CN105142676A - Cftr mrna组合物以及相关方法和用途 - Google Patents
Cftr mrna组合物以及相关方法和用途 Download PDFInfo
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- CN105142676A CN105142676A CN201480015043.5A CN201480015043A CN105142676A CN 105142676 A CN105142676 A CN 105142676A CN 201480015043 A CN201480015043 A CN 201480015043A CN 105142676 A CN105142676 A CN 105142676A
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Abstract
本文提供用于递送CFTR?MRNA以用于诱导包括在哺乳动物肺中CFTR表达的材料、制剂、生产方法和方法。本发明特别用于治疗囊性纤维化。
Description
相关申请
本申请要求2013年3月14日提交的美国临时申请序列No.61/783,663的优先权,所述临时申请的公开内容特此以引用的方式并入。
背景
本发明涉及囊性纤维化跨膜调节因子(CFTR)mRNA组合物、其用途以及其制备和使用方法。
囊性纤维化是由CFTR基因的突变引起的常染色体遗传病症,所述CFTR基因编码据信参与调节上皮细胞中的多种其它离子通道和转运系统的氯离子通道。CFTR的功能损失导致慢性肺病、异常粘液产生并且显著降低预期寿命。通常参见Rowe等,NewEngl.J.Med.352,1992-2001(2005)。
尽管在1989年克隆了CFTR基因,但用于替代CFTR用以治疗囊性纤维化的有效疗法还有待发展。文献记载了在试图诱导肺中CFTR的表达中遇到的许多困难。例如,包含CFTRDNA的病毒载体触发免疫应答并且CF症状在施用后持续。Conese等,J.Cyst.Fibros.10增刊2,S114-28(2011);Rosenecker等,Curr.Opin.Mol.Ther.8,439-45(2006)。DNA、包括CFTRDNA的非病毒递送也据报道触发免疫应答。Alton等,Lancet353,947-54(1999);Rosenecker等,JGeneMed.5,49-60(2003)。此外,非病毒DNA载体遇到另外的问题:核孔复合物的机器通常不引入DNA至细胞核中,在细胞核中将发生转录。Pearson,Nature460,164-69(2009)。
在诱导肺中的CFTR表达中的另一困难源是肺环境本身。肺表面活性剂据报道降低阳离子脂质转移媒介物如Lipofectamine(DOSPA:DOPE)的转染效率。
Ernst等,J.GeneMed.1,331-40(1999)。此外,Rosenecker等,2003,同上鉴别了存在于气道表面液体中的多种抑制性组分,所述组分可干扰聚合物介导的或脂质介导的转染。信使RNA疗法已被提议作为用于诱导治疗性或替代蛋白质的表达的一般方法。先前已报道诱导信使RNA(mRNA)的概念作为在宿主内产生蛋白质的手段(Yamamoto,A.等Eur.J.Pharm.2009,71,484-489;Debus,H.等J.ControlRel.2010,148,334-343)。然而,表观肺特异性困难已被报道用于使用某些脂质复合物制剂进行mRNA递送。例如,携带mRNA或DNA的脂质复合物的体外和体内性能的比较揭示,虽然mRNA组合物在培养的细胞中给出更高的表达,但当鼻内施用至小鼠肺部时仅在DNA组合物的情况下检测到可测量的表达。Andries等,Mol.Pharmaceut.9,2136-45(2012)。
还应当注意,CFTR是相对于模型或报告基因如萤火虫荧光素酶(FFL)的相对较大基因。比较野生型CFTR编码序列(SEQIDNO:2)和FFL编码序列(SEQIDNO:7)的长度。长度的差异可在一些情况下影响稳定性,并且因此影响任何给定剂量的mRNA是否将产生蛋白质表达和将产生多少蛋白质表达。此外,虽然mRNA的体外合成由于不存在正常细胞mRNA和构成不希望的污染物的其它细胞组分而通常优选由细胞合成,但是用较长编码序列如CFTRmRNA体外合成mRNA基本上比用相对较短的编码序列如FFL体外合成mRNA更难以实现。
PCT专利公布WO2007/024708和美国专利公布US2010/0203627及US2011/0035819讨论治疗性施用CFTRmRNA,但既没有提供证明的降低以在施用CFTRmRNA之后在肺中实践功能性CFTR的产生又没有提供足够的指导用于克服与使用体外转录的CFTRmRNA诱导肺中的CFTR表达有关的困难。这些困难包括实现体外合成mRNA的困难和mRNA组合物与肺特异性物质的相互作用特有的困难,研究人员如Andries等(同上)已经发现肺特异性物质使mRNA组合物对于诱导表达无效,甚至在相应的基于DNA的组合物不提供某种水平的表达时。
因此,需要用于治疗囊性纤维化的改进的材料、制剂、产生方法和用于递送CFTRmRNA以用于诱导CFTR表达(包括在哺乳动物肺中)的方法。
发明概述
本发明部分地基于开发可诱导体内功能性CFTR表达的CFTRmRNA和非天然存在的CFTRmRNA的制剂及其施用方法。根据本发明的组合物、方法和用途可以在具有适合于有效治疗囊性纤维化的有利安全性特征的大型哺乳动物的肺中提供CFTR表达。
因此,一方面,本发明提供一种通过递送编码CFTR蛋白的mRNA体内(特别是在需要递送的受试者(例如哺乳动物)的肺中)产生CFTR的方法。在一些实施方案中,编码CFTR蛋白的mRNA直接递送至受试者的肺。如本文所用,“CFTR蛋白”涵盖可用于取代天然存在的CFTR蛋白活性和/或降低与囊性纤维化相关的一种或多种症状的强度、严重性和/或频率的CFTR蛋白的任何全长、片段或部分。例如,根据本发明的适合的CFTR蛋白可具有与野生型人CFTR蛋白(SEQIDNO:1)相同的氨基酸序列。在一些实施方案中,根据本发明的适合的CFTR蛋白可具有与野生型人CFTR蛋白(SEQIDNO:1)至少约70%、75%、80%、85%、90%、95%、96%、97%、98%或99%相同的氨基酸序列。
在一个实施方案中,本发明提供一种在哺乳动物的肺中的上皮细胞中诱导CFTR表达的方法,所述方法包括使所述哺乳动物的肺中的上皮细胞与组合物相接触,其中:所述组合物是包含体外转录的mRNA的药物组合物;所述体外转录的mRNA包含编码SEQIDNO:1的编码序列。在另一个实施方案中,所述体外转录的mRNA包含编码序列,所述编码序列编码与SEQIDNO:1至少约70%、75%、80%、85%、90%、95%、96%、97%、98%或99%相同的氨基酸序列。
在一个实施方案中,本发明提供一种在哺乳动物靶细胞中诱导CFTR表达的方法,所述方法包括使所述哺乳动物靶细胞与组合物相接触,所述组合物包含编码SEQIDNO:1的氨基酸序列的体外转录的mRNA。在另一个实施方案中,所述体外转录的mRNA包含编码序列,所述编码序列编码与SEQIDNO:1至少约70%、75%、80%、85%、90%、95%、96%、97%、98%或99%相同的氨基酸序列。
在另一个实施方案中,本发明提供一种包含编码序列、5’-UTR和3’-UTR的非天然存在的mRNA分子,其中所述编码序列编码SEQIDNO:1的氨基酸序列并且所述编码序列与SEQIDNO:3至少80%相同。在另一个实施方案中,所述编码序列编码与SEQIDNO:1至少约70%、75%、80%、85%、90%、95%、96%、97%、98%或99%相同的氨基酸序列和/或所述编码序列与SEQIDNO:3约50%、55%、60%、65%、70%、75%、80%、85%,、90%、95%、96%、97%、98%或99%相同。
在另一个实施方案中,本发明提供一种包含编码序列、5’-UTR和3’-UTR的非天然存在的mRNA分子,其中所述编码序列编码SEQIDNO:1的氨基酸序列并且所述编码序列相对于SEQIDNO:2的野生型编码序列在表1中所列出的编码序列的位置处包含至少50%、至少55%、至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%或至少95%的表1中所列出的非野生型碱基。
在另一个实施方案中,本发明提供一种包含编码序列、5’-UTR和3’-UTR的非天然存在的mRNA分子,其中所述编码序列编码SEQIDNO:1的氨基酸序列并且所述编码序列相对于SEQIDNO:2的野生型编码序列在表2中所列出的编码序列的对应位置处包含至少50%、至少55%、至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%或至少95%的表2中所列出的非野生型碱基。
在一些实施方案中,本发明提供一种非天然存在的mRNA分子,其包含信号肽的编码序列。在一个具体实施方案中,本发明提供一种非天然存在的mRNA,其包含生长激素前导序列的编码序列。在某些实施方案中,本发明提供一种非天然存在的mRNA,其包含SEQIDNO:18或SEQIDNO:19的编码序列。在一些实施方案中,本发明提供一种非天然存在的mRNA,其包含与SEQIDNO:18或SEQIDNO:19至少约50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、96%、97%、98%或99%相同的编码序列。
在一些实施方案中,本发明提供一种非天然存在的mRNA分子,其包含SEQIDNO:9、SEQIDNO:10、SEQIDNO:11、SEQIDNO:12、SEQIDNO:13、SEQIDNO:14、SEQUDNO:15、SEQIDNO:16或SEQIDNO:17的序列。在一些实施方案中,本发明提供一种非天然存在的mRNA分子,其包含与SEQIDNO:9、SEQIDNO:10、SEQIDNO:11、SEQIDNO:12、SEQIDNO:13、SEQIDNO:14、SEQUDNO:15、SEQIDNO:16或SEQIDNO:17中的任一个至少约50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、96%、97%、98%或99%相同的序列。
在另一个实施方案中,本发明提供一种多核苷酸,其包含与根据本发明的mRNA的序列互补的序列。
在另一个实施方案中,本发明提供一种组合物,其包含根据本发明的多核苷酸、RNA聚合酶和核苷三磷酸。
在另一个实施方案中,本发明提供一种药物组合物,其包含根据本发明的mRNA。
在另一个实施方案中,本发明提供一种负载有根据本发明的药物组合物的雾化或气雾化装置。
在另一个实施方案中,本发明提供一种培养细胞,其包含根据本发明的mRNA和从所述mRNA表达的功能性CFTR。
在另一个实施方案中,本发明提供一种根据本发明的药物组合物用于诱导功能性CFTR的表达的用途。
在另一个实施方案中,本发明提供一种在哺乳动物的肺中的上皮细胞中诱导CFTR表达的方法,所述方法包括使所述上皮细胞与组合物相接触,其中所述组合物是包含根据本发明的mRNA的药物组合物。
在另一个实施方案中,本发明提供一种在哺乳动物靶细胞中诱导CFTR表达的方法,所述方法包括使所述哺乳动物靶细胞与组合物相接触,所述组合物包含根据本发明的mRNA。
在另一个实施方案中,本发明提供一种通过向需要治疗的受试者施用如本文所述的编码CFTR蛋白的mRNA来治疗囊性纤维化的方法。在一个实施方案中,mRNA被施用至所述受试者的肺。在一个实施方案中,通过吸入、雾化、鼻内施用或气雾化施用mRNA。在不同实施方案中,施用mRNA导致受试者的肺中CFTR的表达。
在一个具体实施方案中,本发明提供一种通过向需要治疗的受试者的肺施用包含编码SEQIDNO:1的编码序列的mRNA来治疗囊性纤维化的方法。在一些实施方案中,本发明提供一种通过向需要治疗的受试者的肺施用包含编码序列的mRNA来治疗囊性纤维化的方法,所述编码序列编码与野生型人CFTR蛋白(SEQIDNO:1)至少约70%、75%、80%、85%、90%、95%、96%、97%、98%或99%相同的氨基酸序列。在另一个具体实施方案中,本发明提供一种通过向需要治疗的受试者的肺施用包含SEQIDNO:3的编码序列的mRNA来治疗囊性纤维化的方法。在一些实施方案中,本发明提供一种通过向需要治疗的受试者的肺施用包含编码序列的mRNA来治疗囊性纤维化的方法,所述编码序列与SEQIDNO:3至少约50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、96%、97%、98%或99%相同。
另一方面,本发明提供用于制备如本文所述的编码CFTR蛋白的mRNA的方法。在一个实施方案中,本发明提供一种体外制备CFTRmRNA的方法,所述方法包括使分离的多核苷酸与RNA聚合酶在核苷三磷酸存在下相接触,其中:所述分离的多核苷酸和RNA聚合酶不包含在细胞内;所述分离的多核苷酸是RNA聚合酶的模板;所述分离的多核苷酸包含可操作地连接至模板序列的启动子;所述模板序列包含与编码SEQIDNO:1的序列互补的编码序列互补序列;并且:(a)所述模板序列包含比SEQIDNO:2的互补序列更少的隐蔽性启动子,(b)所述模板序列包含比SEQIDNO:2更少的同向和/或反向重复序列,(c)所述模板序列包含比SEQIDNO:2更少的不利密码子的互补序列,或(d)编码序列互补序列的GC含量低于SEQIDNO:2的GC含量。
在另一个实施方案中,本发明提供一种体外制备CFTRmRNA的方法,所述方法包括使根据本发明的分离的多核苷酸与RNA聚合酶在核苷三磷酸存在下相接触,其中:所述分离的多核苷酸和RNA聚合酶不包含在细胞内;所述分离的多核苷酸是所述RNA聚合酶的模板;并且所述分离的多核苷酸包含可操作地连接至模板序列的启动子,并且所述RNA聚合酶合成包含编码SEQIDNO:1的编码序列的mRNA。
在所述用途和治疗方法的一些实施方案中,体外转录的mRNA是被修饰以包含非天然存在的UTR的编码人CFTR(SEQIDNO:2)的天然存在的或野生型mRNA。在其它实施方案中,体外转录的mRNA是如上所述的非天然存在的mRNA。
本发明的另外目的和优点将在以下描述中部分阐述,并且将从描述中部分地明显,或可通过实践本发明来习得。本发明的目的和优点将通过在所附权利要求书中特别指出的要素和组合来实现并且达成。
应了解上文一般描述与下文详细描述均仅仅是示例性和解释性的,并且不限制所要求的本发明。
并入本说明书中并且构成本说明书的一部分的附图说明本发明的一些实施方案,并且与描述一起用于解释本发明的原理。
附图简述
本发明的上述方面和优点可参考附图从以下详细说明中变得清楚。
图1A.在用人CFTRmRNA转染之后24小时检测人CFTR蛋白的成熟“C”条带。对于未修饰的和修饰的(SNIM)mRNA(包含25%的2-硫代尿苷和5-甲基胞苷)两者均观察到成功的蛋白质产生。使用R&D系统MAB25031抗体进行免疫沉淀并且使用Ab570检测。
图1B.在PEI/未修饰的人CFTRmRNA纳米颗粒暴露后第24小时CFTRKO小鼠肺的蛋白质印迹分析。在大约一小时的过程内通过雾化(PariBoy喷射喷雾器)处理小鼠。进行根据所提供的方法衍生的人CFTR蛋白的免疫沉淀。在所有处理的小鼠中检测到成熟的“C”条带,而在对照小鼠中未观察到。
图2.处理的(4ughCFTRmRNA)和未处理的HEK293T细胞的8-Br-cAMP诱发电流的电流电压图。与未处理的细胞相比,在hCFTRmRNA转染的细胞内诱导较大电流。暴露于特定CFTR蛋白抑制剂CFTRinh-172的处理的细胞表现出Cl-离子电流流动的显著减少(约89%)。
图3.在施加+80mV膜电位时处理的(4ughCFTRmRNA)和未处理的HEK293T细胞的8-Br-cAMP诱发电流的直方图。与未处理的细胞相比,在hCFTRmRNA转染的细胞内诱导较大电流。暴露于特定CFTR蛋白抑制剂CFTRinh-172的处理的细胞表现出Cl-离子电流流动的显著减少(约89%)。
图4.比较天然、毛喉素和GlyH-101暴露的HEK293细胞的特征的电流-电压图。在任何情况下没有观察到电流的显著变化。
图5.处理的(4ughCFTRmRNA)和未处理的HEK293细胞的毛喉素诱发电流的电流电压图。与未处理的细胞相比,在hCFTRmRNA转染的细胞内诱导较大电流。暴露于特定CFTR蛋白抑制剂GlyH-101的处理的细胞表现出Cl-离子电流流动的显著减少(约95%),如在图的右侧上的歩阶图(+100mV)中所展示。
图6.未处理的(PBS)(左)和处理的(右)CFTRKO小鼠肺中的人CFTRmRNA的原位杂交。通过气管内施用使小鼠暴露于30ug的PEI纳米颗粒中的封装未修饰的hCFTRmRNA。在施用后24小时在整个两个肺中观察到大量阳性染色。
图7.在不同放大倍数视图(高达20x放大倍数)下人CFTRmRNA处理的CFTRKO小鼠肺的原位杂交。通过气管内施用使小鼠暴露于30ug的PEI纳米颗粒中的封装未修饰的hCFTRmRNA。
图8.展示人CFTRmRNA处理的(右)CFTRKO小鼠肺的原位杂交的高放大倍数(40x)代表性肺切片。在施用后24小时在靶支气管上皮细胞的顶端细胞质中检测到人CFTRmRNA。通过气管内施用使小鼠暴露于30ug的PEI纳米颗粒中的封装未修饰的hCFTRmRNA。
图9.在施用后六小时(左)和24小时(右)人CFTRmRNA处理的CFTRKO小鼠肺的原位杂交染色的比较。通过气管内施用使小鼠暴露于30ug的PEI纳米颗粒中的封装未修饰的hCFTRmRNA。在6小时内在支气管区域和肺泡区域内的整个两个肺中观察到强烈阳性细胞内染色,同时在施用后24小时仍然观察到显著阳性染色。
图10.未处理的(PBS)(上)和处理的(下)CFTRKO小鼠肺中的人CFTRmRNA的原位杂交。通过气管内施用使小鼠暴露于15ug的C12-200脂质纳米颗粒中的封装未修饰的hCFTRmRNA。在施用后6小时在整个两个肺中观察到大量阳性染色。
图11.展示人CFTRmRNA处理的CFTRKO小鼠肺的原位杂交的高放大倍数(40x)代表性肺切片。在施用后六小时在靶支气管上皮区域(左)以及细胞内肺泡区域(右)的顶端细胞质中检测到人CFTRmRNA。通过气管内施用使小鼠暴露于15ug的C12-200脂质纳米颗粒中的封装未修饰的hCFTRmRNA。
图12.针对hCFTR表达筛选不同细胞系。用hCFTR编码构建体转染的CHO和COS-7(A)以及BHK和PKC(B)细胞的免疫印迹。在转染后24小时制备蛋白质裂解物,并且使用MA1-935作为一级抗体进行筛选。箭头指示推定CFTR。参见实施例6中MA1-935特异性的讨论。
图13.不同抗-人CFTR抗体的交叉反应性。(A)–小鼠抗人CFTRMA1-935(Chemicon);(B)–小鼠抗人CFTRAB570(囊性纤维化基金会);(C)–小鼠抗人CFTRAB596(囊性纤维化基金会);(D)兔抗人CFTRG449(洛克菲勒大学)。箭头指示CFTR。
图14.使用三种不同的抗体(R29、R66/17和R66/16)免疫沉淀人CFTR,接着使用AB596免疫检测。泳道1:T84细胞(阳性对照),泳道2:未处理的猪肺组织(300mg),泳道3:处理的猪肺组织(697mg),泳道4:处理的猪肺组织(163mg)。
图15.在IT喷雾施加各自在实施例6的HGT5001制剂中的20μghCFTRSNIMRNA/10μgFFLSNIMRNA之后24小时小鼠的免疫沉淀和蛋白质印迹。T84细胞用作阳性对照,从而示出hCFTR的成熟糖基化C条带和甘露糖基化B条带。“上清液”残留细胞提取物级分而无免疫沉淀级分。“IP”免疫沉淀级分。
图16.使用MAB25031免疫沉淀来自T84细胞的hCFTR,接着使用AB570(A)和MAB1660(B)免疫检测。
图17.在用不同构建体转染之后72小时免疫沉淀来自NIH3T3细胞的CFTR。
图18.在使用500ug蛋白质和MAB1660(左图和中心图)用不同构建体和使用MAB25031(右图)用增加量的总蛋白质(8mg)转染72小时之后来自NIH3T3细胞的CFTR的免疫沉淀。
图19.在实施例6的PEI制剂中的hCFTRSNIMRNA递送之后使用MAB25031免疫沉淀hCFTR并且随后使用来自猪肺样品的AB570免疫检测。泳道1:来自#2猪的荧光素酶阴性左尾叶的样品,泳道2:来自#1猪的荧光素酶阳性肺区域的样品。
图20.对麻醉的和通气的猪(左)进行雾化。喷雾器同轴连接至通气系统(右,参见白色箭头)。
图21.在用EFlow筛孔喷雾器气雾剂施用在实施例6的PEI制剂中的1mgFFLSNIMRNA之后在来自不同肺区域的猪组织标本的匀浆中测量到荧光素酶表达。将肺标本离体培养过夜,之后进行荧光素酶测量(pg荧光素酶/mg肺组织)。
图22.在气雾剂施用在实施例6的PEI制剂中的1mgFFLSNIMRNA之后来自不同肺区域的代表性猪组织标本中的荧光素酶表达的BLI。将肺标本在测量之前离体培养过夜。
图23.在使用PARIBOY喷射喷雾器气雾剂施用在实施例6的PEI制剂中的1mgFFLSNIMRNA之后来自不同肺区域的代表性猪组织标本中的荧光素酶表达的BLI成像。将肺标本在测量之前离体培养过夜。
图24.在使用Aeroneb筛孔喷雾器气雾剂施用在实施例6的PEI制剂中的各自1mgFFLSNIMRNA和hCFTRmRNA之后来自不同肺区域的代表性猪组织标本中的荧光素酶表达的BLI。将肺标本在测量之前离体培养过夜。
图25.在使用Aeroneb筛孔喷雾器气雾剂施用“SHIRE制剂#3”(HGT5001:DOPE:Chol:DMGPEG2K(50:25:20:5)(摩尔比)中的1mgFFLSNIMRNA之后来自不同肺区域的代表性猪组织标本中的荧光素酶表达的BLI。将肺标本在测量之前离体培养过夜。
图26.来自一个未处理的对照猪的不同肺区域的猪组织标本中的荧光素酶表达的BLI。另一未处理的对照猪显示出相同结果(数据未示出)。
图27.一次处理的#3猪和#6猪的肺标本中的荧光素酶表达的BLI。使用Aeroneb筛孔喷雾器进行气雾剂施用在实施例6的PEI制剂中的各自1mgFFLSNIMRNA和hCFTRSNIMRNA。示出完整猪肺的切片。上三行:#3猪,下三行:#6猪。
图28.两次处理的#4猪和#8猪的肺标本中的荧光素酶表达的BLI。使用Aeroneb筛孔喷雾器进行气雾剂施用在实施例6的PEI制剂中的各自1mgFFLSNIMRNA和hCFTRSNIMRNA。示出完整猪肺的切片。上三行:#4猪,下三行:#8猪。
图29.三次处理的#1猪和#2猪的肺标本中的荧光素酶表达的BLI。使用Aeroneb筛孔喷雾器进行气雾剂施用在实施例6的PEI制剂中的各自1mgFFLSNIMRNA和hCFTR-mRNASNIMRNA。示出完整猪肺的切片。上三行:#1猪,下三行:#2猪。
图30.三次处理的#1猪的肺组织上的荧光素酶IHC。使用Aeroneb筛孔喷雾器进行气雾剂施用在实施例6的PEI制剂中的各自1mgFFLSNIMRNA和hCFTRSNIMRNA。荧光素酶表达出现在微红-粉红颜色(抗荧光素酶pAb1:300,G7451,Promega,RefineAP-试剂盒,chromogen:Newfuchsine)。
图31.使三倍处理的#1猪的高度BLI阳性肺组织经受hCFTRIP/WB。泳道1:T84细胞(阳性对照),泳道2:未处理的猪肺组织(300mg),泳道3:处理的猪肺组织(697mg),泳道4:处理的猪肺组织(163mg)。成熟的复合物-糖基化的hCFTR作为分散的所谓C条带出现。富含甘露糖的hCFTR作为更致密的所谓B条带出现。在T84细胞和hCFTRSNIMRNA处理的#1猪的猪肺组织中观察到hCFTR表达,而在未处理的猪中没有观察到hCFTR表达。
图32.在实施例6的PEI制剂中的hCFTRSNIMRNA递送之后使用MAB25031免疫沉淀hCFTR并且随后使用来自猪肺样品的AB570免疫检测。泳道1:来自#2猪的荧光素酶阴性左尾叶的样品,泳道2:来自#1猪的荧光素酶阳性肺区域的样品。
图33A和33B.用C-末端His10标记的(CO-CFTR-C-His10)和未标记的(CO-CFTR)密码子优化的人CFTRSNIMRNA体外转染HEK293T细胞。在转染后,收集全细胞裂解物,并且使用(A)抗-CFTR抗体#217和(B)抗-His抗体1187通过蛋白质印迹来分析人CFTR表达。将转染的样品与非转染HEK293T对照裂解物(泳道3)相比较。
图33C.用具有生长激素前导序列的编码密码子优化的人CFTR的SNIMRNA和编码C-末端His10标记的密码子优化的人CFTR(CO-CFTR-C-His10)的(GH-CO-CFTR)或SNIMRNA体外转染HEK293T细胞。在转染之后,收集全细胞裂解物,并且使用抗-CFTR抗体#217通过蛋白质印迹来分析人CFTR表达。将转染的样品与非转染HEK293T对照裂解物(泳道3)相比较。
图34.用封装在脂质(cKK-E12)或聚合物(PEI)纳米颗粒制剂内的C-末端His10标记的密码子优化的人CFTRSNIMRNA体内转染CFTR敲除小鼠。在雾化递送各自对应的mRNA制剂之后,收集右和左肺组织裂解物并且使用抗-His抗体1187通过蛋白质印迹分析CFTR表达。对照CFTR敲除肺组织和CFTR-His10HEK293裂解物分别用作阴性对照和阳性对照。
图35.在用注射用水雾化之后收集的猪肺样品中的FFL表达的生物发光检测。
图36.在用于支链25kDaPEI制剂中的1mgFFLSNIMRNA+1mgCO-CFTRSNIMRNA雾化之后收集的猪肺样品中的FFL表达的生物发光检测。
图37.在用于支链25kDaPEI制剂中的1mgFFLSNIMRNA+5mgCO-CFTRSNIMRNA雾化之后收集的猪肺样品中的FFL表达的生物发光检测。
图38.在用于支链25kDaPEI制剂中的1mgFFLSNIMRNA+10mgCO-CFTRSNIMRNA雾化之后收集的猪肺样品中的FFL表达的生物发光检测。
图39.不同组中CFTR表达的相对定量。使条带强度标准化至蛋白质阶梯中的150kDa条带。
图40.通过使用抗-CFTR抗体CFTR免疫组织化学染色在上皮细胞层内检测到并且展示清晰膜定位CFTR信号的具有至少一个上皮细胞的“CFTR阳性”支气管的代表性实例。
图41.在气雾剂递送对照(WFI)或5mgCO-CFTRSNIMRNA之后,猪肺中的CFTR的免疫组织化学染色。
图42.表示在气雾剂递送5mgCO-CFTRSNIMRNA之后通过用抗-CFTR免疫组织化学染色在猪肺中测定的“低”CFTR表达水平。
图43.表示在气雾剂递送5mgCO-CFTRSNIMRNA之后通过用抗-CFTR免疫组织化学染色在猪肺中测定的“中等”CFTR表达水平。
图44.表示在气雾剂递送5mgCO-CFTRSNIMRNA之后通过用抗-CFTR免疫组织化学染色在猪肺中测定的“高”CFTR表达水平。
图45.在气雾剂递送对照(WFI)或10mgCO-CFTRSNIMRNA之后,猪肺中的CFTR的免疫组织化学染色。
图46.每个动物CFTR阳性支气管/细支气管的相对数目的定量。在气雾剂施用后24小时每个组(WFI;和1mg、5mg、10mg人CFTRSNIMRNA)的分析。CFTR表达标准化至150kDa蛋白质标准的信号强度。(WFI=9.4±5.6%,1MG=15.2±6.6%,5MG=25.4±14.1%,10MG=20.9±3.7%;WFI对5MGp=0.0281,WFI对10MGp=0.0174)
图47.示出在通过喷雾器气雾剂递送注射用水之后,猪肺中的(A)泛素C和(B)dapB的多重核酸原位检测。
图48.示出在气雾剂递送于支链25kDaPEI制剂中的1mgFFLSNIMRNA+10mgCO-CFTRSNIMRNA之后,猪肺中的(A)泛素C和(B)dapB的多重核酸原位检测。
图49.示出在通过喷雾器气雾剂递送注射用水之后,猪中的(A)右颅侧和(B)左颅侧的多重核酸原位检测。
图50.示出在气雾剂递送于支链25kDaPEI制剂中的1mgFFLSNIMRNA+1mgCO-CFTRSNIMRNA之后,猪中的(A)右颅侧和(B)左颅侧的多重核酸原位检测。
图51.示出在气雾剂递送于支链25kDaPEI制剂中的1mgFFLSNIMRNA+5mgCO-CFTRSNIMRNA之后,猪中的(A)右颅侧和(B)左颅侧的多重核酸原位检测。
图52.示出在气雾剂递送于支链25kDaPEI制剂中的1mgFFLSNIMRNA+10mgCO-CFTRSNIMRNA之后,猪中的(A)右颅侧和(B)左颅侧的多重核酸原位检测。
图53.示出在暴露于FFL/CO-CFTR-C-His10mRNA封装的cKK-E12脂质纳米颗粒之后通过发光阳性检测处理的猪肺中的活性萤火虫荧光素酶(FFL)蛋白质。使用Pari喷射喷雾器通过雾化用1mgFFL+9mgCO-CFTR-C-His10mRNA封装的脂质纳米颗粒处理猪并且在处理后24小时处死。使用IVIS生物发光计可视化FFL发光。
图54.示出使用给予至猪10、11和12(1mg剂量)的雾化复合物转染的HEK细胞中的hCFTR表达的示例性结果。
图55.示出使用给予至猪13、14和15(5mg剂量)的雾化复合物转染的HEK细胞中和使用给予至猪19、20和21(10mg剂量)的雾化复合物转染的HEK细胞中的hCFTR表达的示例性结果。
图56.示出使用给予至猪16(5mg剂量)、22(10mg剂量)和67(1mg剂量)的雾化复合物转染的HEK细胞中的hCFTR表达的示例性结果。
图57.示出使用给予至猪17、18(5mg剂量)、23、24(10mg剂量)和68、69(1mg剂量)的雾化复合物转染的HEK细胞中的hCFTR表达的示例性结果。
发明详述
定义
如本文所用,术语“多核苷酸”通常用于指核酸(例如,DNA或RNA)。术语多核苷酸、核酸、DNA、RNA和mRNA包括由以下组成的这类分子:标准或未修饰的残基;非标准或修饰的残基;以及标准与非标准残基的混合物。
如本文所用,术语“mRNA”用于指修饰的和未修饰的RNA,包括编码区和非编码区。
如本文所用,术语mRNA的“编码区”通常是指当翻译时导致表达产物如多肽、蛋白质或酶的产生的那一部分。
“非标准核碱基”是不同于天然碱基腺嘌呤(A)、胞嘧啶(C)、鸟嘌呤(G)、胸腺嘧啶(T)或尿嘧啶(U)。非标准核碱基当其在核酸双螺旋中的碱基配对特性和在核酸双螺旋中通过DNA或RNA聚合酶并入基因座(包括局部RNA-DNA螺旋,如在通过RNA聚合酶转录DNA模板期间形成的局部RNA-DNA螺旋)与五种先前列出的核碱基中的一种最相似时是特定核碱基(A、C、G、T或U)的类似物,除了T的类似物通常还将是U的类似物,反之亦然。出于相对于第二序列确定第一序列的百分比同一性的目的,碱基的类似物不是天然碱基的错配;例如,假尿苷匹配尿苷,5-甲基胞苷匹配胞苷等。
与包括但不限于“核苷”、“碱基”、“核苷酸”或“残基”的术语结合使用的术语“非标准”应以如同其与“核碱基”结合使用的相同方式解释。
“GC含量”是在核酸序列中为鸟嘌呤残基、胞嘧啶残基或其类似物的总核碱基残基的分数或百分比。例如,包含准确地30个胞嘧啶,准确地30个鸟嘌呤,准确地一个胞嘧啶类似物以及准确地一个鸟嘌呤类似物的100nt序列具有62%的GC丰度。
如本文所用,“不利密码子”是指与同一氨基酸残基的另一个密码子相比,由哺乳动物细胞不那么有效或快速地翻译的密码子。不利密码子通常包括在密码子的第3或“摇摆”位置中具有A或U的密码子。关于不利密码子的讨论,参见例如美国专利公布2009/0069256A1。
“非天然存在的mRNA分子”是不通过野生型细胞的正常转录和剪接过程产生的mRNA。mRNA可由于其序列(例如,不存在于任何天然存在的CFTRmRNA中的一系列密码子和/或一个或多个UTR)和/或因为它包括非标准核苷酸残基而被限定为非天然存在的。非天然存在的mRNA分子可在体外合成。
在以下表1和表2中的每个中,NWT列指示在CFTR编码序列(参见,例如SEQIDNO:3)中的位置(Pos.)处的非野生型碱基并且WT列指示在同一位置处的野生型碱基(参见,例如SEQIDNO:2或人CFTR的RefSeq输入(登录号NM_000492.3,2013年2月10日,2013,版本,可从GenBank获得;注意NM_000492.3的序列包含非编码序列以使得编码序列出现在位置133至4575,以使得例如以下表中的位置7对应于NM_000492.3序列的位置139)。
非天然存在的CFTRmRNA
除了提供使用天然存在的或野生型CFTRmRNA(以及包含所述mRNA的组合物)体内产生功能性CFTR的方法,本发明还提供编码CFTR蛋白的非天然存在的CFTRmRNA(例如,SEQIDNO:1)。在一些实施方案中,纯化或分离非天然存在的CFTRmRNA。
在其它实施方案中,非天然存在的CFTRmRNA存在于细胞中。在一些实施方案中,包含非天然存在的CFTRmRNA的细胞不合成非天然存在的CFTRmRNA和/或不包含与非天然存在的CFTRmRNA互补的DNA和/或功能性CFTR基因;所述细胞可任选地包含无活性CFTR基因,如具有使基因的表达产物无功能性的无义、错义、移码、插入或缺失突变的CFTR基因。在一些实施方案中,包含非天然存在的CFTRmRNA的细胞还包含从非天然存在的CFTRmRNA翻译的功能性CFTR蛋白。所述细胞可以是例如肺上皮细胞、肝细胞或肾细胞。在一些实施方案中,所述细胞是在细胞培养物中。
CFTR编码序列
在一些实施方案中,根据本发明的CFTRmRNA包含具有比SEQIDNO:2(即,野生型人CFTR的编码序列)更少的隐蔽性启动子的互补序列、比SEQIDNO:2更少的同向和/或反向重复序列、比SEQIDNO:2更少的不利密码子和/或比SEQIDNO:2的GC含量更低的编码序列的GC含量的编码序列。
序列的隐蔽性启动子、同向和/或反向重复序列和/或不利密码子可由本领域的技术人员使用常规方法识别。例如,序列的同向和/或反向重复序列含量可通过序列分析确定(Liu等,JournalofTheoreticalBiology(2014)344:19-30)。序列的隐蔽性启动子含量还可通过序列分析确定,例如构建体内Shine-Dalgarno序列的存在等。
在一些实施方案中,根据本发明的CFTRmRNA是体外转录的,即所述mRNA在不在生物细胞内的人工环境(例如,不含细胞的体外转录系统)中合成。通常,体外转录涉及在适合的反应条件(盐、缓冲液和温度)下提供包含启动子的DNA模板和与所需mRNA互补的序列(其可以是环状或线性的)、RNA聚合酶以及核苷三磷酸。RNA酶抑制剂、还原剂和/或焦磷酸酶可存在于反应混合物中。在一些实施方案中,RNA聚合酶是T7RNA聚合酶。
在一些实施方案中,根据本发明的CFTRmRNA包含编码序列,所述编码序列相对于SEQIDNO:2的野生型编码序列在表1中所列出的编码序列的位置处包含至少50%、至少55%、至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%或至少95%的表1中所列出的非野生型碱基。
表1.可用于编码CFTR的mRNA的编码序列中的非野生型碱基。
在一些实施方案中,根据本发明的CFTRmRNA包含编码序列,所述编码序列相对于SEQIDNO:2的野生型编码序列在表2中所列出的编码序列的相应位置处包含至少50%、至少55%、至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%或至少95%的表2中所列出的非野生型碱基。
表2.可用于编码CFTR的mRNA的编码序列中的非野生型碱基的子集。
在一些实施方案中,本发明包括一种非天然存在的CFTRmRNA,其包含SEQIDNO:3的编码序列。另外的示例性非天然存在的CFTRmRNA编码序列描述于序列简述部分中,例如像SEQIDNO:9、10、11、12、13、14、15、16或17。在一些实施方案中,本发明提供一种CFTRmRNA,其包含与SEQIDNO:3、9、10、11、12、13、14、15、16或17中的任一个至少50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、96%、97%、98%或99%相同的编码序列。在一些实施方案中,非天然存在的CFTRmRNA包含如以下所述的5’UTR、3’UTR、信号肽编码序列或帽或尾结构。
包含与野生型CFTR编码序列不同的编码序列的上述CFTRmRNA可提供关于功效和易于制备的优点。例如,使用包含与CFTR编码序列互补的模板序列的多核苷酸的体外转录反应可提供较大RNA产率;包含所述模板序列的多核苷酸可在宿主细胞中生长期间更稳定(即,不易于突变),从而减少产生在反应中可用的模板所需的纯化的量;并且包含编码序列的mRNA的体内翻译可更高。
信号肽序列
在一些实施方案中,编码CFTR蛋白的mRNA并入编码信号肽的核苷酸序列。如本文所用,术语“信号肽”是指在新合成的蛋白质处存在的可使蛋白质靶向分泌途径的肽。在一些实施方案中,在mRNA的翻译之后,信号肽在易位之后裂解成内质网。信号肽还被称为信号序列、前导序列或前导肽。通常,信号肽是较短(例如,5-30、5-25、5-20、5-15或5-10个氨基酸长)肽。信号肽可存在于新合成的蛋白质的N-末端。不希望受任何具体理论约束,在CFTR编码mRNA上并入信号肽编码序列可促进体内CFTR蛋白质的分泌和/或产生。
用于本发明的适合的信号肽可以是源自不同真核和原核蛋白质、具体地说分泌的蛋白质的异质序列。在一些实施方案中,适合的信号肽是富含亮氨酸的序列。参见YamamotoY等(1989),Biochemistry,28:2728-2732,其以引用的方式并入本文。适合的信号肽可源自人生长激素(hGH)、血清白蛋白前蛋白原、Igκ轻链前体、天青杀素前蛋白原、半胱氨酸蛋白酶抑素S前体、胰蛋白酶原2前体、钾通道阻断剂、α-芋螺毒素lp1.3、α-芋螺毒素、α-半乳糖苷酶、纤维素、天冬氨酸蛋白酶猪笼草蛋白酶(nepenthesin)-1、酸性壳多糖酶、K28前原-毒素、杀伤毒素zygocin前体以及霍乱毒素。示例性信号肽序列描述于Kober,等,Biotechnol.Bioeng.,110:1164-73,2012中,其以引用的方式并入本文。
在一些实施方案中,CFTR编码mRNA可并入编码源自人生长激素(hGH)或其片段的信号肽的序列。编码hGH信号肽的非限制性核苷酸序列在以下示出。
5’人生长激素(hGH)序列(SEQIDNO:18):
AUGGCCACUGGAUCAAGAACCUCACUGCUGCUCGCUUUUGGACUGCUUUGCCUGCCCUGGUUGCAAGAAGGAUCGGCUUUCCCGACCAUCCCACUCUCC
替代5’人生长激素(hGH)序列(SEQIDNO:19):
AUGGCAACUGGAUCAAGAACCUCCCUCCUGCUCGCAUUCGGCCUGCUCUGUCUCCCAUGGCUCCAAGAAGGAAGCGCGUUCCCCACUAUCCCCCUCUCG
在一些实施方案中,根据本发明的mRNA可并入与SEQIDNO:18或SEQIDNO:19具有至少50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、96%、97%、98%或99%或更多同一性的信号肽编码序列。
5’-UTR、3’-UTR、聚腺苷酸尾、帽和非标准核苷酸残基
在一些实施方案中,所述mRNA在其5’-UTR中包含与SEQIDNO:4相同或与SEQIDNO:4至少50%、至少55%、至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少98%或至少99%相同的序列。
在一些实施方案中,所述mRNA在其3’-UTR中包含与SEQIDNO:5相同或与SEQIDNO:5至少50%、至少55%、至少60%、至少65%、至少70%、至少75%、至少80%、至少85%、至少90%、至少95%、至少98%或至少99%相同的序列。
在一些实施方案中,所述mRNA包含聚腺苷酸尾。在一些实施方案中,聚腺苷酸尾具有至少70、100、120、150、200、250、300、400或500个残基的长度。在一些实施方案中,聚腺苷酸尾具有70至100、100至120、120至150、150至200或200至300、300至400或400至500个残基范围内的长度。聚腺苷酸尾可使用多种本领域公认的技术添加。例如,可添加长聚腺苷酸尾以使用聚腺苷酸聚合酶合成或体外转录RNA(Yokoe,等NatureBiotechnology.1996;14:1252-1256)。转录载体还可编码长聚腺苷酸尾。此外,聚腺苷酸尾可通过直接从PCR产物转录来添加。聚腺苷酸还可用RNA连接酶连接至有义RNA的3'端(参见例如,MolecularCloningALaboratoryManual,第2版,Sambrook,Fritsch和Maniatis编辑(ColdSpringHarborLaboratoryPress:1991版))。在一些实施方案中,聚尿苷酸或聚胞苷酸尾可替代或除聚腺苷酸尾之外使用。例如,CFTR编码mRNA可包括3’聚(C)尾结构。mRNA的3'末端上的适合的聚胞苷酸尾通常包括约10至200个胞嘧啶核苷酸(例如,约10至150个胞嘧啶核苷酸、约10至100个胞嘧啶核苷酸、约20至70个胞嘧啶核苷酸、约20至60个胞嘧啶核苷酸或约10至40个胞嘧啶核苷酸)。聚胞苷酸尾可添加至聚腺苷酸尾或可取代聚腺苷酸尾。
在一些实施方案中,所述mRNA包含5’-帽,例如,帽1结构。对于mRNA加帽酶和程序,参见例如Fechter,P.;Brownlee,G.G.“RecognitionofmRNAcapstructuresbyviralandcellularproteins”J.Gen.Virology2005,86,1239-1249;欧洲专利公布2010659A2;U.S.专利号6,312,926。通常如下添加5’帽:首先,RNA末端磷酸酶从5’核苷酸除去一个末端磷酸基团,从而留下两个末端磷酸;然后经由鸟苷酸转移酶将鸟苷三磷酸(GTP)添加至末端磷酸,从而产生5’5’5三磷酸键联;并且然后,通过甲基转移酶将鸟嘌呤的7-氮甲基化帽结构的实例包括但不限于m7G(5')ppp(5'(A,G(5')ppp(5')A和G(5')ppp(5')G。
在一些实施方案中,所述mRNA包含一个或多个非标准核苷酸残基。非标准核苷酸残基可包括例如5-甲基-胞苷(“5mC”)、假尿苷(“ψU”)和/或2-硫代-尿苷(“2sU”)。关于这类残基及其并入mRNA中的讨论,参见,例如美国专利号8,278,036或WO2011012316。在一些实施方案中,mRNA可以是SNIMRNA。如本文所用,SNIMRNA是稳定化非免疫原性信使RNA的首字母缩写词,指代通过体外转录(IVT)产生的信使RNA,包括如在PCT公布WO2011/012316中描述的IVT反应中的修饰的核苷酸的某些优点。用于本文公开的实施例中的SNIMRNA通过IVT产生,其中25%的U残基是2-硫代-尿苷并且25%的C残基是5-甲基胞苷。非标准核苷酸残基的存在可使mRNA比具有相同序列但仅含有标准残基的对照mRNA更稳定和/或免疫原性更低。在其它实施方案中,mRNA可包含选自异胞嘧啶、假异胞嘧啶、5-溴尿嘧啶、5-丙炔基尿嘧啶、6-氨基嘌呤、2-氨基嘌呤、肌苷、二氨基嘌呤和2-氯-6-氨基嘌呤胞嘧啶以及这些修饰的组合和其它核碱基修饰的一个或多个非标准核苷酸残基。某些实施方案还可包括呋喃糖环或核碱基的另外修饰。另外的修饰可包括,例如,糖修饰或取代(例如,一个或多个2′-O-烷基修饰、锁核酸(LNA))。在一些实施方案中,RNA可与另外的多核苷酸和/或肽多核苷酸(PNA)复合或杂交。在糖修饰是2′-O-烷基修饰的实施方案中,这种修饰可包括但不限于2′-脱氧-2′-氟修饰、2′-O-甲基修饰、2′-O-甲氧基乙基修饰和2′-脱氧修饰。在某些实施方案中,这些修饰中的任一种可以0%-100%的核苷酸存在—例如单独地或组合地多于0%、1%、10%、25%、50%、75%、85%、90%、95%或100%的组成核苷酸。
包含CFTRmRNA的组合物
在某些实施方案中,本发明的mRNA分子可作为裸的或未封装的mRNA施用。在一些实施方案中,施用本发明的组合物中的mRNA可通过引入适合的载体促进。在某些实施方案中,载体是基于其以一种或多种mRNA促进靶细胞的转染的能力进行选择。
如本文所用,术语“载体”包括通常意图用于与施用生物活性剂(包括mRNA)结合使用的标准药物载体、媒介物、稀释剂、赋形剂等中的任一种。
在某些实施方案中,在本发明的组合物中采用的载体可包含脂质体囊泡,或有助于mRNA转移至靶细胞和/或组织的其它方式。适合的载体包括但不限于,基于聚合物的载体,如聚乙烯亚胺(PEI)和多结构域-嵌段聚合物、脂质纳米颗粒和脂质体、纳米脂质体、含神经酰胺纳米脂质体、蛋白脂质体、天然和合成衍生的外来体两者、天然的、合成的和半合成的片层体、纳米颗粒、磷光体硅酸钙纳米颗粒、磷酸钙纳米颗粒、二氧化硅纳米颗粒、纳米晶体颗粒、半导体纳米颗粒、干粉、聚(D-精氨酸)、纳米树枝状聚合物、基于淀粉的递送系统、胶束、乳液、溶胶-凝胶、非离子型表面活性剂脂质体囊泡(noisome)、质粒、病毒、磷酸钙核苷酸、适体、肽、肽缀合物、小分子靶向缀合物以及其它媒介标签。还考虑使用生物纳米胶囊和其它病毒衣壳蛋白组件作为适合的载体。(Hum.GeneTher.2008年9月;19(9):887-95)。
在一些实施方案中,载体包含有机阳离子,如阳离子脂质或阳离子有机聚合物。如果存在,阳离子脂质可以是封装mRNA的脂质体囊泡的组分。
在本发明的某些实施方案中,载体使用聚合物作为载体,单独或与其它载体组合来配制。适合的聚合物可包括例如,聚丙烯酸酯、聚氰基丙烯酸烷基酯、聚丙交酯、聚丙交酯-聚乙交酯共聚物、聚己内酯、葡聚糖、白蛋白、明胶、藻酸盐、胶原、壳聚糖、环糊精、鱼精蛋白、聚乙二醇化鱼精蛋白、PLL、聚乙二醇化PLL和聚乙烯亚胺(PEI)。当PEI存在时,其可以是10至40kDa范围内的分子量的支链PEI,例如25kDa支链PEI(Sigma#408727)。适合用于本发明的另外示例性聚合物包括描述于PCT公布WO2013182683中的那些,其内容特此以引用的方式并入。
本发明考虑使用脂质体载体以促进将多核苷酸递送至靶细胞。脂质体(例如,脂质体脂质纳米颗粒)通常适用于研究、工业和医学中的多种应用,尤其是在体内用作诊断性或治疗性化合物的载体(Lasic,TrendsBiotechnol.,16:307-321,1998;Drummond等,Pharmacol.Rev.,51:691-743,1999)并且通常被表征为具有通过一个或多个双层膜与外部介质隔离的内部含水空间的微观囊泡。脂质体的双层膜通常由两亲性分子形成,如合成或天然来源的脂质,其包含空间分离的亲水性和疏水性结构域(Lasic,TrendsBiotechnol.,16:307-321,1998)。脂质体的双层膜也可通过两亲性聚合物和表面活性剂(例如,高分子囊泡、非离子型表面活性剂脂质体囊泡等)来形成。
在某些实施方案中,所述mRNA与脂质纳米颗粒复合以促进递送至靶细胞。在某些实施方案中,本发明的组合物可与采用一种或多种阳离子脂质的多组分脂质混合物组合,另外的脂质如非阳离子脂质(还被称为辅助脂质)、基于胆固醇的脂质和/或聚乙二醇化的脂质形成mRNA封装。
阳离子脂质
在一些实施方案中,适合的脂质纳米颗粒含有阳离子脂质。如本文所用,短语“阳离子脂质”是指在选定pH,如生理pH下具有净正电荷的许多脂质种类中的任何一种。一些阳离子脂质,具体地说被称为可滴定或pH-可滴定阳离子脂质的那些在递送mRNA中特别有效。一些阳离子(例如,可滴定)脂质已经在文献中描述,其中许多可商购获得。用于本发明的组合物和方法中的尤其适合的阳离子脂质包括在国际专利公布WO2010/053572(以及尤其在段落[00225]中描述的C12-200)和WO2012/170930中描述的那些,其均以引用的方式并入本文。在一些实施方案中,使用阳离子脂质cKK-E12(在WO2013/063468中公开),其教义以引用的方式整体并入本文。在一些实施方案中,使用阳离子脂质N-[l-(2,3-二油酰氧基)丙基]-N,N,N-三甲基铵氯化物或“DOTMA”。(Feigner等(Proc.Nat'lAcad.Sci.84,7413(1987);美国专利号4,897,355)。DOTMA可单独配制或可与中性脂质、二油酰磷脂-乙醇胺或“DOPE”或其它阳离子或非阳离子脂质组合成脂质体转移媒介物或脂质纳米颗粒,并且这类脂质体可用于增强将核酸递送至靶细胞中。其它适合的阳离子脂质包括,例如,5-羧基鲸蜡基甘氨酸二十八烷基酰胺或“DOGS”、2,3-二油酰氧基-N-[2(精胺-甲酰胺)乙基]-N,N-二甲基-l-丙铵或“DOSPA”(Behr等Proc.Nat.'lAcad.Sci.86,6982(1989);美国专利号5,171,678;美国专利号5,334,761)、l,2-二油酰基-3-二甲基铵-丙烷或“DODAP”、l,2-二油酰基-3-三甲基铵-丙烷或“DOTAP”。涵盖的阳离子脂质还包括l,2-二硬脂酰氧基-N,N-二甲基-3-氨基丙烷或“DSDMA”、1,2-二油酰氧基-N,N-二甲基-3-氨基丙烷或“DODMA”、1,2-二亚油醇氧基-N,N-二甲基-3-氨基丙烷或“DLinDMA”、l,2-二亚油烯醇氧基-N,N-二甲基-3-氨基丙烷或“DLenDMA”、N-二油酰基-N,N-二甲基铵氯化物或“DODAC”、N,N-二硬脂酰基-N,N-二甲基铵溴化物或“DDAB”、N-(l,2-二肉豆蔻氧基丙-3-基)-N,N-二甲基-N-羟乙基溴化铵或“DMRIE”、3-二甲氨基-2-(胆甾-5-烯-3-β-氧基丁烷-4-氧基)-l-(顺式,顺式-9,12-十八二烯氧基)丙烷或“CLinDMA”、2-[5'-(胆甾-5-烯-3-β-氧基)-3'-氧杂戊氧基)-3-二甲基l-l-(顺式,顺式-9',l-2'-十八二烯氧基)丙烷或“CpLinDMA”、N,N-二甲基-3,4-二油酰氧基苄胺或“DMOBA”、1,2-N,N'-二油酰基氨基甲酰-3-二甲氨基丙烷或“DOcarbDAP”、2,3-二亚油醇氧基-N,N-二甲基丙胺或“DLinDAP”、l,2-N,N'-二亚油醇氨基甲酰-3-二甲氨基丙烷或“DLincarbDAP”、l,2-二亚油醇氨基甲酰-3-二甲氨基丙烷或“DLinCDAP”、2,2-二亚油醇-4-二甲氨基甲基-[l,3]-二氧戊环或“DLin--DMA”、2,2-二亚油醇-4-二甲氨基乙基-[l,3]-二氧戊环或“DLin-K-XTC2-DMA”和2-(2,2-二((9Z,12Z)-十八-9,l2-二烯-1-基)-l,3-二氧戊环-4-基)-N,N-二甲基乙胺(DLin-KC2-DMA))(参见,WO2010/042877;Semple等,NatureBiotech.28:172-176(2010))或其混合物。(Heyes,J.等,JControlledRelease107:276-287(2005);Morrissey,DV.等,Nat.Biotechnol.23(8):1003-1007(2005);PCT公布WO2005/121348A1)。
在某些实施方案中,本发明的组合物和方法使用包含可电离阳离子脂质的脂质纳米颗粒,其描述于2013年3月29日提交的美国临时专利申请61/617,468(以引用的方式并入本文),如,例如,(15Z,18Z)-N,N-二甲基-6-(9Z,12Z)-十八-9,12-二烯-l-基)二十四-15,18-二烯-1-胺(HGT5000)、(15Z,18Z)-N,N-二甲基-6-((9Z,12Z)-十八-9,12-二烯-1-基)二十四-4,15,18-三烯-l-胺(HGT5001)和(15Z,18Z)-N,N-二甲基-6-((9Z,12Z)-十八-9,12-二烯-1-基)二十四-5,15,18-三烯-1-胺(HGT5002)。
在一些实施方案中,存在于这种组合物中的阳离子脂质中的一种或多种包括咪唑、二烷基氨基或胍鎓部分中的至少一种。在优选的实施方案中,一种或多种阳离子脂质不包含季胺。
非阳离子/辅助脂质
在一些实施方案中,适合的脂质纳米颗粒含有一种或多种非阳离子(“辅助”)脂质。如本文所用,短语“非阳离子脂质”是指任何中性、两性离子或阴离子脂质。如本文所用,短语“阴离子脂质”是指在选定pH,如生理pH下携带净负电荷的许多脂质种类中的任何一种。在一些实施方案中,非阳离子脂质是中性脂质,即,在组合物配制和/或施用的条件下不带有净电荷的脂质。非阳离子脂质包括但不限于,二硬脂酰磷脂酰胆碱(DSPC)、二油酰磷脂酰胆碱(DOPC)、二棕榈酰磷脂酰胆碱(DPPC)、二油酰磷脂酰甘油(DOPG)、二棕榈酰磷脂酰甘油(DPPG)、二油酰磷脂酰乙醇胺(DOPE)、棕榈酰油酰磷脂酰胆碱(POPC)、棕榈酰油酰-磷脂酰乙醇胺(POPE)、二油酰基-磷脂酰乙醇胺4-(N-马来酰亚胺甲基)-环已烷-l-羧酸酯(DOPE-mal)、二棕榈酰基磷脂酰基乙醇胺(DPPE)、二肉豆蔻酰基磷酸乙醇胺(DMPE)、二硬脂酰-磷脂酰基-乙醇胺(DSPE)、16-O-单甲基PE、16-O-二甲基PE、18-1-反PE、l-硬脂酰-2-油酰基-磷脂酰乙醇胺(SOPE)或其混合物。
基于胆固醇的脂质
在一些实施方案中,适合的脂质纳米颗粒包含一种或多种基于胆固醇的脂质。例如,适合的基于胆固醇的阳离子脂质包括例如,胆固醇、聚乙二醇化胆固醇、DC-Choi(N,N-二甲基-N-乙基羧酰胺胆固醇)、l,4-双(3-N-油酰氨基-丙基)哌嗪(Gao,等Biochem.Biophys.Res.Comm.179,280(1991);Wolf等BioTechniques23,139(1997);美国专利号5,744,335)或ICE。
聚乙二醇化脂质
在一些实施方案中,适合的脂质纳米颗粒包含一种或多种聚乙二醇化脂质。例如,本发明涵盖与一种或多种阳离子脂质、并且在一些实施方案中其它脂质组合使用聚乙二醇(PEG)-修饰的磷脂和衍生脂质如衍生神经酰胺(PEG-CER),包括N-辛酰基-鞘氨醇-l-[丁二酰(甲氧基聚乙二醇)-2000](C8PEG-2000神经酰胺)。在一些实施方案中,适合的聚乙二醇化脂质包含具有较短酰基链(例如,C14或C18)的PEG-神经酰胺。在一些实施方案中,可使用聚乙二醇化脂质DSPE-PEG-马来酰亚胺-凝集素。其它涵盖的PEG-修饰的脂质包括但不限于与C6-C20长度的烷基链共价连接至脂质的多达5kDa长度的聚乙二醇链。不希望受具体理论约束,考虑添加聚乙二醇化脂质可防止复合体聚集并增加循环寿命以促进脂质体封装的mRNA递送至靶细胞。
在某些实施方案中,所述组合物包含以下脂质组合中的一种:
C12-200、DOPE、胆固醇、DMG-PEG2K;
DODAP、DOPE、胆固醇、DMG-PEG2K;
HGT5000、DOPE、胆固醇、DMG-PEG2K;
HGT5001、DOPE、胆固醇、DMG-PEG2K;
XTC、DSPC、胆固醇、PEG-DMG;
MC3、DSPC、胆固醇、PEG-DMG;
ALNY-100、DSPC、胆固醇、PEG-DSG;
cKK-E12、DOPE、Chol、PEGDMG2K。
在一些实施方案中,脂质:mRNA比可以是5:1(mg:mg)、6:1、7:1、8:1、9:1、10:1以及更高至30:1(mg:mg)或更多。N/P比可以在1.1:1达10:1或更高的范围内。示例性脂质比是40:30:20:10、55:20:20:5、50:25:20:5(阳离子脂质:辅助脂质:chol:PEG脂质)。
在一些实施方案中,根据本发明的药物组合物不包含粘液溶解剂(例如,N-乙酰半胱氨酸、厄多司坦、溴己新(bromheksin)、羧甲司坦、呱芬那辛(guiafenesin)或碘化甘油)。
负载有药物组合物的装置
在一些实施方案中,根据本发明的药物组合物,如包含非天然存在的CFTRmRNA的基于阳离子脂质或基于PEI的组合物提供在用于施用至受试者的呼吸系统的装置内。所述装置可以是,例如,滴注、气雾化或雾化装置。适合的装置包括,例如,PARIBoy喷射喷雾器、Lab喷雾器、或EFlow筛孔喷雾器。或者,可使用干粉吸入器或气雾化装置(如便携式吸入器)。
用途和方法
根据本发明的mRNA用途和方法
除其它事项之外,本发明提供用于体内、特别是在哺乳动物的肺中产生CFTR蛋白的方法。在一些实施方案中,本发明提供在哺乳动物的肺中的上皮细胞中诱导CFTR表达的方法,所述方法包括使所述上皮细胞与包含体外转录的mRNA的药物组合物相接触,其中所述体外转录的mRNA包含编码SEQIDNO:1(野生型人CFTR的氨基酸序列)的编码序列。本发明还提供包含体外转录的mRNA的药物组合物用于在哺乳动物的肺中的上皮细胞中诱导CFTR表达的的用途,其中所述体外转录的mRNA包含编码SEQIDNO:1的编码序列。
本发明还提供在哺乳动物靶细胞中诱导CFTR表达的方法,所述方法包括使所述哺乳动物靶细胞与组合物相接触,所述组合物包含编码SEQIDNO:1的氨基酸序列的体外转录的mRNA。本发明还提供组合物用于在哺乳动物靶细胞中诱导CFTR表达的用途,所述组合物包含编码SEQIDNO:1的氨基酸序列的体外转录的mRNA。
在所述用途和治疗方法的一些实施方案中,体外转录的mRNA是编码人CFTR(SEQIDNO:2)的天然存在的或野生型mRNA。在其它实施方案中,体外转录的mRNA是如上所述的非天然存在的mRNA。
在某些实施方案中,所述体外转录的mRNA包含编码序列,所述编码序列编码与SEQIDNO:2(野生型人CFTRmRNA编码序列)至少65%、70%、75%、80%、85%、88%、90%、92%、95%或100%相同的SEQIDNO:1。
包含编码序列的mRNA可具有比上述mRNA更大的隐蔽性启动子、同向和反向重复序列和/或GC含量,所述编码序列编码与SEQIDNO:2至少65%、70%、75%、80%、85%、90%、95%、99%或100%相同的SEQIDNO:1。观察到在典型生长条件下包含SEQIDNO:2的载体在宿主细胞中频繁地进行插入/缺失/重排突变,从而产生不能直接用于体外转录的不均匀的载体群体。据发现,在如较低温度、柔光和/或低拷贝细胞如的条件下培养宿主细胞减少但并未消除突变的发生。因此,可能可取的是包含与SEQIDNO:2至少65%、70%、75%、80%、85%、90%、95%、99%或100%相同的编码序列的mRNA的体外转录反应使用通过以下方式获得的模板:生长如上所述的载体,收获且线性化所述载体,并且纯化用于在转录反应中使用的所需种类。所述纯化步骤可以是例如尺寸排阻色谱法或弱阴离子交换。
用于根据本发明的用途和方法的体外转录的mRNA可包含5’-UTR、3’UTR、聚腺苷酸、聚尿苷酸和/或聚胞苷酸尾、帽和/或非标准核苷酸残基,如关于所述特征的以上部分中所讨论。
用于使用和方法的药物组合物
用于根据本发明使用的药物组合物可包含mRNA如在前述部分中所讨论用于根据本发明的用途和方法的mRNA和如在以上部分中所讨论关于包含CFTRmRNA的组合物的另外成分。因此,考虑包含以上所讨论的任何载体的药物组合物的使用和/或施用。
在一些优选的实施方案中,药物组合物包含PEI,如具有10-40kDa范围内(例如25kDa)的分子量的支链PEI。
在其它优选的实施方案中,药物组合物包含阳离子脂质、聚乙二醇化脂质和其它脂质(如中性脂质)。所述阳离子脂质、聚乙二醇化脂质和/或其它脂质可选自以上部分中关于包含CFTRmRNA的组合物所列出的那些。
用于在肺中诱导表达的施用途径
在用于在哺乳动物的肺中诱导CFTR表达的方法和用途的一些实施方案中,如上所述的药物组合物通过选自气管内滴注、雾化和气雾化的途径施用。用于施用所述组合物的装置可选自在以上部分中关于负载有药物组合物的装置所列出的装置。
在优选的实施方案中,所述组合物通过雾化或气雾化来施用。一些脂质制剂在试图雾化时可能具有聚集的倾向但是通常可能通过调整配方,例如通过取代阳离子脂质来解决聚集问题。
囊性纤维化的治疗
除其它事项之外,本发明可用于治疗囊性纤维化。在一些实施方案中,本发明提供一种通过向需要治疗的受试者施用如本文所述的编码CFTR蛋白的mRNA或含有所述mRNA的药物组合物来治疗囊性纤维化的方法。所述mRNA或含有所述mRNA的药物组合物可直接施用至受试者的肺。可使用用于肺递送的各种施用途径。在一些实施方案中,mRNA或含有本文所述的mRNA的组合物通过吸入、雾化或气雾化来施用。在不同实施方案中,施用mRNA导致受试者的肺中(例如,肺的上皮细胞中)CFTR的表达。
在一个具体实施方案中,本发明提供一种通过向需要治疗的受试者的肺施用包含编码SEQIDNO:1的编码序列的mRNA来治疗囊性纤维化的方法。在某些实施方案中,本发明提供一种通过向需要治疗的受试者的肺施用包含编码序列的mRNA来治疗囊性纤维化的方法,所述编码序列编码与SEQIDNO:1至少约70%、75%、80%、85%、90%、95%、96%、97%、98%或99%相同的氨基酸序列。在另一个具体实施方案中,本发明提供一种通过向需要治疗的受试者的肺施用包含SEQIDNO:3的编码序列的mRNA来治疗囊性纤维化的方法。在其它实施方案中,本发明提供一种通过向需要治疗的受试者的肺施用包含编码序列的mRNA来治疗囊性纤维化的方法,所述编码序列与SEQIDNO:3至少65%、70%、75%、80%、85%、90%、95%或99%相同。可用于治疗囊性纤维化的另外的示例性非天然存在的CFTRmRNA描述于序列简述部分中,例如像SEQIDNO:9、10、11、12、13、14、15、16或17。在一些实施方案中,可用于治疗囊性纤维化的非天然存在的CFTRmRNA包含与SEQIDNO:3、9、10、11、12、13、14、15、16或17中的任一个至少约50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、96%、97%、98%或99%相同的编码序列。
实施例
以下具体实施例应仅理解为说明性的,而不是以任何方式限定公开内容的剩余部分。无需进一步详述,据信本领域的技术人员可基于本文描述而在最大程度上利用本发明。
除非另外指明,否则用于本文公开的实施例中的CFTRmRNA和SNIMRNA包含具有SEQIDNO:4的序列的5’UTR、具有SEQIDNO:3的序列的编码序列(CDS)以及具有SEQIDNO:5的序列的3’UTR。用于本文公开的实施例中的FFLmRNA和SNIMRNA包含分别具有SEQIDNO:6、7和8的序列的5’UTR、CDS和3’UTR。
实施例1:体外合成的编码CFTR的mRNA
信使RNA合成。通过体外转录从编码基因的质粒DNA模板合成人囊性纤维化跨膜传导调节因子(CFTR)mRNA和萤火虫荧光素酶(FFL)mRNA,接着添加5’帽结构(帽1)(Fechter,P.;Brownlee,G.G.“RecognitionofmRNAcapstructuresbyviralandcellularproteins”J.Gen.Virology2005,86,1239-1249)和如通过凝胶电泳所测定大约200个核苷酸长的3’聚(A)尾。5’和3’非翻译区存在于每种mRNA产物中。
示例性非天然存在的CFTRmRNA包括在序列简述章节中描述的SEQIDNO:9、SEQIDNO:10、SEQIDNO:11、SEQIDNO:12、SEQIDNO:13、SEQIDNO:14、SEQUDNO:15、SEQIDNO:16或SEQIDNO:17。
实施例2:HEK细胞中的CFTR表达和活性
该实施例证明完全功能性CFTR蛋白从递送至细胞的合成人CFTRmRNA表达。
细胞和CFTR转染。将人胚肾HEK293T细胞生长在补充有10%胎牛血清、2mML-谷氨酰胺、100U/ml青霉素和100μg/ml链霉素的DMEM(Invitrogen目录号11965-092)中。在转染前一天,将细胞以50%-60%汇合涂铺在6-孔板上并且在正常组织培养条件(36℃,在5%CO2、95%空气的增湿大气中)下孵育。将60μlLipofectamine2000(Invitrogen目录号11668019)稀释于900μlOptiMem低血清培养基(Invitrogen目录号31985-062)中并且轻轻涡旋。将24μgCFTRmRNA(4μg/板)稀释于900μlOptiMem培养基中。将mRNA立即添加至稀释的Lipofectamine并且在室温下孵育30分钟。将涂铺培养基轻轻地从HEK293T细胞中抽吸出并用1mlOptiMem低血清培养基代替。将300μl的mRNA/Lipofectamine复合物添加至每个孔中,并且允许细胞在正常组织培养条件下静置24小时,之后通过机械脱离再涂铺在聚-L-赖氨酸涂覆的玻璃盖玻片(BDBiosciences,BDBiocoat),以使得所述细胞可容易地转移至记录室中用于电生理记录。将细胞在标准组织培养条件下再孵育最少24小时并且在最终涂铺48小时内使用。
电生理记录。在室温下使用具有5-8MΩ电极的Axopatch200B放大器进行全细胞膜片钳记录。将数据进行数字化(50kHz)并且适当地过滤(5kHz)。对串联电阻进行补偿(70%-80%)以使电压误差最小化。用以下组成的移液管溶液进行电压钳记录:140mMNMDG-Cl;5mMEGTA;1mMMgCl2;10mMHEPES;pH7.2;310mOsm/l。浴溶液含有:140mMNaCl、3mMKCl、2mMMgCl2、2mMCaCl2和10mMHEPES;pH7.3,用D-葡萄糖调节至315mOsm/l。电压钳记录在建立全细胞构型之后3-5分钟开始。
将细胞以-60mV或0mV的保持电位电压钳夹并且一系列正电压和负电压步骤(-80mV至+80mV或-100至+100mV,以20mV增量)注入到所记录的HEK293T细胞中以诱发CFTR诱导的全细胞氯离子(Cl-)电流。将cAMP、8-Br-cAMP(500μM,SigmaAldrich)的膜可渗透类似物施加至所记录的细胞持续4分钟以促进CFTR电流。在每次记录结束时施加“黄金标准”CFTR阻断剂CFTRinh-172(10μM,Sigma)以阻断CFTR诱导的Cl-电流。在未转染的HEK293T细胞中进行对照记录。
测试化合物。使用具有距所记录的细胞大约200μm放置的推顶移液管的DAD-16VC快速灌注系统(ALAScientificInstruments,USA)施加测试化合物。8-Br-cAMP被制备为ddH20中的500mM储备液浓度。CFTRinh-172被制备为DMSO中的10mM储备液。将所有化合物储存在-20℃下并且进行快速除霜且就在使用之前稀释至所需最终浓度。
分析。使用Clampfit(MDSAnalyticalTechnologies)和Excel(Microsoft)软件进行所有分析。所有值是最大诱发峰值电流振幅。数据中的统计差异通过学生t-检验(在适当时配对或未配对)进行评价,并且在P<0.05下被认为是显著的。
体外人CFTR蛋白产生。经由hCFTRmRNA产生人CFTR蛋白是经由在本文所述的HEK293T细胞中转染人CFTRmRNA来实现。收获处理的和未处理的细胞并且在转染后24小时进行免疫沉淀方法。经由蛋白质印迹分析检测人CFTR蛋白证明,完全复合物糖基化的CFTR蛋白(被指定为“C”条带)由合成信使RNA(图1A)产生。
体外人CFTR蛋白活性。为了测定在转染之后产生的合成人CFTRmRNA源性的CFTR蛋白的活性,在HEK293细胞和HEK293T细胞两者中进行全细胞膜片钳测定。使处理的细胞以及对照细胞(未处理的和模拟转染的)经受活化剂(8-Br-cAMP,毛喉素)和抑制剂(CFTRinh-172,GlyH-101)底物以帮助测定电流流动的变化(氯离子转运)。
将HEK293T细胞用4ug的hCFTRmRNA转染,并且在转染后24小时进行分析。进行全细胞钳夹测定以测量电流流动,如由在施加设定电压之后的氯离子转运所表示。作为-80mV至+80mV电压斜升的结果的电流对比电压的曲线(在图2中描绘)证明当比较未处理的细胞对比hCFTRmRNA处理的细胞时的显著电流差异。在暴露于8-Br-cAMP(一种已知的CFTR蛋白活化剂)之后电流的这种增加表明人CFTR蛋白存在于这些细胞中。在用已知的特异性CFTR抑制剂CFTRinh-172处理这些先前转染的细胞之后,相应电流降回至接近对照水平(约89%降低)。在这种抑制剂暴露之后的这种降低有力地支持了人CFTR蛋白的存在。这些结果总体上证明合成hCFTRmRNA可产生活性人CFTR蛋白。
单独地,在HEK293细胞内使用自动系统(IonWorks)进行CFTR全细胞活性测定。如上所述,使处理的细胞以及对照细胞(未处理的和模拟转染的)经受活化剂和抑制剂底物以帮助测定电流流动的变化(氯离子转运)。在这些研究中,毛喉素被用作CFTR蛋白活化剂并且使一部分hCFTRmRNA转染的细胞进一步暴露于不同的特异性CFTR抑制剂,GlyH-101。GlyH-101据信用作CFTR孔阻断剂,其作用于蛋白质的细胞外膜侧。值得注意的是,这种作用机制不同于CFTRinh-172的作用机制,其据报道从CFTR蛋白的细胞内侧起作用。
图4表示用毛喉素以及GlyH-101处理的亲本HEK293细胞系的电流-电压曲线图。未观察到电流的显著变化,从而表明这些特异性CFTR活化剂/抑制剂对存在于细胞系中的内源性蛋白质没有影响。
作为-100mV至+100mV电压斜升的结果的电流对比电压的曲线图(在图5中描绘)证明当比较未处理的HEK293细胞对比hCFTRmRNA处理的细胞时电流存在显著差异。在暴露于毛喉素(一种已知的CFTR蛋白活化剂)之后电流的这种增加指示人CFTR蛋白存在于这些细胞中。在用不同的已知特异性CFTR抑制剂GlyH-101处理这些先前转染的细胞之后,相应电流降回至接近对照水平(约95%降低)。在这种抑制剂暴露之后的这种降低有力地支持了人CFTR蛋白的存在。
总的来说,为两种不同机制的结果的这些抑制数据有利地支持源自合成人CFTR信使RNA的完全功能性CFTR蛋白的身份。
实施例3:CFTR的体内表达
该实施例证明CFTR蛋白由通过肺施用递送的CFTR编码mRNA在体内有效地表达。
制剂方案1。将50mg/mL的C12-200、DOPE、Chol和DMG-PEG2K的乙醇溶液的等分试样混合并且用乙醇稀释至3mL的最终体积。单独地,从1mg/mL储备液制备CFTRmRNA的水性缓冲溶液(10mM柠檬酸盐/150mMNaCl,pH4.5)。将脂质溶液快速注入至水性mRNA溶液中并且振荡以得到于20%乙醇中的最终悬浮液。过滤所得到的纳米颗粒悬浮液,用1xPBS(pH7.4)渗滤,接着用水渗滤,浓缩且储存在2℃-8℃下。最终浓度=1.09mg/mLCFTRmRNA(封装的)。Z平均=80.2nm(Dv(50)=55.5nm;Dv(90)=99.6nm)。
制剂方案2。将2.0mg/mL水溶液PEI(支链,25kDa)的等分试样与CFTRmRNA的水溶液(1.0mg/mL)进行混合。将所得到的复合混合物上下吹吸数次并且在注入之前储存备用20分钟。最终浓度=0.60mg/mLCFTRmRNA(封装的)。Z平均=75.9nm(Dv(50)=57.3nm;Dv(90)=92.1nm)。
经由气管内施用的负载mRNA的纳米颗粒产生的FFL和CFTR蛋白的分析。使用雌性BALB/C小鼠或CFTRKO小鼠进行所有研究。经由直接滴注或雾化(PARIBoy或Aeroneb)相应剂量的封装FFLmRNA来引入FFL样品。使用PARIBoy喷射喷雾器引入CFTRmRNA。将小鼠处死并且在允许表达时间之后用盐水灌注。
气管内施用FFLmRNA。经由MicrosprayerTM(50μL/动物)通过单一气管内气雾剂施用来施用FFL测试材料,同时用腹膜内注射氯胺酮50-100mg/kg与塞拉嗪5-15mg/kg的混合物麻醉动物。
雾化(气雾剂)施用FFLmRNA。经由Lab喷雾器通过单一气雾剂吸入来施用FFL测试材料(达8mL/组的标称剂量体积)。将测试材料递送至含有整个动物组(n=4)的盒子并且连接至氧流动和清除剂系统。
施用CFTRmRNA。以在以下实施例6中描述的方式来制备CFTRmRNA。将四只CFTR敲除小鼠放置在气雾剂室盒子中并且经由雾化(PariBoy喷射喷雾器)暴露于2mg总密码子优化的未修饰的人CFTRmRNA(包含SEQIDNO:3的编码序列)大约一小时的过程。在暴露后24小时处死小鼠。
安乐死。将动物在剂量施用后的代表时间(±5%)通过CO2窒息安乐死,接着进行胸廓切开术和放血。经由心脏穿刺收集全血(最大可获得体积)并且丢弃。
灌注。在放血后,将所有动物用盐水进行心脏灌注。简言之,通过将附接至含有盐水组的10mL注射器的23/21号针插入左心室的管腔中灌注来进行全身心脏内灌注。将右心房切开以提供用于灌注液的排出口。将轻轻且稳定的压力施加至柱塞以在针已经定位在心脏中之后灌注动物。当离开灌注液流动透明(不含可见血液)时确保冲洗溶液的充分流动,从而指示冲洗溶液已经使身体饱和并且程序完成。
组织收集。在灌注后,收获所有动物的肝和肺(右和左)。使选择组经受大约一半的肝和两个(右和左)肺快速冷冻在液氮中并且单独地储存在通常-70℃下。使选择组经受大约一半的肝放置在一个组织学盒/动物中。另外,将肺通过插入至气管中的套管用10%NBF充气。将气管用结扎线打结并且将肺(右和左)和气管完整放置在一个组织学盒/动物中。将所有组织学盒环境储存在10%NBF中24小时并且转移至70%乙醇中。
FFL处理的小鼠中FFL的表达。在组织样品分析之后,在FFL处理的小鼠中检测到FFL表达(数据未示出)。
CFTR敲除小鼠中CFTR的表达。通过CFTRmRNA处理的小鼠肺的免疫沉淀-蛋白质印迹分析来检测CFTR表达。在所有处理的小鼠的左肺和右肺中检测到成熟的“C”条带,而在对照小鼠中未观察到(图1B)。所使用的抗体是MAB25031(R&DSystems)以用于免疫沉淀和SAB4501942(Sigma)以用经由蛋白质印迹分析检测。
在此所示的结果指示,CFTR蛋白可基于mRNA的肺递送成功地在体内表达。此外,CFTRmRNA已经被成功地递送至CFTR敲除小鼠的肺并且导致在肺中的有效蛋白质产生的事实指示基于CFTRmRNA的体内蛋白质产生可用于治疗CFTR蛋白缺乏。
实施例4:使用聚合物纳米颗粒肺递送CFTRmRNA
将人CFTR信使RNA递送至小鼠的肺可经由直接滴注以及雾化来完成。使用原位杂交方法,可在将负载人CFTRmRNA的纳米颗粒气管内施用至小鼠之后成功地检测人CFTRmRNA。可采用基于脂质的纳米颗粒(例如C12-200)以及聚合物纳米颗粒(例如聚乙烯亚胺,PEI)来完成施用。
使用聚合物纳米载体施用CFTRmRNA。将CFTRKO小鼠用负载基于聚乙烯亚胺(PEI)的CFTRmRNA的纳米颗粒经由气管内施用(30ug封装的mRNA)进行处理。将处理的小鼠在施用后六小时和二十四小时处死,并且收获肺且固定在10%中性缓冲福尔马林(NBF)中。原位杂交用于检测外源人CFTRmRNA(图6)。在施用后24小时观察到显著染色,其中在处理的CFTRKO小鼠的两个小鼠肺中具有广泛分布,而对于PBS处理的对照小鼠未观察到染色。
在较高放大倍数下(高达20x放大倍数)对处理的肺的分析揭示在两个肺的整个支气管和肺泡区域中的广泛的阳性细胞内染色(图7)。在进一步放大(40x)之后,观察到靶顶端支气管上皮细胞的细胞质内的阳性染色(图8)。因此可推断,信使RNAAPI被成功地递送至靶顶端支气管上皮细胞。此外,虽然可在施用后6小时观察到显著染色,但在24小时之后仍然观察到hCFTRmRNA的显著阳性检测(图9)。
在施用后24小时在整个两个肺中在支气管和肺泡区域内观察到显著阳性细胞内染色。
实施例5:使用基于脂质的纳米颗粒肺递送CFTRmRNA
使用基于脂质的纳米载体施用CFTRmRNA。如上所述,人CFTRmRNA的成功肺递送可经由基于脂质纳米颗粒的递送媒介物来完成。在此公开利用C12-200作为阳离子脂质组分的负载hCFTRmRNA的阳离子脂质纳米颗粒的实施例。
CFTRKO小鼠的肺内的人CFTRmRNA的成功检测经由原位杂交来实现。将敲除小鼠用封装在基于C12-200的脂质纳米颗粒中的15ug的hCFTRmRNA进行处理并且在施用后6小时处死。当与PBS处理的对照小鼠相比时,在两个肺的整个支气管和肺泡区域中观察到hCFTRmRNA的阳性检测(图10)。
在进一步放大(40x)之后,在支气管上皮细胞以及细胞内末端肺泡区域的顶端细胞质内观察到人CFTRmRNA的阳性检测(图11)。
总的来说,合成人CFTR信使RNA的成功递送可利用聚合物(PEI)和基于脂质纳米颗粒(C12-200)的递送系统两者来实现。这些系统得到小鼠的靶细胞内药物物质的细胞内积聚。此外,在施用后24小时大量hCFTRmRNA存在于这些靶细胞中。
实施例6:使用特异性抗体验证人CFTR表达
在小鼠、猪和培养细胞中用于人CFTR蛋白检测的抗体验证。进行实验以鉴别对于hCFTR蛋白具有特异性的抗体,所述抗体不与小鼠和猪类似物交叉反应并且可供用于未来实验的足够供应。简言之,测试来自学术和商业来源的各种抗hCFTR抗体导致鉴别抗hCFTR抗体的组合,所述抗体能够在免疫沉淀和蛋白质印迹(IP/WB)之后检测人CFTR蛋白而无与鼠或猪CFTR的交叉反应性。因此,基于IP/WB结果来鉴别用于检测hCFTR蛋白而无鼠或猪CFTR的交叉反应性的适合的抗hCFTR抗体。
将细胞用hCFTRmRNA转染并且在转染后24小时使用ProteoExtractTransmembrane试剂盒(Merck)制备蛋白质裂解物,并且使用小鼠抗人CFTR抗体(MA1-935)通过蛋白质印迹针对hCFTR筛选跨膜级分。来自16HBE细胞的裂解物用作阳性对照。图12A呈现来自CHO和COS-7细胞的数据。
将在文献中描述为CFTR-阴性的幼仓鼠肾细胞(BHK)类似于CHO和COS-7细胞进行转染并且通过蛋白质印迹筛选蛋白质裂解物。与先前公开的报道相比,CFTR的明显阳性信号可使用小鼠单克隆抗CFTR抗体观察到(图12B)。为了测试在蛋白质印迹分析中使用的抗体的特异性,来自由EckhardtWolf教授(LudwigMaximiliansUniversity,Munich)友情提供的CFTR敲除猪的猪肾细胞(PKC)用于转染实验中并且针对CFTR表达筛选蛋白质裂解物。如在图12B中显而易见,在PKC细胞中不可检测到CFTR的信号。然而,转染也不导致任何可检测的hCFTR表达。使用荧光素酶作为转染的对照,当与CHO或COS-7细胞相比较时,发现PKC细胞表达荧光素酶的效率低几倍。由于在转染后不可在任何所筛选的细胞系中检测到hCFTR条带强度的显著差异,所以进行具有针对hCFTR的更高灵敏度和特异性的其它hCFTR抗体的广泛筛选。
经由蛋白质印迹进行的抗体筛选。使用ProteoExtractTransmembrane试剂盒(Merck)从人支气管上皮细胞系(BEAS-2B)、人胚肾细胞系(HEK)、小鼠肺和猪肺制备蛋白质裂解物,并且跨膜级分用于使用不同的一级抗体(来自ThermoScientificPierceAntibodies,Rockford,IL,USA的MA1-935;来自CysticFibrosisConsortium,UniversityofPennsylvania,PA,USA的AB596;以及来自CysticFibrosisConsortium,UniversityofPennsylvania,PA,USA的AB570)进行免疫印迹。数据总结为图13。
而MA1-935在所有三个物种中检测到CFTR,AB596检测到人和鼠CFTR而不是猪,并且抗体G449仅特异性地检测到人CFTR。在AB570的情况下,不清楚对鼠和猪样品观察到的稍低分子量条带确实是CFTR还是非特异性产物。在随后的实验(数据未示出)中,发现MA1-935识别不是CFTR的条带。因此,一般而言,MA1-935结果被视为使用其它抗体产生的确认结果,但所使用的仅抗CFTR抗体是MA1-935的实验不被认为是结论性的。
来自组织样品的hCFTR(IP-hCFTR)的免疫沉淀。鉴于所有筛选的抗体产生几个非特异性条带,并且它们中没有一种抗体产生hCFTR的特征带型(C条带表示完全糖基化的蛋白并且B条带表示核心甘露糖基化形式),所以hCFTR的免疫沉淀(IP)和通过蛋白质印迹进行的随后检测被建立以增加检测的灵敏度和特异性,从而增加信噪比。
使用由vanBarneveld等2012,ImmunochemicalanalysisofMutantCFTRinLungexplants,CellPhysiol.Biochem.30,587-595(2012))公开的方案和抗体与BurkhardTümmler教授(MedizinscheHochschuleHannover)合作进行初始IP实验。过表达hCFTR的人结肠癌细胞(T84)用作IP实验的阳性对照。
使用三种不同的抗体(R29、R66/17和R66/16)免疫沉淀hCFTR,接着用AB596免疫检测导致特异性检测来自用如以下实施例8中所描述的hCFTRSNIMRNA的气雾剂处理的猪肺的蛋白质裂解物中的hCFTR(图14)。
HGT5001制剂。在小鼠中进行使用HGT5001:DOPE;Chol;PEGDMG2K(相对量50:25:20:5(mg:mg:mg:mg))(“HGT5001制剂”)的制剂中的hCFTRSNIMRNA的气雾剂实验并且来自在mRNA递送后24小时的分离肺的蛋白质裂解物也通过IP使用与用于猪裂解物的相同抗体和条件进行分析。然而,对于小鼠样品,不可检测到特征性成熟CFTR带型(图15)。
来自体外转染的细胞的hCFTR(IP-hCFTR)的免疫沉淀。使用来自猪的组织材料的初始IP结果提供在体内转录递送后hCFTR检测的技术可行性的证据。然而,因为用于免疫沉淀CFTR中的抗体(R29、R66/17和R66/16)中没有一种是可商购获得的,所以在IP反应中对其它可商购获得的抗体的功效进行筛选。对来自R&D系统的两种抗体(MAB25031和MAB1660)进行了测试。
蛋白质裂解物从T84细胞制备并且500μg的总蛋白用于使用不同浓度的MAB25031抗体的IP反应中。然后使用AB570(囊性纤维化基金会)通过免疫印迹来检测免疫沉淀的hCFTR蛋白的量。在这些条件下AB596导致高得多的背景,并且因此未进一步测试。如在图16A中所揭示,当IP抗体的浓度从2μg/ml增加至4μg/ml时,不存在沉淀的CFTR蛋白的量的进一步增加。检测到完全糖基化形式和仅核心糖基化形式两者(分别是C和B条带)。还在蛋白质印迹中使用MAB1660作为一级抗体筛选相同的免疫沉淀物。然而,使用这种抗体,仅条带C可见(图16B)。
在使用MAB25031抗体成功检测来自T84免疫沉淀物的内源性hCFTR之后,在NIH3T3细胞中进行实验,目的是在转染后检测hCFTR蛋白。用hCFTRSNIMRNA转染NIH3T3细胞。在转染后72小时制备蛋白质裂解物并且使用BCA方法对蛋白质量进行定量。使用2μg/ml的MAB25031抗体从500μg总蛋白质裂解物免疫沉淀人CFTR蛋白,接着使用AB570进行免疫印迹(图17)。然而,不可检测到CFTR。用LacZ编码mRNA转染的细胞作为对照样品针对转染本身对CFTR蛋白的量的作用进行分析。
使用于免疫沉淀中的总蛋白质的量从500μg增加至8mg不会在用AB570免疫检测后产生任何可检测的hCFTR蛋白。还针对免疫沉淀对另一种hCFTR特异性抗体MAB1660(R&D系统)进行筛选(图18)。然而,所述抗体不如MAB25031一样有效地沉淀CFTR。因此,所有未来免疫沉淀用MAB25031进行。
在mRNA转染的样品中hCFTR检测的缺乏可能不一定意味着所测试的mRNA的功能性的缺乏,因为使用荧光素酶作为标记基因的动力学实验已经显示,在转染后24小时观察到mRNA的最大表达。hCFTR检测的缺乏是由于所测试的样品中hCFTR浓度不足或所施加的抗体的特异性的缺乏。
PEI制剂。将所建立的条件针对其在递送至猪(参见实施例7)的如下制备具有25kDa支链PEI的纳米颗粒制剂(“PEI制剂”)中的hCFTRSNIMRNA之后检测hCFTR的可行性进行测试。将所需量的SNIMRNA就在施加之前于注射用水(Braun,Melsungen)中稀释至4ml的总体积并且使用移液管以10的N/P比值快速添加至4ml的支链PEI25kDa的水溶液。通过上下吹吸十次混合所述溶液,并且将其雾化为两个单独的4.0ml级分,使用所指示的喷雾器一个接一个地施用至猪肺。来自1号猪的表达荧光素酶的肺区域的一个样品和来自2号猪的尾叶的另一个样品(其中不可检测到荧光素酶活性,从而指示缺乏mRNA递送和/或表达)被选择作为阳性对照和阴性对照。使用MAB25031(R&D系统)免疫沉淀从这些样品制备的蛋白质裂解物并且使用AB570检测hCFTR蛋白。如图19中所示,荧光素酶表达与hCFTRmRNA的表达相关。来自2号猪的左尾叶的样品(其中不可检测到荧光素酶活性)也是针对hCFTR阴性的(泳道1),而可在来自1号猪的样品(其是针对荧光素酶阳性的)中检测到hCFTR(泳道2)。
实施例7:mRNA的气雾剂递送
封装的mRNA气雾剂递送至猪肺的建立。萤火虫荧光素酶(FFL)SNIMRNA气雾剂施用至猪肺由逐步实验程序建立。在第一步骤中,在受控通气期间将FFLSNIMRNA制剂雾化至麻醉的猪。在第二步骤,在气雾剂施用完成之后立即切除肺并且将肺标本在细胞培养基中孵育过夜,之后通过BLI对肺标本进行离体荧光素酶测量。
GermanLandrace的猪获得自TechnicalUniversityMunich,Weihenstephan,Germany。所述猪具有35-90kg范围内的体重。对一头猪进行每种处理。总计处理五头猪。将第一头猪(90kg重量)使用EFlow筛孔喷雾器用实施例6的PEI制剂中的FFLSNIMRNA进行处理并且测量肺匀浆中的荧光素酶活性。将第二头猪(60kg重量)使用EFlow筛孔喷雾器用实施例6的PEI制剂中的FFLSNIMRNA进行处理并且通过BLI测量肺标本中的荧光素酶活性。将第三头猪(80kg重量)使用PARIBOY喷射喷雾器用实施例6的PEI制剂中的FFLSNIMRNA进行处理并且通过BLI测量肺标本中的荧光素酶活性。将第四头猪(60kg重量)使用Aeroneb筛孔喷雾器用实施例6的PEI制剂中的FFLSNIMRNA/hCFTRmRNA进行处理并且通过BLI测量肺标本中的荧光素酶活性。将第五头猪(35kg重量)使用Aeroneb筛孔喷雾器用实施例6的HGT5001制剂中的FFLSNIMRNA进行处理并且通过BLI测量肺标本中的荧光素酶活性。
猪的镇静通过以下方式来引发:用阿扎哌隆2mg/kg体重、氯胺酮15mg/kg体重、阿托品0.1mg/kg体重的术前用药,并且接着将静脉内管线插入至侧耳静脉。将猪通过静脉内注射丙泊酚(propofol)3-5mg/kg体重视需求进行麻醉。通过以4至8mg/kg体重推注注射异氟烷(2%-3%)与1%丙泊酚以视需求增强麻醉来维持麻醉。麻醉的持续时间是大约1-3小时。将猪经由侧耳静脉用推注注射戊巴比妥(100mg/kg体重)和氯化钾来杀死。将肺切除,并且从不同肺区域收集组织样本,接着在细胞培养基中孵育过夜。为了测量荧光素酶活性,将组织样本均化且在管光度计中分析或在包含D-荧光素底物的培养基浴中孵育且经受离体荧光素酶BLI。
1号猪的细节和结果。实验设置示于图20中。对于气雾剂施用,将EFlow筛孔喷雾器同轴(in-line)连接至呼吸器的通气管。气雾剂施用花费大约60分钟,并且比从使用开放系统的对照实验所预期的时间长。这明显地是由在雾化期间增加的背压引起,如由筛孔喷雾器的储库处的气雾剂流出所证明。将8毫升的实施例6的包含于注射用水中的1mgFFLSNIMRNA的PEI制剂如在WP5中所描述来制备并且在两个单独的4ml部分中一个接一个雾化。在于细胞培养基中过夜孵育之后,在不同肺区域的切除肺标本的组织匀浆中进行荧光素酶测量。将表达值根据肺标本的来源进行绘图(图21)。
结果显示猪肺组织中的成功荧光素酶表达。荧光素酶表达在肺的中心部分中最高,并且朝向肺的更远端区域下降。所述表达模式与根据所选择的通气参数吸入的FFLSNIMRNA-PEI纳米颗粒的预期沉积模式相关。荧光素酶表达的水平在如在WP5中使用实施例6的相同PEI制剂的小鼠实验中所观察到的相同范围中。
2号猪的细节和结果。如在1号猪中一样进行在2号猪中气雾剂施用于实施例6的PEI制剂中的FFLSNIMRNA,但通过生物发光成像(BLI)测量肺标本上的荧光素酶活性。进行该实验以通过BLI建立器官培养的肺标本的离体荧光素酶测量。在处理的猪的不同肺区域的单个组织标本中清楚观察到荧光素酶测量(图22)。从1号猪获得实验证实的结果。
3号猪的细节和结果。在1号猪和2号猪中使用EFlow筛孔喷雾器气雾剂施用揭示了一些技术困难和不充分的雾化时间。因此,使用PARIBOY喷射喷雾器处理3号猪,所述喷雾器经由T形连接件连接至通气管。气雾剂施用比使用EFlow筛孔喷雾器持续更长(大约80分钟)并且气雾剂施用不是令人满意的。在来自处理的猪的不同肺区域的切片肺样品中检测到极低荧光素酶活性(图23)。
4号猪的细节和结果。前述实验的结果证明,在所选择的设置下筛孔喷雾器比喷射喷雾器更适用于气雾剂施用至猪肺。出于这一原因,针对该目的对另一种筛孔喷雾器进行测试,当在开放系统中测试时所述喷雾器令人满意地雾化实施例6的PEI制剂。使用Aeroneb筛孔喷雾器对4号猪进行处理,所述喷雾器同轴连接至呼吸器的管。在所述实验中,1mg的hCFTRmRNA与1mg的FFLSNIMRNA一起在实施例6的PEI制剂中共递送。进行该步骤以就待在实施例8中进行的重复给药而言测试共配制的FFLSNIMRNA/hCFTRmRNA-PEI纳米颗粒的制剂稳定性和喷雾能力。所述制剂是稳定的并且未显示与雾化的不相容性。在处理的猪的不同肺区域的单个组织标本中清楚观察到荧光素酶活性(图24)。
所述实验证实了从1号猪和2号猪获得的结果,但是获得更高的表达水平。所述实验显示,Aeroneb筛孔喷雾器最适合用于将实施例6的PEI制剂递送至猪肺。此外,实验证明FFLSNIMRNA在与hCFTRmRNA一起共递送时仍是活性的。
5号猪的细节和结果。使用于用Aeroneb筛孔喷雾器气雾化的实施例6的HGT5001制剂中的1mg的FFLSNIMRNA处理5号猪。所述制剂可被气雾化而不存在技术困难。在处理的猪的不同肺区域的单个组织标本中清楚观察到荧光素酶活性(图25)。
实验表明,在实施例6的HGT5001制剂中的气雾化的FFLSNIMRNA在猪肺组织中是活性的,但表达水平比用实施例6的PEI制剂处理的猪中低大约15-20倍。
结论。使用Aeroneb筛孔喷雾器用实施例6的PEI制剂获得成功的结果。将四头猪用实施例6的PEI制剂进行处理以鉴别用于气雾剂递送的最优实验设置。结果证明,可通过BLI在猪肺匀浆中检测到荧光素酶表达。荧光素酶表达在肺的中心部分中最高,并且在肺的远端区域中几乎看不到。发现Aeroneb筛孔喷雾器得到最佳结果连同最短递送时间。根据这些实验,将另一头猪用封装在实施例6的HGT5001制剂中的FFLSNIMRNA进行处理。虽然在猪肺的一些部分中清楚地观察到荧光素酶表达,但表达水平低于实施例6的PEI制剂中的FFLSNIMRNA的表达水平。来自该工作包的结果清楚地证明,使用基于各种制剂如基于聚合物(例如,PEI)的制剂和基于脂质(例如HGT5001)的制剂,SNIMRNA递送至作为大型临床前动物模型的猪的肺是可行的。该实施例的结果提供用于成功SNIMRNA递送至大型动物的肺的概念的证据,所述动物通过用于临床实践的喷雾器密切地模拟人患者中的情况。
实施例8:体内mRNA递送(每周剂量)
进行试验来评价在猪中每周一次气雾剂施加的可行性。可行性被定义为在一周的间隔中进行修饰的mRNA的三次气雾剂施加而不诱导肺疾病(不存在高于2级的不良事件)。另外的目的是评价i)动物的痛苦等级,ii)在猪的实验室或临床评估期间发生的不良事件,以及iii)诱导的蛋白质(荧光素酶和hCFTR)的测量。
建立PEI制剂中的SNIMRNA重复气雾剂施用至猪肺。将两头猪的组用实施例6的PEI制剂中的FFLSNIMRNA/hCFTRSNIMRNA以每周间隔一次、两次或三次处理。两头未处理的猪用作对照。将肺在处理后24小时切除并且通过BLI测量分离的肺标本中的离体荧光素酶活性。使用IP/WB对hCFTR蛋白的表达进行分析。进行免疫组织化学(IHC)以用于检测细胞水平的荧光素酶表达。通过测量血清和血液化学中的炎性细胞因子来研究毒理学。对肺样品进行组织病理学。研究方案“Pilotproject:RepeatedapplicationofmodifiedmRNAtoestablishananimalmodelforaerosoltherapyofcysticfibrosisinpigs”在实验开始之前由地方当局批准(动物实验许可Nr.:0-045-12)。
实验设计。在雾化下的雌性大约6周龄(平均约25kg体重)的猪(GermanLandrace)购自TechnicalUniversityMunich,Weihenstephan,Germany。将猪随机分组,并且根据以下方案(表3)进行处理。每两头猪的处理组是如下:
第0组-无处理的对照组
第I组-在第1天气雾剂施用于实施例6的PEI制剂中的1mgFFLSNIMRNA和1mghCFTRSNIMRNA。
第II组-在第1天气雾剂施用于实施例6的PEI制剂中的2mghCFTRSNIMRNA并且在第8天气雾剂施用于实施例6的PEI制剂中的1mgFFLSNIMRNA和1mghCFTRSNIMRNA。
第III组-在第1天和第8天气雾剂施用于实施例6的PEI制剂中的2mghCFTRSNIMRNA(6379-186),在第15天气雾剂施用于实施例6的PEI制剂中的1mgFFLSNIMRNA和1mghCFTRSNIMRNA。
用于处理和评价每组的方案在表3中示出。除了所示的干预,每日对所述猪进行身体检查。
表3.不同处理组的时间-线图。
实验程序。猪的镇静通过以下方式来引发:用阿扎哌隆2mg/kg体重、氯胺酮15mg/kg体重、阿托品0.1mg/kg体重的术前用药,并且接着将静脉内管线插入至侧耳静脉。将猪通过静脉内注射丙泊酚3-5mg/kg体重体重视需求进行麻醉。视需求用连续静脉内输注1%丙泊酚来维持麻醉。通气参数与呼气末二氧化碳匹配并且如果有必要进行调整。使用脉搏血氧定量法、二氧化碳描记、直肠温度探针和反射状态连续监测麻醉、呼吸和心血管参数。动物以10ml/kg/h接受输注平衡电解质溶液。麻醉的持续时间是大约80-120分钟。在开始足够自主呼吸之后对猪拔管。在镇静之后将猪经由侧耳静脉用推注注射戊巴比妥(100mg/kg体重)来处死。将肺切除,并且从不同肺区域收集切片的大约1cm厚的组织样本,接着在细胞培养物中孵育。为了测量荧光素酶活性,将组织样本在包含D-荧光素底物的培养基浴中孵育且经受离体荧光素酶BLI。
通过BLI在处理组中的荧光素酶表达。
对于第0组(无处理的对照组),在肺切片中没有观察到荧光素酶活性(图26)。
对于第I组(气雾剂施用实施例6的PEI制剂中的1mgFFLSNIMRNA和1mghCFTRSNIMRNA),在一次处理的3号猪和6号猪的肺标本中清楚地检测到荧光素酶活性(图27)。荧光素酶表达在肺的中心部分中最高。
对于第II组(在第1天气雾剂施用实施例6的PEI制剂中的2mghCFTRSNIMRNA并且在第8天气雾剂施用实施例6的PEI制剂中的1mgFFLSNIMRNA和1mghCFTRSNIMRNA),在两次处理的4号猪和8号猪的肺标本中清楚地检测到荧光素酶活性(图28)。荧光素酶表达在肺的中心部分中最高。由于在测量那一天的电源中断和所产生的BLI系统的技术问题,所以必须考虑在测量之前将样品在细胞培养基中储存另外10小时。
对于第III组(在第1天和第8天气雾剂施用实施例6的PEI制剂中的2mghCFTRSNIMRNA,在第15天气雾剂施用实施例6的PEI制剂中的1mgFFLSNIMRNA和1mghCFTRSNIMRNA),在三次处理的1号猪和2号猪的肺标本中清楚地检测到荧光素酶活性(图29)。荧光素酶表达在肺的中心部分中最高。
SNIMRNA-PEI纳米颗粒的特性。在雾化之前测量SNIMRNA-PEI制剂的粒度和ζ电位(表X1)。所述SNIMRNA-PEI纳米颗粒能够可重复地形成为具有25-37nm范围内的大小和30-49mV范围内的ζ电位。
表X1.粒度和ζ电位测量值
通过IHC处理组中的荧光素酶表达。对肺切片(SophistolabAG,Eglisau,Switzerland)的组织标本进行针对FFL的IHC,所述切片是通过BLI阳性的且与未处理的猪的肺组织进行比较,并且荧光素酶-阳性小鼠肿瘤组织作为阳性对照。如所预期,在荧光素酶-阳性小鼠肿瘤组织中观察到较强信号,而未处理的猪的肺组织未显示特异性染色。可清楚检测的染色图案可在接受三次处理的1号猪的肺组织中观察到。FFL表达在大和小气道的支气管上皮中最显著(图30)。
通过IP/WB检测处理的猪的肺组织中的hCFTR蛋白。根据由vanBarneveldA等,CellPhysiolBiochem.30,587-95(2012)中描述的方案使三次处理的1号猪的高度BLI-阳性的肺组织经受hCFTRIP/WB(图31)。成熟的复合物-糖基化的hCFTR作为分散的所谓C条带出现。富含甘露糖的hCFTR作为更致密的所谓B条带出现。在T84阳性对照和用实施例6的PEI制剂中的hCFTRSNIMRNA处理的1号猪的肺组织中清楚地观察到hCFTR表达。在未处理的猪中未观察到hCFTR蛋白的表达。使用相同的方案(vanBarneveldA等,同上)比较来自已公开的研究的人肺组织中的hCFTR蛋白表达表明,在hCFTRSNIM气雾剂处理之后,猪肺组织中hCFTR的表达类似于健康人肺中的hCFTR表达。
通过使用不同组的抗体用于通过IP/WB检测处理的猪肺中的hCFTR蛋白来进一步证实这一发现(参见实施例6)。来自1号猪的表达荧光素酶的肺区域的一个样品和来自2号猪的尾叶的另一个样品(其中不可检测到荧光素酶活性,从而指示缺乏mRNA递送和/或表达)被选择作为阳性对照和阴性对照。使用MAB25031(R&D系统)免疫沉淀从这些样品制备的蛋白质裂解物并且使用AB570检测hCFTR蛋白。如图32中所示,荧光素酶表达与hCFTRmRNA的表达相关。来自2号猪的左尾叶的样品(其中不可检测到荧光素酶活性)也是针对hCFTR阴性的(泳道1),而可在来自1号猪的样品(其是针对荧光素酶阳性的)中检测到hCFTR(泳道2)。
毒理学:肺样品的初步组织学评定。进行在三只动物安乐死之后所取得的样品的组织学评定。在包埋于石蜡切片中之后,将肺样品用苏木精-曙红染色以用于形态评价。所述发现在来自三头猪的样品中一致,其中两头(1号猪和2号猪)接受三次气雾剂施加,并且第三(7号猪)头是无气雾剂施加的未处理的对照。
毒理学:痛苦。在所述第一处理之后第2-4天,仅2号猪和1号猪显示出轻微痛苦症状。因此,三周内的三次气雾剂施加仅引起轻微痛苦。
毒理学:不良事件。通过实验室参数(血液、MBS和BAL)且通过猪的身体检查(被定义为该试验中的第二目标)来分析不良事件(AE)的种类和频率。
在由研究方案限定的时间点取得血清和全血样品。选择指示以显示器官特异性病理学(血液、骨髓、肝、肌肉和肾)的十二种代表性参数(血红蛋白、血细胞比容、AP、ALT、AST、CK、胆红素、肌酸酐、葡萄糖、钾、血小板和白细胞),并且从根据VCOG(2011版)分类的VetMedLab,Ludwigsburg,Germany获得测试结果。
所述结果显示,在猪中未观察到严重的不良事件(AE)(3、4、或5级AE将被定性为严重)。在气雾剂施加实施例6的PEI制剂中的SNIMRNA之后,不存在实验室参数的受损。对于一些参数(例如CK或肝酶)的略微变化,更可能的是,这些变化由实验程序本身(例如肌肉内注射和麻醉)所引起。也不可检测到来自重复施加的不利作用,即使在第三次施加之后,第3组的猪未显示高于2级AE的AE。即使1级或2级AE很少见并且显示实施例6的PEI制剂中的SNIMRNA的气雾剂施加不相关。
除了重复的血液样品外,评定两个其它参数以评价肺的病理过程:i)支气管肺泡灌洗液(BALF)-在安乐死后取得,和ii)微生物学样品(MBS)(来自气管的涂片–在麻醉期间取得)。在尸体剖检期间从每头猪取得BALF并且储存在-80℃下以用于进一步检查。在每次气雾剂施加之前取得气管涂片并且进行微生物检查。这些检查揭示广谱病原体,包括支气管炎博德特氏菌(Bordetellabronchiospectica)(猪呼吸道的常见病原体)和大肠杆菌(Escherichiacoli)。将猪用托拉菌素(tulathromycin)肌肉内注射(1ml10%)进行一次处理。
身体检查。除了实验室参数之外,在气雾剂施加之间的观察时期中对猪进行身体检查(关于细节,参见研究方案的附录1的1.1.2和附录4)。因为没有系统用于记录、分级和将AE的属性分配至干预或针对猪定义其它事项,所以使用针对狗和猫建立的常见毒理学标准(CTC)系统(在2011由VOCG公开)。为了对实验室参数分级,使用物种特异性ULN(正常值上限)和LLN(正常值下限)。在以下六种AE类别内进行临床评定:
(1)过敏性/免疫事件;(2)肺的/呼吸的;(3)全身临床症状;(4)皮肤病学的/皮肤;(5)胃肠道的;和(6)肺的/呼吸的。
结果表明,在猪中未观察到严重AE(无3、4或5级)。在气雾剂施加PEI制剂中的SNIMRNA之后,不存在通过身体检查评定的参数受损。第3组的两头猪显示三个呼吸参数(支气管痉挛/气喘、喉水肿和呼吸困难)中的1级和2级AE,但这些轻度或中度发现被限制至一天或两天。因为这些观察结果仅在这两头猪中在第一麻醉/插管/气雾剂施加之后发生而不在这两头猪或在任何其它猪中在第二或第三气雾剂施加之后发生,所以这些发现不太可能由在研究中的物质引起。
结论。该实施例的结果证明,编码FFL和hCFTRSNIMRNA的PEI制剂可成功地重复雾化至猪肺而无每个处理循环之后的活性损失并且无不良事件。在肺组织的中心部分中发现荧光素酶表达,但在远端肺区域中几乎检测不到。荧光素酶表达的区域模式与根据用于控制通气的设置的实施例6的PEI制剂的预期沉积模式相关。对来自处理的猪的选定肺样品的免疫组织化学显示主要在大和小气道的支气管上皮中的荧光素酶表达。IP/WB清楚地证明在处理的猪肺中成熟人CFTR的复合物-糖基化C条带的表达,其在未处理的猪肺和荧光素酶阴性肺标本中不存在。当与使用用于hCFTR蛋白检测的相同方案的公开的报道相比时,在hCFTRSNIMRNA气雾剂处理之后猪肺组织中的hCFTR表达与健康人肺中的hCFTR表达相当。1级或2级不良事件很少见并且显示与PEI制剂中的SNIMRNA的气雾剂施加不相关。因此,在用SNIMhCFTRmRNA处理的猪肺中成功地证明了hCFTR蛋白的表达。
实施例9:含有信号肽的CFTR编码mRNA。
该实施例证明CFTR蛋白可从具有信号肽编码序列的CFTR编码mRNA有效地表达。
信使RNA合成。对于所述实验,使用标准方法通过从质粒DNA模板体外转录合成C-末端His10标记的密码子优化的人囊性纤维化跨膜传导调节因子(CO-CFTR-C-His10)(SEQIDNO:15)、具有生长激素信号序列前导序列的密码子优化的人CFTR(GH-CO-CFTR)(SEQIDNO:16)和密码子优化的人CFTR(CO-CFTR)(SEQIDNO:17)SNIMRNA。细胞和CFTR转染。将人胚肾HEK293T细胞生长在补充有10%胎牛血清、2mML-谷氨酰胺、100U/ml青霉素和100μg/ml链霉素的DMEM(Invitrogen目录号11965-092)中。在转染前一天,将细胞以50%-60%汇合涂铺在6-孔板上并且在正常组织培养条件(36℃,在5%CO2、95%空气的增湿大气中)下孵育。在转染准备中,将60μlLipofectamine2000(Invitrogen目录号11668019)稀释于OptiMem低血清培养基(Invitrogen目录号31985-062)中,并且轻轻涡旋。对于所述实验,将4μg的CO-CFTR,GH-CO-CFTR或CO-CFTR-C-His10SNIMRNA稀释于900μl的OptiMem培养基中。将mRNA立即添加至稀释的并且在室温下孵育30分钟。将涂铺培养基轻轻地抽吸出并且用1mlOptiMem低血清培养基和300μl的每种相应mRNA/复合物代替。在标准组织培养条件下孵育细胞。
蛋白质印迹分析。在转染后大约48,将细胞从其相应板中除去并且裂解。使全细胞裂解物经受SDS-PAGE分离并且通过蛋白质印迹进行探测。如图33中所示,在CO-CFTR、GH-CO-CFTR和人CO-CFTR-C-His10mRNA转染后检测到由抗CFTR(A&B)或抗His(C)抗体稳健表达人CFTR蛋白(图33)。
实施例10:体内CO-CFTR-C-His10mRNA递送至CFTR敲除小鼠
经由气管内施用的负载mRNA的纳米颗粒产生的人CFTR蛋白的分析。使用CFTRKO小鼠进行所有研究。使用PARIBoy喷射喷雾器引入CFTRmRNA制剂或媒介物对照。将小鼠处死并且用盐水灌注,在一段预定时间段之后,允许从mRNA表达蛋白质。
信使RNA合成。在所述实施例中,通过从质粒DNA模板体外转录合成C-末端His10标记的密码子优化的人囊性纤维化跨膜传导调节因子(CO-CFTR-C-His10)SNIMRNA和密码子优化的FFLSNIMRNA。
PEI制剂。对于所述方法,使用基于聚合物和脂质两者的纳米颗粒制剂评价CFTR敲除小鼠的肺中的CO-CFTR-C-His10mRNA的递送和表达。如下制备具有25kDa支链PEI的聚合物纳米颗粒制剂。将所需量的SNIMRNA就在施加之前于注射用水(Braun,Melsungen)中稀释至4ml的总体积并且使用移液管以10的N/P比值快速添加至4ml的支链PEI25kDa的水溶液。通过上下吹吸十次混合所述溶液,并且将其雾化为两个单独的4.0ml级分,使用所指示的喷雾器一个接一个地施用至小鼠肺。
cKK-E12制剂。对于基于脂质的纳米颗粒实验,使用于cKK-E12:DOPE:Chol:PEGDMG2K(相对量50:25:20:5(mg:mg:mg:mg)的制剂中的CO-CFTR-C-His10SNIMRNA产生脂质制剂。使用所指示的喷雾器将溶液雾化至小鼠肺。
雾化(气雾剂)施用人CO-CFTR-C-His10mRNA。经由PARIBoy喷射喷雾器通过单一气雾剂吸入来施用CFTR测试材料(达8mL/组的标称剂量体积)。将测试材料递送至含有整个动物组(n=4)的盒子并且连接至氧流动和清除剂系统。
施用人CO-CFTR-C-His10mRNA。以上述方式制备CFTRmRNA。将四只CFTR敲除小鼠放置在气雾剂室盒子中并且经由雾化(PariBoy喷射喷雾器)暴露于2mg总密码子优化的未修饰的人CFTRmRNA(包含SEQIDNO:3的编码序列)大约一小时的时程。在暴露后24小时处死小鼠。
安乐死。将动物在剂量施用后的代表时间(±5%)通过CO2窒息安乐死,接着进行胸廓切开术和放血。经由心脏穿刺收集全血(最大可获得体积)并且丢弃。
灌注。在放血后,将所有动物用盐水进行心脏灌注。简言之,通过将附接至含有盐水组的10mL注射器的23/21号针插入左心室的管腔中灌注来进行全身心脏内灌注。将右心房切开以提供用于灌注液的排出口。将轻轻且稳定的压力施加至柱塞以在针已经定位在心脏中之后灌注动物。当离开灌注液流动透明(不含可见血液)时确保冲洗溶液的充分流动,从而指示冲洗溶液已经使身体饱和并且程序完成。
组织收集。在灌注后,收获所有动物的肺(右和左)。将两个(右和左)肺快速冷冻在液氮中,并且单独储存在通常-70℃下。
CFTR敲除小鼠中来自CO-CFTR-C-His10mRNA的人CFTR的表达。通过从CFTRmRNA处理的小鼠肺收集的组织裂解物的蛋白质印迹分析来检测CFTR表达。对于基于脂质的制剂和基于聚合物的制剂两者,在所有处理的小鼠的左肺和右肺中检测成熟的“C”条带(图34)。通过与如实施例9中所描述从HEK293T人CO-CFTR-C-His10阳性细胞收集的裂解物相比较来验证成熟“C”条带的表达。相比之下,在从野生型未处理的对照小鼠收集的裂解物中未观察到可检测的信号(图34)。总之,这些数据表明基于聚合物的制剂和基于脂质的制剂(如上述cKK-E12制剂)可有效用于CFTRmRNA的肺递送(例如经由吸入)并且一旦递送,密码子优化的CFTRmRNA就可有效地表达人CFTR蛋白。
实施例11:体内剂量递增研究
PEI封装的mRNA气雾剂递送至猪肺的剂量递增。通过逐步实验程序建立在不同浓度下的萤火虫荧光素酶(FFL)SNIMRNA和密码子优化的人CFTR(CO-CFTR)SNIMRNA的组合气雾剂施用至猪肺。在第一步骤中,在受控通气期间将FFL/CO-CFTRSNIMRNA制剂雾化至麻醉的猪。在第二步骤中,在气雾剂施用完成之后在镇静24小时后将动物通过经由侧耳静脉推注注射戊巴比妥(100mg/kg体重)和氯化钾处死。将肺切除且切片成大约1cm厚的组织标本。为了测量荧光素酶活性,将组织样本在包含D-荧光素底物的培养基浴中孵育且经受离体荧光素酶BLI。在BLI之后,取得来自荧光素酶阳性和荧光素酶阴性区域的样品以用于组织病理学、免疫组织化学和原位杂交。将残余标本休克冷冻在液氮中,并且随后储存在-80℃下直到通过IP/WB和Elisa分析。
信使RNA合成。在所述实施例中,使用标准方法通过从质粒DNA模板体外转录合成密码子优化的人囊性纤维化跨膜传导调节因子(CO-CFTR)SNIMRNA、密码子优化的FFLmRNASNIMRNA。
实验设计。GermanLandrace的猪获得自TechnicalUniversityMunich,Weihenstephan,Germany。所述猪具有35-90kg范围内的体重。使用年龄和体重两者匹配的猪设计本研究以控制可变性。对于4个分组研究的每个实验组建立6头猪(3头雄性和3头雌性)的单个组。第一组用单独注射用水(WFI)处理,所述注射用水使用Aeroneb筛孔喷雾器施用。第二组用1mgFFLSNIMRNA和1mg的密码子优化的人CFTR(CO-CFTR)SNIMRNA于以下所述的PEI制剂中的溶液使用Aeroneb筛孔喷雾器进行处理。第三组接受于以下所述的PEI制剂中的1mg的FFLSNIMRNA和5mg的密码子优化的人CFTR(CO-CFTR)SNIMRNA。第四组用于以下所述的PEI制剂中的1mg的FFLSNIMRNA和10mg的密码子优化的人CFTR(CO-CFTR)SNIMRNA处理。用于处理的方案和每组的评价在以下表4中示出。
表4.用于剂量递增研究的实验设计
mRNA–PEI制剂。就在处理动物之前进行以下所述的示例性标准化配制程序。
材料:
注射泵(混合装置):
制造商:KDScientific
类型:KDS-210-CE
注射器:
制造商:B.Braun
类型:Omnifix,20mL或30mL/LuerLockSolo
Ref.:4617207V
管:
制造商:B.Braun
类型:SafeflowExtensionSet
Ref.:4097154
针:
制造商:B.Braun
类型:Sterican,20Gx11/2‘’
Ref.:4657519
混合阀:
制造商:B.Braun
类型:DiscofixC3SC
Ref.:16494C
注射用水:
制造商:B.Braun
类型:Aqua
Ref.:82423E
用于制备含有8mL体积中的1mghCFTRSNIMRNA和1mgFFLSNIMRNAN/P10的聚合复合物的示例性方法:将3mL注射用水和3mLRNA储备溶液(c:1mg/mL于水中;1.5mLFFLmRNA+1.5mLCFTRmRNA)填充至15mLfalcon管中。在第二falcon管中,将5.61mL注射用水与0.39mLbrPEI储备溶液(c:10mg/mL于水中)进行混合。将两个20mL注射器固定在混合装置中。它们中的每一个通过管连接至针。使用注射泵的抽取功能,将一个注射器填充RNA溶液并且另一个填充PEI溶液。(设置:直径:20.1mm,流速:5mL/分钟,体积:5.9mL)。将针除去,并且将管连接至混合阀。重要的是将含有RNA溶液的注射器连接至阀的倾斜位置。为了控制出口直径,连接针。使用注射泵的输注功能进行混合(设置:直径:20.1mm,流速:40mL/分钟,体积:5.8mL)。为了实现可再现的多分散性指数,在混合期间将样品人工分级分离。将直到流动稳定的最初几μL(100-200μL)和有时含有气泡的最后几μL收集在单独的管中。将混合物在室温下孵育30分钟以用于聚合复合物形成并且之后储存在冰上。对于不同剂量,如表5中所示对所述参数进行修改和适配。
表5:用于不同混合体积的示例性体积和设置
V(抽取)和V(输注)指定用于分别吸入和分配mRNA和PEI组分的注射器泵上的设置。
转染HEK细胞以检查雾化复合物的功能性。在雾化后,将复合物的等分试样(80μl)用于转染HEK细胞。在转染前一天,将1x106个细胞涂铺在6孔板中。在转染当天,从细胞中除去培养基,将细胞用PBS洗涤细胞一次,接着每孔添加80μl的复合物连同920μl的无血清MEM培养基。对于每种复合物,制备三个重复的孔。在标准细胞培养条件下将细胞与复合物一起孵育4小时。在孵育结束时,将含有复合物的培养基除去并且每孔添加含血清的MEM培养基(1ml)。将板在标准细胞培养条件下孵育。在转染后24小时,使用用于动物组织的相同方案和缓冲剂制备蛋白质裂解物,不包括均化步骤。汇集来自三个孔的细胞以用于分析。使用用R24.1抗体(R&DSystems)免疫沉淀和用217、432和596抗体(全部来自CysticFibrosisConsortium,UniversityofPennsylvania,PA,USA)的组合蛋白质印迹来检测人CFTR的表达。对于在猪中雾化的所有复合物可检测到hCFTR(参见图54-57)。
气雾剂施加。将气雾剂(WFI单独44ml;于WFI中修饰的mRNAPEI制剂:8、24和44ml)雾化并且经由筛孔喷雾器吸入至麻醉的猪中。猪的镇静通过以下方式来引发:用阿扎哌隆2mg/kg体重、氯胺酮15mg/kg体重、阿托品0.1mg/kg体重术前用药,并且接着将静脉内管线插入至侧耳静脉。将猪通过静脉内注射丙泊酚3-5mg/kg体重视需求进行麻醉。通过以4至8mg/kg体重推注注射异氟烷(2%-3%)与1%丙泊酚以视需求增强麻醉来维持麻醉。麻醉的持续时间是大约1-3小时。在完成气雾化之后经由侧耳静脉24小时将猪用推注注射戊巴比妥(100mg/kg体重)和氯化钾来处死。将肺切除并且从不同肺区域收集组织样本。使储存的样品经受不同评定方法,如生物发光、组织病理学、IP/蛋白质印迹和Elisa。
生物发光分析。为了测量荧光素酶活性,将组织样本均化且在管光度计中分析或在包含D-荧光素底物的培养基浴中孵育且经受离体荧光素酶BLI。所述数据显示,当与来自第1组(WFI媒介物对照)的对照肺组织样品相比较时,对于2-4组(分别1mg、5mg和10mg)中的每个观察到较强生物发光信号(图35-38)。
通过蛋白质印迹和免疫组织化学进行CFTR表达分析。将FFL阳性组织样品切除(对于一组内的每头猪最少10个样品)并且通过免疫沉淀/蛋白质印迹(IP-WB)和免疫组织化学针对人CFTR进行分析。简言之,从猪肺如下制备蛋白质裂解物:300-400mg之间的肺组织用于分析。使用LysingMatrixA(MPBiomedicals,Ref:6910-500)和匀浆器“FastPrep24”(MPBiomedicals)将组织在含有蛋白酶抑制剂的基础缓冲液(20mMTris,150mMNaCl,pH8.0)中均化。将整个组织混合物转移至新的2ml安全锁定预冷却的Eppendorf管并且添加25μl碘乙酰胺(Sigma:I6125)和1μlOmni裂解物(1:5稀释于Omni裂解缓冲液中)(Epicenter:OC7810K)。然后将样品在冰上孵育5分钟,接着添加26μl的10%SDS溶液。将样品在振荡器上在4℃下进一步孵育60分钟。在孵育之后,将260μl的裂解缓冲液(850μl基础缓冲液+10%TritonX-100+5%脱氧胆酸钠)添加至样品并且将它们在4℃下在振荡器上孵育90分钟。最后,将蛋白质裂解物在13,000rpm下在4℃下离心10-20分钟,并且将上清液转移至新的Eppendorf管中。使用BCA蛋白质测定(Pierce)定量蛋白质浓度。将样品等分成含有10mg总蛋白质并且用基础缓冲液将最终体积调整至1ml/样品。基于实施例6中呈现的数据,使用抗体R24.1进行CFTR的免疫沉淀并且接着使用自CysticFibrosisConsortium,UniversityofPennsylvania,PA,USA获得的三种不同抗体(抗体217、432、596)的三重组合进行CFTR的蛋白质印迹免疫检测。为了控制不同动物之间的组内可变性和CFTR表达的可变性,将对应于150kDa的蛋白质大小标准中的标记物条带设置为参考并且将不同组的条带强度标准化至该值。如图39中所示,来自第1组的对照猪分析的仅16%的组织样品产生高于基线的CFTR表达水平。相比之下,第3组和第4组(其分别代表5mg和10mg处理组)各自产生大于30%的其肺组织样品针对高于基线的CFTR表达水平测试为阳性(图39)。此外,在第3组和第4组内观察到的CFTR表达的增加比对照几乎高两倍。
通过定量CFTR-阳性支气管和细支气管来进行CFTR免疫组织化学的分析。如果在展示清晰膜定位的CFTR信号的上皮细胞层内检测到至少一个上皮细胞,则支气管/细支气管被认为是阳性的。“阳性”样品的代表性图像在图40中描绘。分别利用单一抗体或多达三种抗体的组合通过评定可用抗体针对CFTR的特异性来优化用于CFTR免疫组织化学的条件。在抗体596的孵育之后观察到清楚的CFTR特异性信号。数据证明,在所有四组的肺组织切片中检测到CFTR阳性上皮细胞,从而证明通过免疫组织化学程序检测人和猪CFTR(图41和45)。虽然对于第3组观察到低(图42)、中(图43)和高(图44)CFTR表达水平,但总体发现证明,与媒介物对照相比,密码子优化的人CFTRSNIMRNA的5mg处理产生较大数目的CFTR阳性细胞和总体CFTR信号强度。数据还示出在10mg处理后CFTR表达的进一步增强,从而证明清楚的剂量响应作用(图45)。CFTR阳性支气管/细支气管的绝对和相对数量的定量进一步支持这些发现,从而揭示与媒介物对照相比,用5或10mg的人CFTRSNIMRNA处理的动物中的显著更高数目(图46)。在用人CFTRSNIMRNA处理后,指示CFTR表达水平的总体上升。
通过原位杂交(ISH)进行CFTR表达分析。将FFL阳性组织样品切除(对于一组内的每头猪最少10个样品)并且使用(AdvancedCellDiagnostic)“ZZ”探针技术经受人工原位杂交分析。基于密码子优化的人CFTRSNIMRNA(SEQIDNO:17)的密码子优化的序列产生探针。简言之,测定是被设计来显现固定在载玻片上的福尔马林固定、石蜡包埋的(FFPE)组织中的单个RNA分子/细胞的原位杂交测定。将每个包埋的组织样品根据制造商方案进行预处理,并且与靶特异性人CFTR特异性RNA探针一起孵育。所述hCFTR探针显示结合CFTR,其中与人、小鼠、大鼠、猪和猴具有交叉反应性。一旦结合,将所述探针通过一系列6轮连续扩增杂交至一连串信号放大分子。然后将所述样品用对于信号放大表达盒特异性的HRP标记的探针进行处理,并且通过使用3,3’-二氨基联苯胺(DAB)彩色可视化测定。对于泛素C具有特异性的探针用作阳性对照(图47A和48A),而dapB用作阴性对照(图47B和48B)。将阳性CFTR信号与未处理的和媒介物对照处理的猪肺组织的阳性CFTR信号进行比较(图49A和49B)。在标准亮场显微镜下可视化染色的样品。数据证明,当与媒介物对照相比较时,用1mg的密码子优化的人CFTRSNIMRNA处理在第2组的右(A)和左(B)肺组织两者中产生CFTR表达的显著增加(图49和图50A和图50B)。此外,对于5mg和10mg处理组观察到CFTR表达的进一步增加,如由在针对第3组和第4组分析的右(A)和左(B)肺样品内观察到的显著增加染色所证明(图51和图52A和图52B)。总之,这些数据有力地支持经由吸入有效递送mRNA和在肺及其各种组织的两叶内表达人CFTR。
结论。所述结果证明,荧光素酶和CFTRmRNA两者可有效地体内递送至肺组织。在从肺的右叶和左叶两者内的不同区域收集的各种组织样品中观察到荧光素酶表达。因此表明雾化是用于施用mRNA的有效方法并且产生相当均匀的分布。此外,除了荧光素酶,CFTRmRNA也可有效地递送至肺,从而导致增强的蛋白质表达。通过IP-WB、免疫组织化学和原位杂交验证表达和蛋白质活性。每种方法清楚地证明在肺的组织内mRNA递送和CFTR表达和/或活性的剂量依赖性增加。总之,实验强调用于将CFTRmRNA递送至人受试者的肺的总体可实践性和可行性并且证明体内CFTR蛋白质产生用于治疗用途的有效性。
实施例12:肺中的体内表达
所述实施例证明在气雾剂递送负载mRNA的纳米颗粒之后肺中的成功体内表达。使用获得自TechnicalUniversityMunich,Weihenstephan,Germany的GermanLandrace的猪进行所有研究。所述猪具有35-90kg范围内的体重。使用Pari喷射喷雾器引入FFL/CO-CFTR-C-His10mRNA制剂或媒介物对照。将猪处死并且用盐水灌注,在一段预定时间段之后,允许从mRNA表达蛋白质。
信使RNA合成。在所述实施例中,通过从质粒DNA模板体外转录来合成密码子优化的萤火虫荧光素酶(CO-FFL)mRNA。
cKK-E12制剂。对于基于脂质的纳米颗粒实验,使用封装于cKK-E12:DOPE:Chol:PEGDMG2K(相对量40:30:25:5(摩尔比))的制剂中的1mgFFL+9mg的CO-CFTR-C-His10mRNA产生脂质制剂。使用所指示的喷雾器将溶液雾化至猪肺。
气雾剂施加。将气雾剂(盐水或CO-FFLcKK-E12制剂)雾化并且吸入至麻醉的猪中。猪的镇静通过以下方式来引发:用阿扎哌隆2mg/kg体重、氯胺酮15mg/kg体重、阿托品0.1mg/kg体重术前用药,并且接着将静脉内管线插入至侧耳静脉。将猪通过静脉内注射丙泊酚3-5mg/kg体重视需求进行麻醉。通过以4至8mg/kg体重推注注射异氟烷(2%-3%)与1%丙泊酚以视需求增强麻醉来维持麻醉。麻醉的持续时间是大约1-3小时。将猪通过侧耳静脉用推注注射戊巴比妥(100mg/kg体重)和氯化钾来杀死。将肺切除,并且从不同肺区域收集组织样本,接着在细胞培养基中孵育过夜。使所储存的样品经受生物发光检测。
生物发光分析。为了测量荧光素酶活性,将组织样本均化且在管光度计中分析或在包含D-荧光素底物的培养基浴中孵育且经受离体荧光素酶BLI。当与(B)来自对照猪的对照肺组织样品(盐水媒介物对照)相比时,对于(A)FFL/CO-CFTR-C-His10mRNA处理的猪中的每个观察到强的生物发光信号(图53A和53B)。
这些数据说明,通过气雾剂施用,FFL/CFTRmRNA被成功地递送至肺并且在肺中表达。
序列简述
SEQIDNO1.野生型CFTR氨基酸序列。
SEQIDNO2.野生型CFTRmRNA编码序列。
SEQIDNO3.非天然存在的CFTRmRNA编码序列#1。
SEQIDNO4.CFTRmRNA5’-UTR。
SEQIDNO5.CFTRmRNA3’-UTR#1。
SEQIDNO6.FFL5’UTR。
SEQIDNO7.FFL编码序列。
SEQIDNO8.FFL3’UTR。
SEQIDNO9.非天然存在的CFTRmRNA编码序列#2。
SEQIDNO10.非天然存在的CFTRmRNA编码序列#3。
SEQIDNO11.非天然存在的CFTRmRNA编码序列#4。
SEQIDNO12.非天然存在的CFTRmRNA编码序列#5。
SEQIDNO13.非天然存在的CFTRmRNA编码序列#6。
SEQIDNO14.非天然存在的CFTRmRNA编码序列#7。
SEQIDNO15.密码子优化的人CFTRC-末端His10融合mRNA编码序列。
SEQIDNO16.具有生长激素前导序列的密码子优化的人CFTRmRNA编码序列
SEQIDNO17.密码子优化的CFTRmRNA
SEQIDNO18.mRNA前导序列#1
SEQIDNO19.mRNA前导序列#2
SEQIDNO20.CFTRmRNA3’-UTR#2
SEQIDNO:1
MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWDRELASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEERSIAIYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISIGQLVSLLSNNLNKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLALFQAGLGRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQTELKLTRKAAYVRYFNSSAFFFSGFFVVFLSVLPYALIKGIILRKIFTTISFCIVLRMAVTRQFPWAVQTWYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEEGFGELFEKAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTGAGKTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKACQLEEDISKFAEKDNIVLGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDFSSKLMGCDSFDQFSAERRNSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKRKNSILNPINSIRKFSIVQKTPLQMNGIEEDSDEPLERRLSLVPDSEQGEAILPRISVISTGPTLQARRRQSVLNLMTHSVNQGQNIHRKTTASTRKVSLAPQANLTELDIYSRRLSQETGLEISEEINEEDLKECFFDDMESIPAVTTWNTYLRYITVHKSLIFVLIWCLVIFLAEVAASLVVLWLLGNTPLQDKGNSTHSRNNSYAVIITSTSSYYVFYIYVGVADTLLAMGFFRGLPLVHTLITVSKILHHKMLHSVLQAPMSTLNTLKAGGILNRFSKDIAILDDLLPLTIFDFIQLLLIVIGAIAVVAVLQPYIFVATVPVIVAFIMLRAYFLQTSQQLKQLESEGRSPIFTHLVTSLKGLWTLRAFGRQPYFETLFHKALNLHTANWFLYLSTLRWFQMRIEMIFVIFFIAVTFISILTTGEGEGRVGIILTLAMNIMSTLQWAVNSSIDVDSLMRSVSRVFKFIDMPTEGKPTKSTKPYKNGQLSKVMIIENSHVKKDDIWPSGGQMTVKDLTAKYTEGGNAILENISFSISPGQRVGLLGRTGSGKSTLLSAFLRLLNTEGEIQIDGVSWDSITLQQWRKAFGVIPQKVFIFSGTFRKNLDPYEQWSDQEIWKVADEVGLRSVIEQFPGKLDFVLVDGGCVLSHGHKQLMCLARSVLSKAKILLLDEPSAHLDPVTYQIIRRTLKQAFADCTVILCEHRIEAMLECQQFLVIEENKVRQYDSIQKLLNERSLFRQAISPSDRVKLFPHRNSSKCKSKPQIAALKEETEEEVQDTRL(SEQIDNO:1)
SEQIDNO:2AUGCAGAGGUCGCCUCUGGAAAAGGCCAGCGUUGUCUCCAAACUUUUUUUCAGCUGGACCAGACCAAUUUUGAGGAAAGGAUACAGACAGCGCCUGGAAUUGUCAGACAUAUACCAAAUCCCUUCUGUUGAUUCUGCUGACAAUCUAUCUGAAAAAUUGGAAAGAGAAUGGGAUAGAGAGCUGGCUUCAAAGAAAAAUCCUAAACUCAUUAAUGCCCUUCGGCGAUGUUUUUUCUGGAGAUUUAUGUUCUAUGGAAUCUUUUUAUAUUUAGGGGAAGUCACCAAAGCAGUACAGCCUCUCUUACUGGGAAGAAUCAUAGCUUCCUAUGACCCGGAUAACAAGGAGGAACGCUCUAUCGCGAUUUAUCUAGGCAUAGGCUUAUGCCUUCUCUUUAUUGUGAGGACACUGCUCCUACACCCAGCCAUUUUUGGCCUUCAUCACAUUGGAAUGCAGAUGAGAAUAGCUAUGUUUAGUUUGAUUUAUAAGAAGACUUUAAAGCUGUCAAGCCGUGUUCUAGAUAAAAUAAGUAUUGGACAACUUGUUAGUCUCCUUUCCAACAACCUGAACAAAUUUGAUGAAGGACUUGCAUUGGCACAUUUCGUGUGGAUCGCUCCUUUGCAAGUGGCACUCCUCAUGGGGCUAAUCUGGGAGUUGUUACAGGCGUCUGCCUUCUGUGGACUUGGUUUCCUGAUAGUCCUUGCCCUUUUUCAGGCUGGGCUAGGGAGAAUGAUGAUGAAGUACAGAGAUCAGAGAGCUGGGAAGAUCAGUGAAAGACUUGUGAUUACCUCAGAAAUGAUUGAAAAUAUCCAAUCUGUUAAGGCAUACUGCUGGGAAGAAGCAAUGGAAAAAAUGAUUGAAAACUUAAGACAAACAGAACUGAAACUGACUCGGAAGGCAGCCUAUGUGAGAUACUUCAAUAGCUCAGCCUUCUUCUUCUCAGGGUUCUUUGUGGUGUUUUUAUCUGUGCUUCCCUAUGCACUAAUCAAAGGAAUCAUCCUCCGGAAAAUAUUCACCACCAUCUCAUUCUGCAUUGUUCUGCGCAUGGCGGUCACUCGGCAAUUUCCCUGGGCUGUACAAACAUGGUAUGACUCUCUUGGAGCAAUAAACAAAAUACAGGAUUUCUUACAAAAGCAAGAAUAUAAGACAUUGGAAUAUAACUUAACGACUACAGAAGUAGUGAUGGAGAAUGUAACAGCCUUCUGGGAGGAGGGAUUUGGGGAAUUAUUUGAGAAAGCAAAACAAAACAAUAACAAUAGAAAAACUUCUAAUGGUGAUGACAGCCUCUUCUUCAGUAAUUUCUCACUUCUUGGUACUCCUGUCCUGAAAGAUAUUAAUUUCAAGAUAGAAAGAGGACAGUUGUUGGCGGUUGCUGGAUCCACUGGAGCAGGCAAGACUUCACUUCUAAUGAUGAUUAUGGGAGAACUGGAGCCUUCAGAGGGUAAAAUUAAGCACAGUGGAAGAAUUUCAUUCUGUUCUCAGUUUUCCUGGAUUAUGCCUGGCACCAUUAAAGAAAAUAUCAUCUUUGGUGUUUCCUAUGAUGAAUAUAGAUACAGAAGCGUCAUCAAAGCAUGCCAACUAGAAGAGGACAUCUCCAAGUUUGCAGAGAAAGACAAUAUAGUUCUUGGAGAAGGUGGAAUCACACUGAGUGGAGGUCAACGAGCAAGAAUUUCUUUAGCAAGAGCAGUAUACAAAGAUGCUGAUUUGUAUUUAUUAGACUCUCCUUUUGGAUACCUAGAUGUUUUAACAGAAAAAGAAAUAUUUGAAAGCUGUGUCUGUAAACUGAUGGCUAACAAAACUAGGAUUUUGGUCACUUCUAAAAUGGAACAUUUAAAGAAAGCUGACAAAAUAUUAAUUUUGAAUGAAGGUAGCAGCUAUUUUUAUGGGACAUUUUCAGAACUCCAAAAUCUACAGCCAGACUUUAGCUCAAAACUCAUGGGAUGUGAUUCUUUCGACCAAUUUAGUGCAGAAAGAAGAAAUUCAAUCCUAACUGAGACCUUACACCGUUUCUCAUUAGAAGGAGAUGCUCCUGUCUCCUGGACAGAAACAAAAAAACAAUCUUUUAAACAGACUGGAGAGUUUGGGGAAAAAAGGAAGAAUUCUAUUCUCAAUCCAAUCAACUCUAUACGAAAAUUUUCCAUUGUGCAAAAGACUCCCUUACAAAUGAAUGGCAUCGAAGAGGAUUCUGAUGAGCCUUUAGAGAGAAGGCUGUCCUUAGUACCAGAUUCUGAGCAGGGAGAGGCGAUACUGCCUCGCAUCAGCGUGAUCAGCACUGGCCCCACGCUUCAGGCACGAAGGAGGCAGUCUGUCCUGAACCUGAUGACACACUCAGUUAACCAAGGUCAGAACAUUCACCGAAAGACAACAGCAUCCACACGAAAAGUGUCACUGGCCCCUCAGGCAAACUUGACUGAACUGGAUAUAUAUUCAAGAAGGUUAUCUCAAGAAACUGGCUUGGAAAUAAGUGAAGAAAUUAACGAAGAAGACUUAAAGGAGUGCCUUUUUGAUGAUAUGGAGAGCAUACCAGCAGUGACUACAUGGAACACAUACCUUCGAUAUAUUACUGUCCACAAGAGCUUAAUUUUUGUGCUAAUUUGGUGCUUAGUAAUUUUUCUGGCAGAGGUGGCUGCUUCUUUGGUUGUGCUGUGGCUCCUUGGAAACACUCCUCUUCAAGACAAAGGGAAUAGUACUCAUAGUAGAAAUAACAGCUAUGCAGUGAUUAUCACCAGCACCAGUUCGUAUUAUGUGUUUUACAUUUACGUGGGAGUAGCCGACACUUUGCUUGCUAUGGGAUUCUUCAGAGGUCUACCACUGGUGCAUACUCUAAUCACAGUGUCGAAAAUUUUACACCACAAAAUGUUACAUUCUGUUCUUCAAGCACCUAUGUCAACCCUCAACACGUUGAAAGCAGGUGGGAUUCUUAAUAGAUUCUCCAAAGAUAUAGCAAUUUUGGAUGACCUUCUGCCUCUUACCAUAUUUGACUUCAUCCAGUUGUUAUUAAUUGUGAUUGGAGCUAUAGCAGUUGUCGCAGUUUUACAACCCUACAUCUUUGUUGCAACAGUGCCAGUGAUAGUGGCUUUUAUUAUGUUGAGAGCAUAUUUCCUCCAAACCUCACAGCAACUCAAACAACUGGAAUCUGAAGGCAGGAGUCCAAUUUUCACUCAUCUUGUUACAAGCUUAAAAGGACUAUGGACACUUCGUGCCUUCGGACGGCAGCCUUACUUUGAAACUCUGUUCCACAAAGCUCUGAAUUUACAUACUGCCAACUGGUUCUUGUACCUGUCAACACUGCGCUGGUUCCAAAUGAGAAUAGAAAUGAUUUUUGUCAUCUUCUUCAUUGCUGUUACCUUCAUUUCCAUUUUAACAACAGGAGAAGGAGAAGGAAGAGUUGGUAUUAUCCUGACUUUAGCCAUGAAUAUCAUGAGUACAUUGCAGUGGGCUGUAAACUCCAGCAUAGAUGUGGAUAGCUUGAUGCGAUCUGUGAGCCGAGUCUUUAAGUUCAUUGACAUGCCAACAGAAGGUAAACCUACCAAGUCAACCAAACCAUACAAGAAUGGCCAACUCUCGAAAGUUAUGAUUAUUGAGAAUUCACACGUGAAGAAAGAUGACAUCUGGCCCUCAGGGGGCCAAAUGACUGUCAAAGAUCUCACAGCAAAAUACACAGAAGGUGGAAAUGCCAUAUUAGAGAACAUUUCCUUCUCAAUAAGUCCUGGCCAGAGGGUGGGCCUCUUGGGAAGAACUGGAUCAGGGAAGAGUACUUUGUUAUCAGCUUUUUUGAGACUACUGAACACUGAAGGAGAAAUCCAGAUCGAUGGUGUGUCUUGGGAUUCAAUAACUUUGCAACAGUGGAGGAAAGCCUUUGGAGUGAUACCACAGAAAGUAUUUAUUUUUUCUGGAACAUUUAGAAAAAACUUGGAUCCCUAUGAACAGUGGAGUGAUCAAGAAAUAUGGAAAGUUGCAGAUGAGGUUGGGCUCAGAUCUGUGAUAGAACAGUUUCCUGGGAAGCUUGACUUUGUCCUUGUGGAUGGGGGCUGUGUCCUAAGCCAUGGCCACAAGCAGUUGAUGUGCUUGGCUAGAUCUGUUCUCAGUAAGGCGAAGAUCUUGCUGCUUGAUGAACCCAGUGCUCAUUUGGAUCCAGUAACAUACCAAAUAAUUAGAAGAACUCUAAAACAAGCAUUUGCUGAUUGCACAGUAAUUCUCUGUGAACACAGGAUAGAAGCAAUGCUGGAAUGCCAACAAUUUUUGGUCAUAGAAGAGAACAAAGUGCGGCAGUACGAUUCCAUCCAGAAACUGCUGAACGAGAGGAGCCUCUUCCGGCAAGCCAUCAGCCCCUCCGACAGGGUGAAGCUCUUUCCCCACCGGAACUCAAGCAAGUGCAAGUCUAAGCCCCAGAUUGCUGCUCUGAAAGAGGAGACAGAAGAAGAGGUGCAAGAUACAAGGCUUUAG(SEQIDNO:2)
SEQIDNO:3
AUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAA(SEQIDNO:3)
SEQIDNO:4
GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG(SEQIDNO:4)
SEQIDNO:5
CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC(SEQIDNO:5)
SEQIDNO:6
GGGAUCCUACC(SEQIDNO:6)
SEQIDNO:7
AUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCACUCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCCUGGUGCCCGGCACCAUCGCCUUUACCGACGCACAUAUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUCAGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGCCAGGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGAAGAGCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGUGUA(SEQIDNO:7)
SEQIDNO:8
UUUGAAUU(SEQIDNO:8)
SEQIDNO:9
AUGCAGAGAAGCCCCCUGGAAAAGGCCAGCGUGGUGUCCAAGCUGUUCUUCAGCUGGACCAGACCCAUCCUGAGAAAGGGCUACAGACAGAGACUGGAACUGAGCGACAUCUACCAGAUCCCCAGCGUGGACAGCGCCGACAACCUGAGCGAGAAGCUGGAAAGAGAGUGGGACAGAGAGCUGGCUAGCAAGAAGAACCCCAAGCUGAUCAACGCCCUGAGGCGGUGCUUCUUCUGGCGGUUUAUGUUCUACGGCAUCUUCCUGUACCUGGGCGAAGUGACAAAGGCCGUGCAGCCCCUGCUCCUGGGCAGAAUCAUUGCCAGCUACGACCCCGACAACAAAGAGGAAAGAUCUAUCGCCAUCUACCUGGGCAUCGGCCUGUGCCUGCUGUUCAUCGUGCGGACACUGCUGCUGCACCCCGCCAUCUUCGGCCUGCACCACAUCGGCAUGCAGAUGAGAAUCGCCAUGUUCAGCCUGAUCUACAAGAAAACCCUGAAGCUGAGCAGCAGGGUGCUGGACAAGAUCAGCAUCGGACAGCUGGUGUCCCUGCUGAGCAACAACCUGAACAAGUUCGACGAGGGACUGGCCCUGGCUCACUUCGUGUGGAUCGCUCCACUGCAGGUCGCCCUGCUGAUGGGCCUGAUCUGGGAGCUGCUGCAGGCCAGCGCUUUCUGCGGCCUGGGCUUUCUGAUUGUGCUGGCCCUGUUUCAGGCUGGCCUGGGCAGGAUGAUGAUGAAGUACAGGGACCAGAGAGCCGGCAAGAUCAGCGAGAGACUGGUCAUCACCAGCGAGAUGAUCGAGAACAUCCAGAGCGUGAAGGCCUACUGCUGGGAAGAGGCCAUGGAAAAGAUGAUCGAAAACCUGAGACAGACCGAGCUGAAGCUGACCAGAAAGGCCGCCUACGUGCGGUACUUCAACAGCAGCGCCUUCUUCUUCUCCGGCUUCUUCGUGGUGUUCCUGUCCGUGCUGCCCUACGCCCUGAUCAAGGGCAUCAUCCUGAGGAAGAUCUUCACCACCAUUUCUUUCUGCAUCGUGCUGAGAAUGGCCGUGACCAGACAGUUCCCCUGGGCCGUGCAGACUUGGUACGACAGCCUGGGCGCCAUCAACAAGAUCCAGGACUUCCUGCAGAAGCAGGAGUACAAGACCCUCGAGUACAACCUGACCACCACCGAGGUGGUCAUGGAAAACGUGACCGCCUUCUGGGAGGAAGGCUUCGGCGAGCUGUUCGAGAAGGCCAAGCAGAACAACAACAACAGAAAGACCAGCAACGGCGACGACUCCCUGUUCUUCUCCAACUUCUCCCUGCUGGGCACCCCCGUGCUGAAGGACAUCAACUUCAAGAUCGAGAGAGGCCAGCUGCUCGCCGUGGCCGGCUCUACAGGCGCUGGCAAGACCUCUCUGCUGAUGGUCAUCAUGGGCGAGCUGGAACCCAGCGAGGGCAAGAUCAAGCACAGCGGCAGAAUCAGCUUCUGCAGCCAGUUCAGCUGGAUCAUGCCCGGCACCAUCAAAGAGAACAUCAUCUUCGGCGUGUCCUACGACGAGUACAGAUACAGAAGCGUGAUCAAGGCCUGCCAGCUGGAAGAGGACAUCAGCAAGUUCGCCGAGAAGGACAACAUCGUGCUGGGCGAGGGCGGCAUCACCCUGUCUGGCGGCCAGAGAGCCAGAAUCAGCCUGGCCAGAGCCGUGUACAAGGACGCCGACCUGUACCUGCUGGACAGCCCCUUCGGCUACCUGGACGUGCUGACCGAGAAAGAGAUCUUCGAGAGCUGCGUGUGCAAGCUGAUGGCCAACAAGACCAGAAUCCUGGUCACCAGCAAGAUGGAACACCUGAAGAAGGCCGACAAGAUCCUGAUCCUGCACGAGGGCAGCAGCUACUUCUACGGCACAUUCAGCGAGCUGCAGAACCUGCAGCCCGACUUCAGCAGCAAACUGAUGGGCUGCGACAGCUUCGACCAGUUCAGCGCCGAGAGAAGAAACAGCAUCCUGACCGAGACACUGCACAGAUUCAGCCUGGAAGGCGACGCCCCCGUGUCUUGGACCGAGACAAAGAAGCAGAGCUUCAAGCAGACCGGCGAGUUCGGCGAGAAGAGAAAGAACUCCAUCCUGAACCCCAUCAACAGCAUCCGGAAGUUCAGCAUCGUGCAGAAAACCCCCCUGCAGAUGAACGGCAUCGAAGAGGACAGCGACGAGCCCCUGGAAAGACGGCUGAGCCUGGUGCCUGACAGCGAGCAGGGCGAGGCCAUCCUGCCUAGAAUCAGCGUGAUCAGCACCGGCCCCACCCUGCAGGCUAGAAGGCGGCAGAGCGUGCUGAACCUGAUGACCCACAGCGUGAACCAGGGCCAGAACAUCCACCGCAAGACCACCGCCAGCACCAGAAAGGUGUCCCUGGCUCCUCAGGCCAACCUGACCGAGCUGGACAUCUACAGCAGAAGGCUGAGCCAGGAAACCGGCCUGGAAAUCAGCGAGGAAAUCAACGAAGAGGACCUGAAAGAGUGCUUCUUCGACGACAUGGAAUCCAUCCCCGCCGUGACCACCUGGAACACCUACCUGCGGUACAUCACCGUGCACAAGAGCCUGAUCUUCGUGCUGAUCUGGUGCCUGGUCAUCUUCCUGGCCGAGGUGGCCGCCAGCCUGGUGGUGCUGUGGCUCCUGGGAAACACCCCUCUGCAGGACAAGGGCAACAGCACCCACAGCAGAAACAACAGCUACGCCGUGAUCAUCACCUCCACCAGCUCCUACUACGUGUUCUACAUCUACGUGGGCGUGGCCGACACCCUGCUGGCUAUGGGCUUCUUCAGAGGCCUGCCCCUGGUGCACACCCUGAUCACCGUGUCCAAGAUCCUGCACCAUAAGAUGCUGCACAGCGUGCUGCAGGCUCCCAUGAGCACCCUGAACACACUGAAGGCUGGCGGCAUCCUGAACAGGUUCAGCAAGGAUAUCGCCAUCCUGGACGACCUGCUGCCUCUGACCAUCUUCGACUUCAUCCAGCUGCUGCUGAUCGUGAUCGGCGCUAUCGCCGUGGUGGCCGUGCUGCAGCCCUACAUCUUCGUGGCCACCGUGCCCGUGAUCGUGGCCUUCAUUAUGCUGAGAGCCUACUUUCUGCAGACCAGCCAGCAGCUGAAGCAGCUGGAAAGCGAGGGCAGAAGCCCCAUCUUCACCCACCUCGUGACCAGCCUGAAGGGCCUGUGGACCCUGAGAGCCUUCGGCAGACAGCCCUACUUCGAGACACUGUUCCACAAGGCCCUGAACCUGCACACCGCCAACUGGUUUCUGUACCUGUCCACCCUGAGAUGGUUCCAGAUGAGGAUCGAGAUGAUCUUCGUCAUCUUCUUUAUCGCCGUGACCUUCAUCUCUAUCCUGACCACCGGCGAGGGCGAGGGAAGAGUGGGAAUCAUCCUGACCCUGGCCAUGAACAUCAUGAGCACACUGCAGUGGGCCGUGAACAGCAGCAUCGACGUGGACAGCCUGAUGAGAAGCGUGUCCAGAGUGUUCAAGUUCAUCGACAUGCCUACCGAGGGCAAGCCCACCAAGAGCACCAAGCCCUACAAGAACGGCCAGCUGAGCAAAGUGAUGAUCAUCGAGAACAGCCACGUCAAGAAGGACGACAUCUGGCCCAGCGGCGGACAGAUGACCGUGAAGGACCUGACCGCCAAGUACACAGAGGGCGGCAACGCUAUCCUGGAAAACAUCAGCUUCAGCAUCAGCCCAGGCCAGAGAGUGGGCCUGCUGGGGAGAACAGGCAGCGGCAAGUCUACCCUGCUGUCCGCCUUCCUGAGACUGCUGAACACCGAGGGCGAGAUCCAGAUCGAUGGCGUGUCCUGGGACUCCAUCACCCUGCAGCAGUGGCGCAAGGCCUUCGGCGUGAUCCCCCAGAAGGUGUUCAUCUUCAGCGGCACCUUCAGAAAGAACCUGGACCCCUACGAGCAGUGGUCCGACCAGGAAAUCUGGAAGGUCGCCGAUGAAGUGGGCCUGAGAUCCGUGAUCGAGCAGUUCCCCGGCAAGCUGGACUUCGUGCUGGUGGACGGCGGCUGCGUGCUGAGCCACGGCCACAAGCAGCUGAUGUGUCUGGCCCGCUCCGUGCUGAGCAAGGCUAAGAUUCUGCUGCUGGACGAGCCUAGCGCCCACCUGGACCCUGUGACCUACCAGAUCAUCAGAAGGACCCUGAAGCAGGCCUUCGCCGACUGCACCGUGAUCCUGUGCGAGCACAGAAUCGAGGCCAUGCUGGAAUGCCAGCAGUUCCUGGUCAUCGAAGAGAACAAAGUGCGGCAGUACGACAGCAUCCAGAAGCUGCUGAACGAGAGAAGCCUGUUCAGACAGGCCAUCAGCCCCAGCGACAGAGUGAAGCUGUUCCCCCACCGCAACAGCAGCAAGUGCAAGAGCAAGCCCCAGAUCGCCGCCCUGAAAGAAGAGACUGAGGAAGAGGUGCAGGACACCAGACUGUGA(SEQIDNO:9)
SEQIDNO:10
AUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCCCGCUGCAAGUCGCACUGCUUAUGGGACUGAUUUGGGAACUGUUGCAGGCCAGCGCCUUUUGCGGCCUGGGAUUUCUCAUUGUGCUUGCACUUUUCCAAGCAGGGCUCGGCAGAAUGAUGAUGAAGUACAGGGACCAGAGAGCCGGAAAGAUCUCAGAACGGCUCGUGAUUACUUCAGAAAUGAUCGAGAACAUUCAAUCGGUGAAAGCGUACUGCUGGGAAGAGGCGAUGGAAAAGAUGAUCGAAAACCUCAGACAGACCGAGUUGAAGCUGACCCGGAAGGCCGCGUACGUCAGAUACUUCAACAGCAGCGCUUUCUUCUUCUCGGGCUUCUUCGUCGUGUUCCUGUCGGUGCUGCCGUAUGCCCUCAUUAAGGGAAUUAUCUUGCGGAAGAUCUUUACUACUAUCUCAUUUUGCAUCGUCCUUCGGAUGGCGGUCACUCGGCAGUUCCCGUGGGCCGUGCAGACCUGGUACGACAGCCUCGGGGCCAUCAACAAGAUCCAAGACUUUCUCCAAAAGCAAGAGUACAAAACCCUCGAAUACAACCUCACCACUACUGAAGUGGUCAUGGAAAACGUGACCGCCUUUUGGGAAGAAGGCUUCGGAGAACUGUUCGAGAAGGCGAAGCAAAACAACAAUAAUCGCAAGACUAGCAACGGGGAUGACUCACUGUUCUUCAGCAAUUUCUCACUGCUCGGCACCCCGGUGCUUAAGGACAUCAACUUCAAGAUUGAACGCGGACAGCUCUUGGCGGUGGCCGGAUCCACCGGAGCAGGAAAGACUAGCCUGCUGAUGGUGAUCAUGGGUGAGCUGGAACCGUCCGAAGGCAAAAUCAAGCACUCCGGCAGAAUCAGCUUCUGCUCGCAGUUUUCGUGGAUCAUGCCAGGAACCAUCAAAGAGAACAUCAUCUUUGGAGUCUCAUACGAUGAGUACCGCUACAGAAGCGUGAUUAAGGCCUGCCAGCUUGAAGAGGACAUCUCCAAGUUCGCGGAAAAGGACAACAUCGUGCUGGGUGAGGGAGGGAUCACGUUGUCGGGCGGUCAGAGAGCCCGCAUUUCGCUGGCACGGGCUGUGUACAAGGAUGCGGAUCUUUACCUUCUGGACUCGCCAUUCGGUUACCUCGACGUGCUGACCGAAAAAGAAAUCUUCGAGAGCUGCGUGUGUAAGCUGAUGGCUAAUAAGACUAGAAUCCUCGUGACGUCCAAAAUGGAACAUCUUAAGAAGGCGGAUAAGAUUCUCAUUCUUCACGAGGGGUCGAGCUACUUCUACGGGACUUUUAGCGAGCUGCAGAAUUUGCAGCCGGACUUCAGCUCAAAGCUCAUGGGCUGCGACUCGUUCGAUCAGUUCAGCGCCGAACGGCGCAAUUCGAUCUUGACGGAAACCCUGCACAGAUUCUCGCUGGAGGGAGAUGCACCUGUCUCGUGGACCGAAACCAAGAAGCAGUCCUUCAAGCAGACGGGAGAGUUCGGAGAAAAGCGGAAGAACUCAAUCCUCAACCCAAUCAACUCCAUUCGCAAAUUCUCAAUCGUGCAGAAAACUCCACUGCAGAUGAACGGUAUCGAAGAGGAUUCGGACGAGCCACUUGAGCGGAGACUGUCGCUGGUGCCAGAUUCAGAACAGGGGGAGGCAAUCCUGCCGCGCAUUUCCGUGAUCAGCACUGGGCCGACCCUCCAAGCUAGACGCAGGCAAUCAGUGCUGAAUCUCAUGACCCACUCCGUCAACCAGGGACAGAAUAUCCACCGCAAGACCACCGCGUCGACUAGAAAGGUGUCAUUGGCACCGCAAGCAAAUUUGACUGAACUUGACAUCUACUCACGGCGCCUCUCCCAAGAAACCGGAUUGGAAAUCUCCGAAGAGAUUAACGAAGAAGAUUUGAAAGAGUGUUUCUUCGACGAUAUGGAGUCGAUCCCCGCAGUGACCACUUGGAAUACGUAUCUUCGGUACAUCACCGUGCACAAGAGCCUGAUCUUCGUCCUCAUCUGGUGCCUGGUGAUCUUUCUGGCCGAAGUCGCCGCUUCGCUGGUCGUGCUGUGGCUGCUCGGUAAUACCCCGCUCCAAGACAAAGGCAAUUCCACUCACUCGCGCAACAACAGCUACGCUGUGAUUAUCACGUCAACCUCGUCGUACUAUGUGUUCUACAUCUACGUGGGAGUCGCGGACACUCUGCUCGCUAUGGGCUUCUUUCGCGGACUGCCCCUGGUCCACACUCUCAUCACGGUGAGCAAGAUCCUCCAUCAUAAGAUGCUCCAUUCCGUGCUGCAGGCCCCGAUGAGCACUCUCAACACUCUGAAGGCGGGUGGAAUCUUGAACAGAUUUUCCAAAGACAUCGCGAUUCUGGACGAUCUGCUCCCACUCACUAUCUUCGACUUCAUCCAACUGCUGCUGAUCGUCAUCGGAGCUAUCGCCGUGGUGGCUGUCCUCCAGCCGUAUAUCUUCGUGGCCACUGUGCCGGUGAUUGUCGCUUUCAUCAUGUUGCGCGCGUACUUCUUGCAAACCUCGCAGCAACUCAAGCAACUGGAGUCCGAGGGCCGGAGCCCAAUCUUUACCCAUCUGGUGACUUCACUGAAAGGUCUGUGGACCCUCCGCGCCUUUGGUCGCCAGCCUUACUUCGAAACUCUCUUUCACAAAGCACUGAAUCUCCACACUGCAAACUGGUUCUUGUACCUGUCCACCCUGCGGUGGUUCCAAAUGCGGAUCGAGAUGAUCUUUGUCAUCUUCUUCAUCGCCGUGACUUUUAUCUCCAUCCUCACCACCGGCGAGGGAGAGGGGAGAGUGGGAAUCAUCCUGACGCUGGCGAUGAAUAUCAUGUCCACUUUGCAGUGGGCCGUCAAUUCGAGCAUCGACGUGGAUUCGCUGAUGCGCAGCGUGUCGCGCGUGUUCAAGUUCAUCGAUAUGCCCACCGAAGGUAAACCCACCAAGAGCACGAAGCCUUACAAGAACGGGCAGCUCUCAAAGGUGAUGAUUAUCGAGAACUCCCAUGUGAAGAAGGACGACAUCUGGCCAUCCGGAGGACAGAUGACCGUGAAGGACCUGACCGCCAAAUACACGGAGGGCGGAAAUGCAAUCCUCGAAAACAUCUCGUUCUCCAUCUCGCCUGGCCAAAGGGUGGGACUUUUGGGACGCACUGGAUCCGGAAAGAGCACCCUGCUUAGCGCCUUCUUGAGGCUCUUGAACACCGAGGGCGAAAUCCAGAUCGAUGGCGUGUCGUGGGAUUCGAUCACCCUGCAGCAGUGGAGAAAGGCCUUCGGGGUGAUCCCGCAAAAAGUGUUCAUCUUCUCCGGAACGUUUCGGAAAAACCUUGACCCAUACGAACAAUGGUCGGAUCAAGAGAUUUGGAAGGUCGCCGACGAAGUGGGGCUGCGCUCCGUGAUCGAGCAGUUUCCGGGAAAACUGGACUUCGUCUUGGUCGACGGCGGAUGCGUCCUGUCCCACGGACAUAAGCAGCUGAUGUGCCUGGCCCGCAGCGUCCUUUCAAAAGCUAAGAUCCUGCUGCUGGAUGAACCUUCAGCACACCUCGACCCGGUCACCUACCAGAUCAUCAGACGGACCCUGAAACAGGCCUUUGCGGAUUGUACUGUGAUCUUGUGUGAACACCGCAUUGAAGCCAUGCUGGAGUGCCAGCAGUUCCUGGUCAUCGAAGAGAACAAAGUGCGGCAGUACGAUUCCAUCCAAAAACUGCUCAAUGAGCGGUCCCUGUUCAGACAGGCAAUUAGCCCGAGCGACAGGGUCAAAUUGUUCCCCCAUAGAAAUUCGUCGAAAUGUAAGUCAAAGCCUCAGAUCGCGGCACUGAAAGAAGAAACUGAAGAAGAGGUGCAAGACACCAGACUGUGA(SEQIDNO:10)
SEQIDNO:11
AUGCAGAGAAGCCCACUGGAAAAGGCGUCGGUGGUGUCAAAGCUGUUCUUUAGCUGGACCAGACCUAUCUUGCGGAAGGGAUACCGCCAACGCCUGGAGCUGUCGGACAUCUACCAGAUUCCGUCAGUGGAUUCAGCAGACAAUCUCUCCGAAAAGCUGGAACGCGAAUGGGACAGAGAGUUGGCGUCAAAGAAGAACCCAAAGUUGAUCAAUGCCCUGCGCCGCUGCUUCUUCUGGCGGUUCAUGUUCUACGGAAUCUUUCUGUACCUCGGCGAAGUCACCAAGGCUGUGCAACCGCUUCUGCUGGGACGCAUCAUCGCCUCAUACGACCCGGACAACAAGGAAGAACGCUCCAUCGCAAUCUACCUCGGGAUCGGCCUCUGCCUGCUGUUUAUCGUGCGGACGCUGCUGCUCCAUCCAGCCAUUUUCGGACUGCACCACAUUGGCAUGCAAAUGCGGAUCGCCAUGUUCAGCCUGAUCUACAAAAAGACCCUGAAGUUGAGCUCACGGGUGUUGGAUAAGAUUUCGAUCGGACAGCUGGUGUCGCUGCUCUCCAACAACCUCAACAAGUUUGACGAAGGCCUGGCACUGGCCCACUUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAGGAAGGAUUCGGCGAAUUGUUCGAAAAGGCUAAGCAGAACAACAACAAUCGGAAAACCUCCAAUGGGGACGAUUCGCUGUUCUUCUCGAAUUUCUCCCUGCUGGGAACGCCCGUGCUUAAAGACAUCAACUUCAAGAUCGAACGGGGCCAGCUGCUCGCGGUCGCGGGCAGCACUGGAGCGGGAAAGACUUCCCUGCUCAUGGUCAUCAUGGGAGAGCUGGAGCCCUCGGAGGGCAAAAUCAAGCACUCGGGGAGGAUCUCAUUUUGCAGCCAGUUCUCGUGGAUCAUGCCCGGUACUAUCAAAGAAAACAUCAUCUUUGGAGUCAGCUAUGACGAGUACCGCUACCGGUCGGUGAUCAAGGCCUGCCAGCUGGAAGAAGAUAUCUCCAAGUUCGCCGAAAAGGACAACAUUGUGCUGGGAGAAGGUGGAAUCACUCUCUCGGGAGGCCAGCGCGCACGGAUCUCACUCGCAAGGGCCGUGUACAAGGAUGCCGAUUUGUACCUGUUGGAUUCGCCGUUCGGUUAUCUUGAUGUCCUCACUGAGAAAGAGAUUUUUGAGUCGUGCGUCUGUAAGCUGAUGGCCAACAAAACCCGCAUCCUGGUGACCUCGAAGAUGGAGCACUUGAAGAAGGCCGACAAAAUCCUUAUCCUCCAUGAGGGUAGCUCAUACUUCUACGGCACCUUUUCGGAACUGCAGAAUCUGCAGCCCGACUUCUCAUCAAAACUGAUGGGAUGUGACUCGUUCGAUCAGUUCUCGGCGGAGCGGCGGAACUCGAUCCUCACCGAAACUCUCCACCGGUUCAGCCUCGAGGGAGAUGCCCCAGUCAGCUGGACCGAAACUAAGAAGCAGUCCUUCAAACAGACCGGAGAGUUCGGAGAAAAACGCAAGAACUCCAUCCUCAAUCCAAUCAACAGCAUCCGCAAGUUCAGCAUCGUGCAGAAAACUCCACUUCAGAUGAACGGAAUCGAAGAGGAUAGCGACGAGCCGCUUGAGCGGAGAUUGUCACUGGUGCCGGACAGCGAGCAAGGGGAAGCGAUUCUGCCGCGGAUCUCCGUGAUCUCGACUGGCCCUACCCUCCAAGCUCGCAGACGCCAGAGCGUGCUGAAUCUCAUGACCCACUCAGUCAACCAGGGACAAAACAUCCAUAGAAAGACCACCGCUUCAACCCGGAAAGUGUCACUUGCACCGCAGGCAAACCUGACCGAACUCGACAUCUACAGCAGACGGCUCUCACAAGAAACUGGAUUGGAGAUCAGCGAAGAGAUCAACGAAGAAGAUCUCAAAGAAUGCUUCUUCGACGAUAUGGAGUCCAUCCCAGCAGUCACUACGUGGAAUACCUACCUCCGCUACAUCACUGUGCACAAGAGCCUGAUUUUCGUGUUGAUCUGGUGCCUGGUCAUCUUCUUGGCCGAGGUGGCCGCGAGCCUCGUGGUCCUCUGGCUGCUCGGCAAUACGCCGCUGCAAGAUAAGGGAAAUUCCACGCAUAGCAGAAACAACUCAUACGCAGUGAUCAUCACUAGCACUUCAUCGUACUACGUGUUCUACAUCUACGUGGGGGUGGCCGAUACUCUGUUGGCAAUGGGAUUCUUUAGAGGGCUGCCUCUGGUGCAUACUCUGAUCACUGUGUCCAAGAUCCUCCACCACAAGAUGCUCCACUCCGUGCUUCAGGCCCCUAUGUCAACUCUCAACACCCUCAAGGCCGGAGGUAUUCUUAAUCGCUUUUCCAAGGACAUCGCCAUUCUCGAUGACUUGCUUCCCCUGACUAUCUUCGACUUUAUCCAGUUGCUGCUGAUUGUGAUCGGCGCUAUUGCCGUCGUCGCAGUGCUGCAACCGUACAUCUUUGUGGCUACCGUCCCAGUCAUUGUGGCCUUCAUCAUGCUCAGGGCAUACUUUCUCCAGACCAGCCAGCAGCUCAAGCAGCUCGAAUCCGAAGGCAGAUCGCCGAUCUUCACCCACCUCGUCACUUCGCUCAAGGGCCUCUGGACCCUGCGCGCCUUCGGUCGCCAGCCGUAUUUCGAAACCCUGUUCCAUAAAGCACUGAACCUCCAUACUGCGAACUGGUUUCUCUACCUUUCAACCCUGAGGUGGUUCCAGAUGAGAAUCGAGAUGAUCUUUGUGAUCUUCUUUAUCGCUGUGACGUUCAUCUCCAUUCUCACUACCGGCGAGGGAGAGGGCAGAGUGGGGAUUAUCCUCACGCUGGCCAUGAAUAUCAUGAGCACGCUGCAGUGGGCCGUCAAUAGCAGCAUCGACGUGGACUCCCUGAUGCGGUCCGUGUCGAGAGUGUUUAAGUUCAUCGAUAUGCCUACUGAAGGGAAACCGACCAAGUCGACCAAGCCGUACAAGAAUGGGCAGCUGAGCAAGGUGAUGAUUAUUGAGAACUCCCAUGUGAAGAAGGACGACAUCUGGCCCAGCGGAGGCCAGAUGACCGUGAAGGACUUGACCGCUAAGUACACUGAGGGUGGAAAUGCCAUUCUUGAGAAUAUCAGCUUCUCGAUCUCGCCGGGACAACGCGUGGGAUUGCUCGGGCGCACUGGCAGCGGCAAAUCCACCCUGCUUAGCGCUUUUCUGAGGCUGCUGAACACUGAAGGUGAAAUUCAAAUCGAUGGAGUGUCGUGGGAUAGCAUCACCCUUCAACAGUGGCGCAAGGCCUUCGGCGUGAUCCCUCAAAAGGUCUUUAUCUUCUCGGGGACGUUCCGGAAAAAUCUCGACCCCUACGAACAGUGGUCAGACCAAGAGAUUUGGAAAGUCGCAGAUGAGGUCGGACUGCGCUCAGUGAUCGAACAGUUUCCGGGUAAACUUGACUUCGUGCUCGUCGAUGGAGGUUGCGUCCUGUCCCACGGACAUAAGCAGCUGAUGUGUCUGGCGCGCUCGGUCCUCUCCAAAGCGAAGAUCCUGCUGCUCGAUGAACCGUCCGCCCACCUUGAUCCAGUGACCUAUCAGAUCAUUCGGAGAACUUUGAAGCAAGCCUUCGCUGACUGCACCGUCAUCCUCUGCGAACACCGGAUCGAGGCAAUGCUGGAGUGCCAACAGUUUCUGGUCAUCGAAGAAAACAAAGUGCGCCAGUAUGACUCGAUCCAAAAACUUCUGAACGAGCGCUCCCUCUUCCGGCAGGCAAUCAGCCCAUCCGACCGCGUGAAGUUGUUCCCUCAUCGGAAUAGCUCCAAAUGCAAAUCGAAGCCGCAGAUCGCUGCCUUGAAAGAAGAAACCGAAGAAGAAGUCCAAGACACUAGGUUGUAG(SEQIDNO:11)
SEQIDNO:12
AUGCAGCGGUCCCCUCUGGAGAAGGCUUCCGUGGUCAGCAAGCUGUUCUUCUCGUGGACCAGACCUAUCCUCCGCAAGGGAUACCGCCAGCGCCUGGAGCUGUCAGAUAUCUACCAGAUCCCAAGCGUGGACUCAGCCGACAAUCUGAGCGAAAAGCUGGAACGGGAGUGGGACCGGGAGCUCGCCUCCAAGAAGAAUCCGAAGUUGAUCAAUGCGCUGCGCAGAUGCUUCUUCUGGCGGUUUAUGUUUUACGGCAUCUUUCUGUAUCUCGGAGAAGUGACCAAAGCCGUGCAGCCGCUGCUCUUGGGUAGGAUCAUUGCUUCGUACGACCCGGACAACAAAGAAGAACGCUCCAUCGCCAUCUACCUCGGAAUCGGUCUGUGCCUGCUCUUUAUCGUGCGCACUCUCCUGCUGCAUCCGGCGAUCUUCGGACUGCACCACAUCGGCAUGCAAAUGCGGAUCGCAAUGUUCUCACUGAUCUACAAAAAGACUCUGAAGCUCAGCUCCAGAGUGCUGGAUAAGAUCUCGAUCGGGCAACUCGUCAGCCUGCUGUCGAACAAUCUGAAUAAGUUCGACGAAGGGUUGGCCCUCGCACAUUUCGUGUGGAUCGCACCGCUGCAAGUGGCGCUCCUGAUGGGACUCAUUUGGGAACUGCUCCAAGCCAGCGCGUUUUGCGGACUCGGAUUCCUGAUCGUGCUCGCCCUGUUCCAAGCCGGACUGGGGCGCAUGAUGAUGAAGUACCGCGAUCAGCGGGCAGGAAAGAUCUCCGAGCGGUUGGUGAUCACUUCCGAAAUGAUCGAGAAUAUUCAGUCCGUGAAGGCCUACUGCUGGGAAGAAGCUAUGGAAAAGAUGAUUGAAAACUUGCGGCAAACUGAGCUGAAAUUGACUCGCAAAGCGGCAUACGUCCGCUACUUCAAUAGCAGCGCCUUCUUCUUUUCGGGCUUUUUCGUGGUGUUUCUGAGCGUGCUGCCCUACGCUCUGAUCAAGGGAAUCAUCCUCCGGAAAAUCUUCACCACCAUUUCGUUCUGUAUCGUGUUGCGCAUGGCCGUGACUCGCCAGUUCCCCUGGGCGGUGCAGACCUGGUACGACAGCUUGGGGGCAAUCAAUAAGAUUCAAGACUUCUUGCAAAAGCAGGAGUACAAGACUCUGGAGUACAACCUGACCACCACUGAAGUCGUGAUGGAGAACGUGACCGCCUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUGGUGACUUCCAAAAUGGAGCAUCUCAAGAAGGCGGACAAGAUCCUGAUUCUGCAUGAGGGAUCAAGCUAUUUCUACGGAACUUUUUCCGAGCUGCAGAACCUCCAGCCGGAUUUUAGCUCCAAGCUGAUGGGUUGCGACUCAUUCGACCAAUUCUCGGCUGAGCGGCGGAACUCAAUCCUGACCGAAACCCUGCAUCGCUUCUCCCUUGAGGGAGAUGCCCCGGUGUCGUGGACUGAGACUAAAAAGCAGUCGUUUAAGCAAACUGGCGAAUUCGGCGAAAAGCGGAAGAAUAGCAUCCUCAACCCAAUCAACAGCAUUCGGAAGUUCAGCAUCGUCCAAAAGACCCCGCUCCAGAUGAACGGCAUUGAAGAGGACUCAGACGAGCCAUUGGAAAGACGCCUGUCACUGGUCCCAGAUUCGGAGCAGGGUGAAGCAAUUCUGCCUCGGAUCUCGGUCAUCUCGACUGGCCCCACUCUCCAAGCUCGGCGGAGACAGAGCGUGCUUAACUUGAUGACCCACUCCGUGAACCAGGGUCAGAACAUCCACCGCAAAACCACCGCCUCCACCAGGAAGGUGUCACUGGCCCCUCAAGCCAAUCUGACUGAGUUGGAUAUCUACUCCAGAAGGCUCAGCCAGGAAACCGGACUGGAAAUCUCGGAAGAGAUCAACGAAGAGGAUCUCAAAGAGUGUUUCUUCGACGACAUGGAAUCAAUCCCUGCUGUCACUACUUGGAACACCUAUCUCCGCUACAUUACCGUGCACAAGUCACUCAUCUUCGUCCUGAUCUGGUGCCUCGUGAUCUUCCUGGCCGAGGUCGCAGCAUCGCUGGUCGUGCUGUGGCUGCUCGGCAACACCCCACUCCAAGACAAAGGCAACAGCACCCAUUCCCGCAACAACUCCUACGCGGUGAUCAUCACUUCAACUUCGUCCUACUACGUCUUUUACAUCUACGUGGGCGUGGCGGACACGCUCCUGGCUAUGGGGUUCUUUCGCGGGCUGCCUCUUGUCCACACGCUCAUCACUGUGUCAAAGAUUCUCCACCACAAAAUGCUGCACUCCGUGCUCCAGGCCCCUAUGUCGACUUUGAACACGCUUAAGGCCGGAGGCAUCCUUAACAGAUUCUCGAAAGAUAUCGCGAUCUUGGACGAUCUUCUGCCGCUGACUAUCUUUGACUUCAUCCAACUCCUGCUGAUCGUCAUCGGUGCCAUCGCAGUGGUCGCGGUGCUCCAACCGUACAUUUUCGUGGCGACUGUGCCGGUGAUCGUGGCGUUCAUCAUGCUGCGGGCUUACUUUCUUCAGACCUCACAGCAGCUGAAGCAACUCGAAUCGGAGGGUAGAUCACCAAUCUUUACCCACCUCGUCACCUCGCUGAAGGGACUCUGGACCCUGCGCGCAUUUGGACGGCAACCGUACUUCGAGACUCUCUUCCAUAAGGCCCUGAAUCUGCAUACGGCGAAUUGGUUUCUUUACCUCUCGACGCUCCGCUGGUUCCAGAUGCGCAUUGAGAUGAUUUUCGUCAUCUUUUUCAUCGCGGUGACCUUCAUCUCCAUCCUCACCACGGGUGAGGGAGAGGGCAGAGUCGGAAUUAUCCUCACUCUGGCCAUGAACAUCAUGUCCACUCUGCAGUGGGCCGUCAACUCAUCCAUUGACGUGGACUCGCUGAUGCGCUCCGUGUCGAGAGUGUUCAAGUUCAUCGAUAUGCCGACCGAGGGAAAGCCAACUAAGUCGACCAAGCCGUACAAAAACGGACAGCUGAGCAAGGUCAUGAUCAUCGAAAACUCCCACGUGAAAAAGGAUGACAUCUGGCCGUCCGGUGGACAGAUGACGGUGAAGGAUCUGACUGCGAAGUACACUGAGGGAGGGAAUGCCAUCCUCGAAAACAUCUCAUUCUCAAUCUCCCCUGGACAGAGGGUCGGGCUGCUGGGCCGCACUGGCUCGGGGAAGUCGACUCUUCUUUCGGCAUUUCUGCGCUUGCUCAAUACCGAGGGAGAAAUCCAGAUCGAUGGAGUGUCAUGGGACUCGAUCACCCUGCAGCAGUGGCGCAAGGCUUUUGGCGUCAUCCCGCAAAAGGUGUUCAUCUUCUCGGGCACUUUUAGAAAGAAUCUGGAUCCCUACGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCAGACGAAGUGGGCCUCCGGUCCGUGAUUGAACAGUUUCCGGGAAAGCUCGACUUCGUGCUUGUGGACGGAGGAUGUGUGCUGAGCCACGGCCACAAACAGCUCAUGUGCCUGGCUCGGUCGGUCCUGUCGAAAGCAAAGAUCCUGCUGCUGGACGAACCGUCGGCACACCUCGAUCCAGUGACGUACCAGAUCAUCCGGCGGACCCUGAAGCAGGCCUUCGCAGACUGCACUGUCAUUUUGUGUGAACACAGAAUCGAAGCUAUGUUGGAGUGCCAGCAGUUCCUGGUCAUCGAAGAAAACAAAGUCCGCCAGUACGAUUCGAUUCAGAAGCUGCUGAACGAACGGAGCCUCUUCAGACAGGCGAUCAGCCCCAGCGAUCGGGUCAAGUUGUUCCCGCAUCGGAACAGCAGCAAGUGUAAGUCAAAGCCUCAGAUCGCUGCACUCAAAGAAGAGACUGAAGAAGAAGUGCAAGACACCAGACUCUGA(SEQIDNO:12)
SEQIDNO:13
AUGCAGCGCUCGCCUCUGGAGAAAGCCUCAGUCGUGUCAAAACUGUUCUUUAGCUGGACUCGCCCGAUUCUCCGGAAGGGUUAUAGACAGCGCUUGGAGCUCUCCGACAUCUACCAAAUCCCUUCCGUGGACUCCGCCGACAACCUGUCGGAGAAGCUCGAACGCGAGUGGGACCGGGAACUCGCGUCCAAAAAGAAUCCAAAACUCAUUAAUGCACUGCGCCGCUGCUUCUUCUGGCGCUUUAUGUUUUACGGUAUCUUUCUCUACCUGGGCGAGGUGACGAAAGCAGUGCAGCCGCUCCUGCUUGGCAGAAUUAUCGCCUCGUACGAUCCGGAUAACAAAGAAGAACGCUCAAUCGCUAUCUACCUCGGUAUCGGAUUGUGCCUGCUUUUCAUCGUGCGCACCCUGUUGCUGCACCCGGCGAUUUUCGGACUCCACCACAUCGGAAUGCAAAUGAGAAUUGCAAUGUUCUCAUUGAUCUACAAAAAGACCCUUAAACUGUCGUCCCGCGUCCUCGACAAGAUUUCAAUCGGCCAGCUGGUGUCGCUUCUUUCGAAUAAUCUUAACAAGUUCGAUGAAGGACUCGCGCUCGCCCAUUUCGUGUGGAUCGCACCACUUCAAGUCGCACUGCUCAUGGGACUGAUUUGGGAGUUGCUGCAGGCUUCCGCCUUUUGCGGCCUGGGAUUCCUGAUCGUCCUGGCUUUGUUCCAGGCUGGACUGGGCAGAAUGAUGAUGAAGUACCGGGACCAGCGGGCAGGAAAGAUCAGCGAAAGGCUCGUGAUCACUAGCGAAAUGAUCGAGAACAUCCAAUCCGUCAAGGCGUACUGCUGGGAAGAAGCGAUGGAGAAGAUGAUCGAAAAUCUUCGCCAGACCGAACUCAAACUCACUAGAAAGGCUGCCUACGUGCGCUACUUUAACAGCUCAGCAUUUUUCUUCUCCGGAUUUUUCGUGGUGUUCCUGUCGGUGCUGCCAUACGCCCUGAUCAAGGGGAUCAUUCUUCGCAAAAUCUUCACCACGAUCUCAUUCUGCAUUGUCCUCCGGAUGGCCGUGACGCGGCAGUUCCCUUGGGCAGUGCAAACUUGGUACGAUUCGCUGGGGGCCAUUAACAAGAUUCAAGAUUUUCUUCAAAAGCAGGAGUACAAAACCCUGGAGUACAAUCUGACCACUACGGAAGUCGUGAUGGAAAACGUGACUGCUUUUUGGGAGGAAGGCUUCGGCGAACUUUUUGAAAAGGCAAAGCAAAACAAUAACAACAGAAAGACGUCAAACGGCGAUGACUCGCUGUUCUUCUCCAAUUUCUCCCUGCUCGGCACCCCUGUGCUGAAGGACAUCAACUUCAAAAUUGAACGCGGACAGCUGCUGGCCGUGGCGGGAUCGACCGGGGCUGGGAAAACCUCGUUGUUGAUGGUGAUCAUGGGAGAACUCGAACCCUCGGAGGGAAAGAUUAAGCAUAGCGGACGGAUCAGCUUCUGUUCCCAGUUCUCGUGGAUCAUGCCGGGAACCAUUAAGGAAAACAUCAUCUUCGGCGUGUCCUACGACGAGUACCGGUAUAGGUCGGUGAUCAAGGCCUGCCAGUUGGAAGAGGACAUCUCCAAGUUCGCUGAGAAGGACAACAUCGUGCUCGGUGAAGGGGGCAUUACUCUGUCCGGUGGCCAGCGCGCGAGAAUUUCGCUGGCUCGCGCGGUGUACAAAGAUGCGGAUCUCUAUCUGCUGGAUUCGCCCUUCGGAUACCUCGAUGUCCUCACGGAGAAGGAGAUCUUCGAAUCGUGCGUGUGCAAGUUGAUGGCGAACAAGACUAGGAUCCUGGUCACUUCCAAGAUGGAGCACUUGAAGAAGGCCGAUAAGAUCUUGAUCCUCCAUGAAGGAUCGAGCUACUUUUACGGAACUUUCUCAGAGCUGCAGAACUUGCAGCCGGACUUCUCAAGCAAACUGAUGGGUUGCGACUCGUUCGACCAGUUUUCGGCAGAACGGCGGAACUCGAUCCUGACUGAGACUCUGCAUCGCUUUUCGCUGGAAGGCGAUGCCCCUGUGUCCUGGACUGAAACCAAGAAGCAAUCCUUCAAACAAACUGGAGAAUUCGGAGAAAAGCGGAAGAACUCCAUCCUUAACCCCAUCAAUAGCAUCCGGAAGUUCUCAAUCGUCCAAAAGACCCCGCUGCAGAUGAAUGGCAUCGAAGAAGAUAGCGACGAACCUCUUGAAAGACGGCUGUCCUUGGUGCCAGACUCAGAACAGGGAGAAGCUAUCCUGCCGCGGAUCUCCGUGAUCAGCACCGGACCGACUCUGCAGGCUCGCAGACGCCAGAGCGUGCUCAACCUGAUGACCCACUCCGUGAACCAGGGACAAAACAUCCAUAGAAAGACCACGGCCUCCACCAGAAAAGUCUCCCUGGCACCGCAAGCCAACCUGACUGAACUGGACAUCUACAGCAGAAGGCUCAGCCAAGAAACCGGACUGGAGAUUUCAGAAGAAAUCAACGAGGAAGAUCUUAAAGAGUGCUUCUUCGACGACAUGGAAUCGAUCCCAGCCGUGACCACUUGGAAUACCUAUCUGAGAUACAUCACCGUGCACAAAUCCCUGAUCUUCGUGCUGAUCUGGUGCCUGGUGAUCUUCCUGGCUGAGGUGGCCGCCUCACUGGUGGUGCUUUGGUUGCUGGGGAAUACGCCGCUCCAAGACAAGGGAAACUCCACGCACUCCAGAAACAACUCGUACGCCGUGAUCAUCACGUCGACUUCGUCGUACUACGUGUUCUACAUCUACGUCGGUGUGGCAGACACUCUCUUGGCGAUGGGCUUUUUCCGGGGACUGCCACUGGUCCACACCCUGAUCACCGUGUCCAAAAUCUUGCACCACAAGAUGCUCCACAGCGUGCUGCAAGCCCCGAUGAGCACCCUGAAUACCCUCAAAGCGGGAGGCAUCCUCAACAGAUUCAGCAAGGACAUCGCCAUCCUCGACGACCUGUUGCCCCUGACCAUCUUCGAUUUCAUCCAGCUUCUUCUCAUCGUGAUCGGGGCAAUCGCUGUCGUGGCGGUGCUGCAGCCGUACAUCUUCGUGGCGACUGUGCCAGUGAUCGUCGCCUUUAUCAUGCUGCGGGCCUACUUUCUCCAAACUUCCCAACAGCUGAAACAACUGGAGUCGGAGGGCCGCAGCCCUAUCUUCACCCAUCUGGUGACCAGCCUCAAAGGACUGUGGACUCUGAGGGCUUUCGGGAGGCAGCCAUACUUCGAGACUCUCUUUCACAAGGCCCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGCAAGGCGUUCGGAGUCAUUCCCCAAAAGGUGUUCAUCUUUUCGGGAACCUUCCGCAAGAAUCUGGAUCCGUACGAACAGUGGAGCGACCAAGAGAUUUGGAAAGUGGCAGAUGAAGUGGGAUUGCGGAGCGUCAUCGAACAGUUUCCGGGAAAGCUCGAUUUCGUCCUUGUGGACGGUGGAUGUGUGCUGUCGCACGGCCAUAAGCAGCUGAUGUGUCUCGCCCGCUCGGUGCUGUCAAAGGCGAAGAUCCUCUUGCUGGAUGAGCCAUCAGCCCAUCUGGACCCGGUGACGUACCAGAUCAUUAGACGGACGCUGAAACAGGCAUUCGCGGACUGCACUGUGAUCCUCUGUGAACAUCGGAUCGAGGCCAUGCUGGAGUGUCAACAAUUCUUGGUCAUCGAAGAGAACAAAGUGCGGCAGUACGACAGCAUCCAAAAGCUGCUGAACGAGAGGUCCCUCUUCCGCCAGGCCAUCUCCCCAUCCGACCGGGUCAAGCUGUUCCCUCACCGCAACAGCUCAAAGUGCAAAUCCAAACCCCAGAUCGCAGCGCUGAAAGAAGAAACUGAAGAAGAAGUGCAAGACACUAGACUGUGA(SEQIDNO:13)
SEQIDNO:14
AUGCAAAGGUCCCCAUUGGAGAAGGCCUCAGUGGUGUCGAAGCUGUUCUUCUCGUGGACCAGGCCUAUCCUCCGGAAGGGAUACAGACAGCGGCUGGAACUGUCCGAUAUCUACCAGAUCCCCAGCGUGGACAGCGCCGAUAAUCUCAGCGAAAAGCUGGAACGGGAAUGGGACCGCGAACUCGCUUCGAAGAAGAACCCGAAGCUGAUUAAUGCUCUGCGGAGAUGUUUCUUUUGGCGGUUCAUGUUUUACGGAAUCUUUCUGUACUUGGGAGAGGUCACGAAGGCUGUGCAGCCUCUGCUGCUGGGACGGAUUAUCGCGUCGUAUGACCCCGACAAUAAGGAAGAACGCAGCAUCGCAAUCUACCUGGGCAUCGGAUUGUGCCUGCUGUUCAUCGUGAGAACUCUCCUGCUGCAUCCAGCCAUCUUCGGACUCCACCACAUUGGAAUGCAGAUGAGAAUCGCAAUGUUCUCCCUGAUCUACAAGAAAACGCUCAAGCUCAGCAGCCGCGUGCUCGAUAAGAUCAGCAUCGGUCAAUUGGUGUCCCUGCUGUCGAAUAACCUCAACAAGUUCGACGAAGGGUUGGCCCUCGCUCACUUCGUGUGGAUCGCACCUCUGCAAGUGGCCCUGCUGAUGGGACUGAUUUGGGAGCUGCUGCAGGCUUCCGCUUUCUGCGGCCUGGGAUUUCUUAUCGUGCUUGCUCUGUUCCAGGCGGGACUGGGACGCAUGAUGAUGAAGUACCGGGACCAACGGGCUGGAAAGAUCAGCGAACGGCUGGUGAUCACUUCCGAAAUGAUUGAGAAUAUCCAGUCAGUCAAGGCGUACUGCUGGGAAGAGGCUAUGGAAAAGAUGAUUGAAAAUCUGAGACAAACCGAGCUGAAGCUGACUCGGAAAGCGGCCUACGUCAGAUACUUCAAUAGCUCAGCUUUCUUUUUCUCGGGGUUUUUCGUCGUGUUCCUGUCGGUGCUUCCCUAUGCCCUGAUUAAGGGCAUCAUUCUGCGCAAGAUCUUCACUACGAUCUCAUUCUGCAUCGUGCUGCGCAUGGCUGUGACCAGACAAUUCCCGUGGGCCGUGCAAACCUGGUACGAUUCACUGGGAGCCAUCAACAAGAUCCAAGACUUUCUCCAAAAACAGGAGUAUAAGACCCUGGAGUACAACCUGACUACUACCGAGGUGGUGAUGGAGAACGUGACUGCGUUUUGGGAAGAAGGGUUCGGCGAACUGUUUGAAAAGGCCAAGCAGAACAAUAACAACAGAAAGACUUCAAACGGAGAUGACUCGCUGUUCUUUUCGAACUUCAGCCUGCUGGGUACCCCAGUGUUGAAAGAUAUCAACUUCAAGAUUGAGAGAGGACAGCUGCUGGCUGUGGCGGGAUCCACCGGAGCAGGAAAAACUUCACUCCUGAUGGUGAUCAUGGGAGAACUCGAACCGUCAGAGGGGAAGAUUAAACACUCGGGAAGAAUCUCAUUUUGCUCCCAAUUUUCAUGGAUUAUGCCGGGAACCAUUAAAGAAAACAUUAUCUUCGGCGUGUCCUACGACGAGUACCGCUACAGAUCGGUGAUCAAAGCAUGCCAGCUGGAAGAGGACAUCUCGAAAUUCGCUGAAAAAGACAAUAUCGUGCUCGGGGAAGGCGGCAUCACCCUCAGCGGAGGACAACGGGCACGGAUUUCGCUCGCACGCGCAGUCUACAAAGACGCCGAUCUCUACCUCUUGGACAGCCCAUUCGGGUAUCUGGACGUGCUCACCGAGAAAGAGAUCUUCGAAAGCUGCGUCUGCAAGCUCAUGGCCAACAAGACCCGCAUCCUCGUGACGUCGAAGAUGGAACAUCUUAAGAAGGCUGACAAGAUUCUCAUUCUCCAUGAAGGGAGCUCAUACUUCUACGGCACCUUUUCCGAGCUCCAGAAUCUGCAACCGGACUUCUCGUCCAAGCUGAUGGGCUGCGAUUCGUUUGAUCAGUUCUCCGCCGAGCGGAGAAACAGCAUUCUGACGGAAACCCUGCACCGGUUCUCGCUGGAAGGCGAUGCACCGGUGUCGUGGACCGAAACUAAGAAGCAAUCGUUCAAGCAGACGGGAGAGUUUGGAGAGAAGCGGAAAAACUCCAUCCUCAACCCGAUCAACAGCAUCCGGAAGUUCAGCAUCGUGCAAAAGACCCCGCUCCAGAUGAAUGGCAUUGAAGAGGACUCCGACGAACCUUUGGAACGCAGACUGAGCCUCGUGCCGGAUUCAGAACAGGGAGAAGCCAUUCUGCCACGGAUCUCCGUGAUCAGCACUGGGCCAACUCUCCAAGCACGGCGGAGGCAGUCCGUGCUGAAUCUUAUGACGCACAGCGUGAACCAAGGGCAGAACAUCCAUAGAAAAACGACCGCUUCGACCAGGAAAGUCUCCCUCGCCCCACAAGCUAACCUCACGGAACUGGAUAUCUACUCCCGCAGACUGUCGCAAGAGACUGGCCUUGAGAUCUCCGAAGAGAUUAACGAAGAAGAUCUCAAAGAAUGUUUCUUCGAUGAUAUGGAAUCAAUCCCGGCAGUGACCACUUGGAACACCUACUUGCGCUAUAUCACUGUGCACAAAAGCCUUAUCUUCGUCCUCAUCUGGUGCCUCGUCAUCUUCCUGGCUGAGGUCGCAGCCUCGCUGGUCGUGCUCUGGUUGCUCGGAAACACUCCGCUGCAGGAUAAGGGGAAUUCGACUCACUCGCGGAACAAUUCGUACGCUGUCAUUAUCACCUCGACGUCGUCAUACUACGUGUUUUACAUCUACGUGGGAGUGGCUGACACUCUGUUGGCUAUGGGGUUCUUUCGCGGCCUGCCACUGGUCCAUACUCUCAUUACUGUGUCCAAAAUCCUUCAUCACAAGAUGUUGCAUUCAGUGCUGCAAGCACCGAUGUCCACCCUCAAUACCCUUAAGGCUGGCGGGAUUCUCAACCGCUUCUCGAAAGACAUCGCCAUCCUCGAUGAUCUUCUGCCUCUCACCAUCUUUGAUUUCAUCCAGCUGCUCCUGAUCGUGAUCGGAGCGAUUGCCGUGGUGGCAGUGUUGCAGCCGUACAUCUUUGUCGCAACUGUGCCGGUCAUCGUCGCCUUCAUCAUGCUGCGCGCCUACUUCUUGCAAACGUCACAGCAACUGAAGCAGCUUGAAUCCGAGGGAAGAUCACCUAUCUUCACCCACCUCGUGACUUCGCUGAAGGGGCUGUGGACGCUGCGCGCAUUUGGAAGGCAACCGUACUUCGAGACUUUGUUCCACAAGGCGCUCAAUCUUCACACUGCCAAUUGGUUCUUGUACCUGUCAACGCUGAGAUGGUUUCAGAUGCGGAUCGAAAUGAUCUUCGUGAUCUUCUUUAUCGCGGUGACUUUCAUCUCGAUCCUGACUACCGGAGAGGGAGAAGGACGGGUGGGUAUUAUCCUCACUCUGGCGAUGAACAUCAUGUCGACGCUUCAGUGGGCGGUGAAUAGCUCAAUCGAUGUCGACUCGCUGAUGCGCUCCGUGAGCCGGGUGUUUAAGUUCAUCGACAUGCCAACUGAAGGGAAGCCGACCAAGUCGACCAAACCGUACAAAAACGGACAGCUCUCCAAGGUGAUGAUUAUCGAGAAUUCCCACGUGAAAAAGGACGACAUCUGGCCAUCCGGUGGACAGAUGACCGUGAAGGACCUGACCGCGAAGUACACUGAGGGAGGCAACGCAAUCCUUGAGAACAUCAGCUUCUCCAUCUCGCCCGGUCAGAGGGUGGGCCUUCUUGGCCGGACCGGAUCGGGAAAGUCCACUCUUCUGUCGGCCUUUCUUCGCCUCUUGAAUACUGAAGGGGAAAUCCAGAUCGACGGAGUGUCGUGGGAUAGCAUCACUCUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAA(SEQIDNO:14)
SEQIDNO:15
AUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUCACCAUCACCAUCACCAUCACCAUCACCAUUAA(SEQIDNO:15)
SEQIDNO:16
AUGGCCACUGGAUCAAGAACCUCACUGCUGCUCGCUUUUGGACUGCUUUGCCUGCCCUGGU UGCAAGAAGGAUCGGCUUUCCCGACCAUCCCACUCUCCAUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAA(SEQIDNO:16)
SEQIDNO:17
AUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAA(SEQIDNO:17)
SEQIDNO:18
AUGGCCACUGGAUCAAGAACCUCACUGCUGCUCGCUUUUGGACUGCUUUGCCUGCCCUGGUUGCAAGAAGGAUCGGCUUUCCCGACCAUCCCACUCUCC(SEQIDNO):18)
SEQIDNO:19
AUGGCAACUGGAUCAAGAACCUCCCUCCUGCUCGCAUUCGGCCUGCUCUGUCUCCCAUGGCUCCAAGAAGGAAGCGCGUUCCCCACUAUCCCCCUCUCG(SEQIDNO:19)
SEQIDNO:20
CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGCU
等效物
根据在本说明书内引用的参考文献的教导,最充分地理解本说明书。本说明书内的实施方案提供本发明的实施方案的说明,并且不应解释为限制本发明的范围。本领域的技术人员能够容易地认识到,许多其它实施方案由本发明涵盖。本公开中所引用的所有公布和专利均以引用的方式全部并入。在以引用的方式并入的材料与本说明书相矛盾或不一致的的情况下,本说明书将优先于任何这种材料。本文中任何参考文献的引用并不承认此类参考文献是本发明的现有技术。
除非另外指出,否则表示本说明书(包括权利要求)所用的成分的量、反应条件等的所有数字应理解为近似值并且可取决于寻求通过本发明获得的所需特性而变化。在最低限度并且不试图限制对权利要求书范围的等价范围的原则的应用,每个数值参数均应当根据有效数字的数值和普通四舍五入方法来解读。在本说明书中具有不同数量的有效数字的数值系列的叙述不应被解释为暗示具有更少给定有效数字的数值具有与具有更多给定有效数字的数值相同的精度。
当在权利要求和/或说明书中连同术语“包含”一起使用时,使用措辞“一个(a)”或“一种(an)”可意味“一个”,但它也符合“一个或多个”、“至少一个”及“一个或一个以上”的含义。除非明确指明仅仅指代替代物,或替代物相互排斥,否则权利要求书中术语“或”的使用用于意指“和/或”,但是本公开支持仅仅指代替代物和“和/或”的定义。
除非另外指出,否则在一系列要素之前的术语“至少”应被理解为是指所述系列中的每一种要素。本领域普通技术人员仅使用常规实验将认识到或能够确定本文描述的本发明的具体实施方案的许多等效方案。所述等效物意图由以下权利要求涵盖。
除非另外定义,否则本文使用的所有技术术语和科学术语具有与通过本发明所属领域中的普通技术人员通常理解的相同的含义。虽然在本发明的实践或测试中可使用类似或等同于本文所描述的那些方法和材料的任何方法和材料,但现在描述优选方法和材料。
本文所讨论的出版物仅为其在本申请的申请日之前的公开而提供。本文没有任何内容被解释为承认本发明没有资格先于现有发明的这种出版物。此外,所提供的出版日期可与实际出版日期不同,这可能需要独立证实。
从本文公开的本发明的说明书和实践考虑,本发明的其它实施方案对于本领域技术人员来说将是显而易见的。本说明书和实施例意在仅被认为是示例性的,并且本发明的真实范围和精神由以下权利要求书指示。
Claims (47)
1.一种在哺乳动物的肺中的上皮细胞中诱导CFTR表达的方法,所述方法包括使所述哺乳动物的肺中的上皮细胞与组合物相接触,其中:
所述组合物是包含体外转录的mRNA的药物组合物;
所述体外转录的mRNA包含编码SEQIDNO:1的编码序列。
2.一种在哺乳动物靶细胞中诱导CFTR表达的方法,所述方法包括使所述哺乳动物靶细胞与组合物相接触,所述组合物包含编码SEQIDNO:1的氨基酸序列的体外转录的mRNA。
3.如权利要求1或权利要求2所述的方法,其中:
(a)所述体外转录的mRNA序列包含比SEQIDNO:2更少的隐蔽性启动子的互补序列,
(b)所述体外转录的mRNA序列包含比SEQIDNO:2更少的同向和/或反向重复序列,
(c)所述编码序列包含比SEQIDNO:2更少的不利密码子,和/或
(d)所述编码序列的GC含量比SEQIDNO:2的GC含量更低。
4.一种包含编码序列、5’-UTR和3’-UTR的非天然存在的mRNA分子,其中所述编码序列编码SEQIDNO:1的氨基酸序列并且所述编码序列与SEQIDNO:3至少80%相同。
5.一种包含编码序列、5’-UTR和3’-UTR的非天然存在的mRNA分子,其中所述编码序列编码SEQIDNO:1的氨基酸序列并且所述编码序列相对于SEQIDNO:2的野生型编码序列在表1中列出的所述编码序列的位置处包含至少50%、至少60%、至少70%、至少80%、至少90%或至少95%的表1中列出的非野生型碱基。
6.一种包含编码序列、5’-UTR和3’-UTR的非天然存在的mRNA分子,其中所述编码序列编码SEQIDNO:1的氨基酸序列并且所述编码序列相对于SEQIDNO:2的野生型编码序列在表2中列出的所述编码序列的对应位置处包含至少50%、至少60%、至少70%、至少80%、至少90%或至少95%的表2中列出的非野生型碱基。
7.如权利要求4至6中任一项所述的mRNA分子,其中所述编码序列与SEQIDNO:3至少85%、至少90%、至少95%、至少98%或至少99%相同。
8.如权利要求4所述的mRNA分子,其中所述编码序列与SEQIDNO:3相同。
9.如权利要求4至8中任一项所述的mRNA分子,其中所述5’-UTR包含SEQIDNO:4和/或所述3’-UTR包含SEQIDNO:5。
10.如权利要求4至9中任一项所述的mRNA分子,其还包含至少70、100、120、150、200或250个残基长度的聚腺苷酸尾。
11.如权利要求4至10中任一项所述的mRNA分子,其还包含5’帽。
12.如权利要求4至11中任一项所述的mRNA分子,其中至少一种组成核苷酸是锁核酸残基。
13.如权利要求4至12中任一项所述的mRNA分子,其中所述mRNA包含至少一种非标准核碱基。
14.如权利要求13所述的mRNA分子,其中所述非标准核碱基选自5-甲基-胞苷、假尿苷和2-硫代-尿苷中的一种或多种。
15.如权利要求4至14中任一项所述的mRNA分子,其用于在哺乳动物或哺乳动物细胞中诱导功能性CFTR表达。
16.一种多核苷酸,其包含与如权利要求4至15中任一项所述的mRNA的序列互补的序列。
17.如权利要求16所述的多核苷酸,其中所述多核苷酸是包含脱氧核糖核苷酸残基的线性或环状多核苷酸。
18.一种组合物,其包含如权利要求16或权利要求17所述的多核苷酸、RNA聚合酶以及核苷三磷酸。
19.一种药物组合物,其包含如权利要求4至15中任一项所述的mRNA。
20.如权利要求19所述的药物组合物,其还包含选自聚乙烯亚胺(PEI)、鱼精蛋白、聚乙二醇化鱼精蛋白、PLL、聚乙二醇化PLL或阳离子脂质的有机阳离子,其中所述有机阳离子与所述mRNA非共价复合。
21.如权利要求20所述的药物组合物,其中所述有机阳离子是阳离子脂质并且所述组合物还包含中性脂质、聚乙二醇化脂质和/或胆固醇。
22.如权利要求21所述的药物组合物,其中所述阳离子脂质选自DODAP(1,2-二油酰基-3-二甲基铵丙烷)、DLinDMA、DLin-KC2-DMA、C12-200、HGT4003、HGT5000、HGT5001、MC3、ICE、包含二烷基氨基部分的阳离子脂质、包含咪唑部分的阳离子脂质及包含胍鎓部分的阳离子脂质。
23.如权利要求21或22中任一项所述的药物组合物,其中所述中性脂质存在于所述组合物中并且选自DSPC(1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱)、DPPC(1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱)、DOPE(1,2-二油酰基-sn-甘油-3-磷酸乙醇胺)、DPPE(1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺)、DMPE(1,2-二肉豆蔻酰基-sn-甘油-3-磷酸乙醇胺)以及DOPG(1,2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油))。
24.如权利要求21至23中任一项所述的药物组合物,其中所述聚乙二醇化脂质存在于所述组合物中并且包含共价连接至达5kDa长度的聚(乙二醇)链的C6-C20长度的一个或多个烷基链。
25.如权利要求20所述的药物组合物,其中所述有机阳离子是具有10kDa至40kDa范围内的分子量的支链PEI。
26.一种雾化或气雾化装置,其负载有如权利要求19至25中任一项所述的药物组合物。
27.一种培养细胞,其包含如权利要求4至15中任一项所述的mRNA和从所述mRNA表达的功能性CFTR。
28.如权利要求27所述的培养细胞,其不包含编码野生型人CFTR的基因组DNA或编码野生型人CFTR的cDNA。
29.如权利要求19至25中任一项所述的药物组合物的用途,其用于诱导功能性CFTR的表达。
30.一种在哺乳动物的肺中的上皮细胞中诱导CFTR表达的方法,所述方法包括使所述上皮细胞与组合物相接触,其中所述组合物是包含如权利要求4至15中任一项所述的mRNA的药物组合物。
31.一种在哺乳动物靶细胞中诱导CFTR表达的方法,所述方法包括使所述哺乳动物靶细胞与组合物相接触,所述组合物包含如权利要求4至15中任一项所述的mRNA。
32.如权利要求1至3或29至31中任一项所述的方法,其中所述组合物还包含选自聚乙烯亚胺(PEI)的有机阳离子或阳离子脂质,其中所述有机阳离子与所述体外转录的mRNA非共价复合。
33.如权利要求1至3或29至32中任一项所述的方法,其中所述组合物不包含粘液溶解剂。
34.如权利要求1至3或29至33中任一项所述的方法,其中所述有机阳离子是阳离子脂质并且所述组合物还包含中性脂质、聚乙二醇化脂质和/或胆固醇。
35.如权利要求1至3或29至34中任一项所述的方法,其中所述阳离子脂质选自选自DOTAP(1,2-二油酰基-3-三甲基铵丙烷)、DODAP(1,2-二油酰基-3-二甲基铵丙烷)、DOTMA(1,2-二-O-十八碳烯基-3-三甲基铵丙烷)、DLinDMA、DLin-KC2-DMA、C12-200、HGT4003、HGT5000、HGT5001、MC3、ICE、包含二烷基氨基部分的阳离子脂质、包含咪唑部分的阳离子脂质以及包含胍鎓部分的阳离子脂质。
36.如权利要求1至3或29至35中任一项所述的方法,其中所述中性脂质存在于所述组合物中并且选自DSPC(1,2-二硬脂酰基-sn-甘油-3-磷酸胆碱)、DPPC(1,2-二棕榈酰基-sn-甘油-3-磷酸胆碱)、DOPE(1,2-二油酰基-sn-甘油-3-磷酸乙醇胺)、DPPE(1,2-二棕榈酰基-sn-甘油-3-磷酸乙醇胺)、DMPE(1,2-二肉豆蔻酰基-sn-甘油-3-磷酸乙醇胺)及DOPG(,2-二油酰基-sn-甘油-3-磷酸-(1'-rac-甘油))。
37.如权利要求1至3或29至36中任一项所述的方法,其中所述聚乙二醇化脂质存在于所述组合物中并且包含共价连接至达5kDa长度的聚(乙二醇)链的C6-C20长度的一个或多个烷基链。
38.如权利要求1至3或29至37中任一项所述的方法,其中所述有机阳离子是具有10kDa至40kDa范围内的分子量的支链PEI。
39.如权利要求1至3或29至38中任一项所述的方法,其中所述组合物经由雾化或气雾化施用。
40.一种体外制备CFTRmRNA的方法,所述方法包括使分离的多核苷酸与RNA聚合酶在核苷三磷酸存在下相接触,其中:
所述分离的多核苷酸和RNA聚合酶不包含在细胞内;
所述分离的多核苷酸是所述RNA聚合酶的模板;
所述分离的多核苷酸包含可操作地连接至模板序列的启动子;
所述模板序列包含与编码SEQIDNO:1的序列互补的编码序列互补序列;以及:
(a)所述模板序列包含比SEQIDNO:2的互补序列更少的隐蔽性启动子,(b)所述模板序列包含比SEQIDNO:2更少的同向和/或反向重复序列,(c)所述模板序列包含比SEQIDNO:2更少的不利密码子的互补序列,或(d)所述编码序列互补序列的GC含量比SEQIDNO:2的GC含量更低。
41.一种体外制备CFTRmRNA的方法,所述方法包括使如权利要求16或17中任一项所述的分离的多核苷酸与RNA聚合酶在核苷三磷酸存在下相接触,其中:
所述分离的多核苷酸和RNA聚合酶不包含在细胞内;
所述分离的多核苷酸是所述RNA聚合酶的模板;以及
所述分离的多核苷酸包含可操作地连接至模板序列的启动子,
并且所述RNA聚合酶合成包含编码SEQIDNO:1的编码序列的mRNA。
42.如权利要求40至41中任一项所述的方法,其中所述RNA聚合酶是T7RNA聚合酶。
43.如权利要求40至42中任一项所述的方法,其中所述核苷三磷酸包含假尿苷三磷酸、5-甲基-胞苷三磷酸和2-硫代-尿苷三磷酸中的一种或多种。
44.如权利要求40至43中任一项所述的方法,其还包括分离包含编码SEQIDNO:1的编码序列的所述mRNA。
45.如权利要求44所述的方法,其还包括将5’帽添加至所述分离的mRNA。
46.如权利要求45所述的方法,其还包括通过使所述加帽的分离的mRNA与包含一种或多种有机阳离子的一种或多种药学上可接受的载体相接触来配制药物组合物。
47.如权利要求46所述的方法,其中所述一种或多种有机阳离子包括聚乙烯亚胺(PEI)、鱼精蛋白、聚乙二醇化鱼精蛋白、PLL、聚乙二醇化PLL或阳离子脂质。
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