成。
27.权利要求25的方法,其中3'臂区域的最5’端核酸残基是胞嘧啶残基或鸟嘌呤残基。
28.权利要求25的方法,其中3'臂区域至少40%、50%、60%、70%、80%或90%是尿嘧啶残基。
29.权利要求25的方法,其中3'臂区域包含至少7个连续尿嘧啶残基。
30.权利要求25的方法,其中5'臂区域还包含位于末端三磷酸根和聚尿嘧啶核心之间的一个或多个核酸残基。
31.权利要求25的方法,其中5'臂区域由末端三磷酸根组成,并且其中末端三磷酸根直接连接至聚尿嘧啶核心的5'端。
32.权利要求25的方法,其中末端三磷酸根、5'臂区域的一个或多个核酸残基和聚尿嘧啶核心在丙型肝炎病毒中不天然地一起出现。
33.权利要求25的方法,其中含有PAMP的核酸分子在受试者的癌细胞中诱导视黄酸诱导型基因I(RIG-I)样受体(RLR)与TRIM16的相互作用。
34.权利要求33的方法,其中RLR与TRIM16的相互作用在癌细胞中诱导细胞死亡信号传导。
35.权利要求33的方法,其中RLR是RIG-I。
36.权利要求25的方法,还包括向受试者施用有效量的干扰素(IFN)。
37.权利要求25的方法,还包括向受试者施用包含可操作地连接至启动子序列的编码TRIM16的序列的核酸,其中启动子序列能够在受试者的癌细胞中诱导所编码的TRIM16的表达。
38.权利要求25的方法,其中癌症是选自胰腺癌、膀胱癌、结肠直肠癌、乳腺癌、前列腺癌、肾癌、肝细胞癌、肺癌、卵巢癌、宫颈癌、胃癌、食道癌、头颈癌、黑素瘤、神经内分泌癌、CNS癌、脑瘤、骨癌和软组织肉瘤的实体瘤癌。
39.权利要求25的方法,其中癌症是血液学癌症。
40.权利要求25的方法,还包括向受试者施用用于癌症的免疫疗法。
41.权利要求40的方法,其中免疫疗法包括癌症特异性抗体或其功能片段、免疫检查点抑制剂和过继性细胞疗法,包括CAR T细胞疗法。
42.权利要求25的方法,其中受试者是人。
43.治疗组合物,包含权利要求1-8中任一项所述的含有病原体相关分子模式(PAMP)的核酸分子和干扰素(IFN)的组合。
44.权利要求43的治疗组合物,还包含可操作地连接至启动子的编码TRIM16的核酸。
45.权利要求43的治疗组合物,其中将组合物配制于脂质体中。
46.治疗组合物,包含权利要求1-8中任一项所述的含有病原体相关分子模式(PAMP)的核酸分子和可操作地连接至启动子的编码TRIM16的核酸的组合。
47.权利要求46的治疗组合物,还包含干扰素(IFN)。
48.权利要求47的治疗组合物,其中将组合物配制于脂质体中。
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US201862625623P | 2018-02-02 | 2018-02-02 | |
US62/625,623 | 2018-02-02 | ||
PCT/US2019/016413 WO2019152884A1 (en) | 2018-02-02 | 2019-02-01 | Compositions and methods for inducing tripartite motif-containing protein 16 (trim16) signaling |
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US20220396797A1 (en) * | 2019-11-15 | 2022-12-15 | Infectious Disease Research Institute | Rig-i agonist and adjuvant formulation for tumor treatment |
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AU2019215191A1 (en) | 2020-09-17 |
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