CN110392581A - 用于预防或治疗乙型肝炎的药物组合物 - Google Patents
用于预防或治疗乙型肝炎的药物组合物 Download PDFInfo
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Abstract
本发明涉及一种用于治疗或预防乙型肝炎的药物组合物,以及一种根据HBV是否与候选材料形成G‑四链体筛选乙型肝炎治疗剂的方法。作为活性成分,药物组合物包含:一种或多种选自以下的寡核苷酸:SEQ ID NO:1、2或6的核酸序列或者其互补核酸序列表示的寡核苷酸;以及在寡核苷酸上具有至少一个化学修饰的寡核苷酸。所述药物组合物与HBV形成G‑四链体并减少HBV的cccDNA(共价闭合环状DNA),因此可以用于乙型肝炎的治疗或预防。
Description
技术领域
本发明涉及一种通过给药用于预防或治疗乙型肝炎的寡核苷酸以及包括所述寡核苷酸的药物组合物治疗乙型肝炎的方法。
背景技术
在病毒感染中,乙型肝炎病毒(下文中称作HBV)对人类危害最大,困扰全世界超过3.5亿人。当个体感染HBV时,其可以引起肝病如慢性肝炎、肝硬化、肝细胞癌等,并且在严重情况下,病毒性肝病可以导致死亡。HBV具有DNA基因组,并且其是到目前为止已知具有最小基因组的病毒之一(Ganem and Prince N Engl J Med(2004)350,1118-1129)。
目前,因为可以预防HBV感染的疫苗的开发,新感染率已显著下降,但是在不发达国家感染状况仍十分严重。此外,有许多患者在疫苗接种之前感染HBV,引起大量社会问题。
HBV是具有3.2kb双链DNA基因组的病毒,并且DNA被衣壳蛋白包围,而衣壳蛋白被表面蛋白包围。HBV具有突出特征,其表现出亲肝性(hepatotropism),在非细胞毒性状态下持续感染,并且具有非常严格的宿主向性;其对人和黑猩猩以外的动物没有传染性(Ganemand Prince N Engl J Med(2004)350,1118-1129)。
HBV感染之后,衣壳分解,基因递送至核,在那里双链DNA转化为cccDNA(共价闭合环状DNA)。cccDNA在HBV生命周期中具有重要作用,用作HBV转录的模板。最近的研究已证实cccDNA通过组蛋白衣壳化,并且可以通过组蛋白的各种修饰进行调节(Pollicino etal.Gastroenterology(2006)130,823-837)。已知作为游离微小染色体存在的cccDNA是慢性感染的主要原因,不仅因为它产生HBV的所有RNA,而且还因为目前的抗HBV治疗剂不可以消除cccNDA(Urban et al.J Hepatol(2010)52,282-284)。
产生自cccDNA的病毒RNA产生核心、表面、聚合酶等,并且基于在细胞质中可以转化为基因组DNA的前基因组RNA进行衣壳化。已从前基因组RNA成功转化为DNA的HBV病毒粒子出芽。出芽之后,病毒粒子通过感染或再感染外周肝细胞稳定增殖(Urban et al.JHepatol(2010)52,282-284)。
所有目前使用的乙型肝炎治疗剂都是核酸衍生物,并且它们在前基因组RNA通过衣壳中的聚合酶转化为DNA时通过干扰病毒的新DNA链来工作,并且最终终止合成。因此,所有目前可用的靶向这个部分的治疗剂在HBV聚合酶的RT(逆转录酶)结构域的活性位点中发生突变时诱导耐药性,因此,由于长期治疗中的这些耐药突变,难以提供完全治愈(Zoulimand Locarnini,Gastroenterology(2009)137,1593-1608e1591-1592)。
目前FDA批准作为慢性乙型肝炎治疗剂的核酸类似物包括拉米夫定、阿德福韦、恩替卡韦、替比夫定、克来夫定和替诺福韦,并且因为这些全部是聚合酶抑制剂,它们不可以完全治愈慢性肝炎。
在2014年,Lucifora等人报道IFN-α和淋巴毒素b受体(LTbR)可以上调APOBEC3A或APOBEC3B并选择性地去除cccDNA而不凋亡。但是,其可能难以实际应用,因为必须大量使用。
构建基于细胞的cccDNA测定并筛选85,000种化合物。结果,据发现命名为CCC-0975和CCC-0346的两种二取代磺酰胺(DSS)能够在一定程度上减少cccDNA,但是效果仍不足以作为药物开发,而且,作用机制未知(Cai et al.Antimicrob Agents Chemother.(2012)Aug;56(8):4277-88)。
因此,需要新治疗以治疗HBV感染。在这方面,本发明人分析了HBV的基因组,由此发现了能够形成G-四链体的部分并开发了调节HBV基因的活性和去除HBV的cccDNA的技术。G-四链体是基于4个鸟嘌呤之间的键合形成的四链螺旋DNA结构(Metifiot et al.NucleicAcids Res.2014Nov10;42(20):12352-66.Epub 2014Oct 20)。
发明内容
技术问题
本发明的一个目的是提供一种用于治疗或预防乙型肝炎的药物组合物,作为活性成分,其包括:至少一种选自以下的寡核苷酸:SEQ ID NO:1、2或6的核酸序列或者其互补核酸序列表示的寡核苷酸;以及在寡核苷酸上具有至少一个化学修饰的寡核苷酸。
此外,本发明的另一目的是提供一种治疗或预防乙型肝炎的方法,包括:向个体给药有效剂量的药物组合物。
此外,本发明的另一目的是提供一种筛选乙型肝炎的治疗剂的方法,包括:使乙型肝炎病毒(HBV)与候选材料接触并证实HBV是否与候选材料形成G-四链体。
技术方案
在一方面,提供一种用于治疗或预防乙型肝炎的药物组合物,作为活性成分,其包括:至少一种选自以下的寡核苷酸:SEQ ID NO:1、2或6的核酸序列或者其互补核酸序列表示的寡核苷酸;以及在寡核苷酸上具有至少一个化学修饰的寡核苷酸。
在一些实施方案中,具有化学修饰的寡核苷酸可以具有至少一个化学修饰的核苷间连接(internucleoside linkage)。
在一些实施方案中,具有化学修饰的核苷间连接的寡核苷酸可以具有这样的化学修饰,其中用硫代磷酸(phosphorothioate)、二硫代磷酸(phosphorodithioate)、磷酰胺(phosphoramidate)或硼烷磷酸(boranophosphate)化学修饰核苷酸的磷酸基团。
在一些实施方案中,具有化学修饰的寡核苷酸可以具有至少一个化学修饰的糖部分。
在一些实施方案中,可以修饰糖部分,从而用甲氧基乙基(MOE)、二甲基氨基氧基乙氧基(DMAOE)、二甲基氨基乙氧基乙基(DMAEOE)、甲基(OMe)、氨基丙氧基(AP)或氟(F)取代核苷酸中戊糖的2'位置处的-H基团,或者可以用F-ANA取代糖部分。
在一些实施方案中,糖部分可以以LNA(锁核酸)或PNA(肽核酸)的形式化学修饰。
在一些实施方案中,寡核苷酸可以是这样的状态,其中GalNAc(N-乙酰半乳糖胺)通过接头结合至3'或5'端。
在一些实施方案中,具有化学修饰的寡核苷酸可以具有两个或更多个选自以下的化学修饰:核苷间连接的化学修饰和糖部分的化学修饰。
在一些实施方案中,具有两个或更多个化学修饰的寡核苷酸可以具有这样的化学修饰,其中用硫代磷酸、二硫代磷酸、磷酰胺或硼烷磷酸化学修饰核苷酸的磷酸基团,并且
可以进一步具有这样的化学修饰,其中用甲氧基乙基(MOE)、二甲基氨基氧基乙氧基(DMAOE)、二甲基氨基乙氧基乙基(DMAEOE)、甲基(OMe)、氨基丙氧基(AP)或氟(F)取代核苷酸中戊糖的2'位置处的-H基团,或者可以用F-ANA取代核苷酸的糖部分。
在一些实施方案中,具有两个或更多个化学修饰的寡核苷酸可以具有化学修饰,其中用硫代磷酸、二硫代磷酸、磷酰胺或硼烷磷酸化学修饰核苷酸的磷酸基团,并且
可以进一步具有这样的化学修饰,其中糖部分以LNA(锁核酸)或PNA(肽核酸)的形式化学修饰。
在一些实施方案中,具有两个或更多个化学修饰的寡核苷酸可以具有化学修饰,其中用硫代磷酸、二硫代磷酸、磷酰胺或硼烷磷酸化学修饰核苷酸的磷酸基团,并且
可以进一步是这样的状态,其中GalNAc(N-乙酰半乳糖胺)通过接头结合至3'或5'端。
在一些实施方案中,寡核苷酸可以与HBV形成G-四链体。
在一些实施方案中,用于治疗或预防乙型肝炎的药物组合物可以减少HBV的cccDNA(共价闭合环状DNA)。
在一些实施方案中,用于治疗或预防乙型肝炎的药物组合物可以进一步包括药学可接受的载体。
在一些实施方案中,药学可接受的载体可以包括壳聚糖纳米颗粒、胶体分散系统、大分子复合物、纳米胶囊、纳米颗粒、微球、珠和水包油乳剂、胶束、混合胶束或脂质体,但不限于此。
在一些实施方案中,药学可接受的载体可以是壳聚糖纳米颗粒,并且壳聚糖可以具有50kDa-190kDa的分子量。
在一些实施方案中,用于治疗或预防乙型肝炎的药物组合物可以口服或肠胃外给药至个体。
在一些实施方案中,用于治疗或预防乙型肝炎的药物组合物可以腹腔内、静脉内、经皮、舌下、肌肉内、鼻内或皮下给药至个体。
本文公开的寡核苷酸可以用于预防和/或治疗乙型肝炎,以及用于制造其治疗药物。
在另一方面,提供一种用于治疗或预防乙型肝炎的方法,包括:向个体给药有效剂量的用于治疗或预防乙型肝炎的药物组合物,所述药物组合物,作为活性成分,包括至少一种选自以下的寡核苷酸:SEQ ID NO:1、2或6的核酸序列或者其互补核酸序列表示的寡核苷酸;以及在寡核苷酸上具有至少一个化学修饰的寡核苷酸。
在一些实施方案中,具有化学修饰的寡核苷酸可以具有至少一个化学修饰的核苷间连接。
在一些实施方案中,具有化学修饰的核苷间连接的寡核苷酸可以具有这样的化学修饰,其中用硫代磷酸、二硫代磷酸、磷酰胺或硼烷磷酸化学修饰核苷酸的磷酸基团。
在一些实施方案中,具有化学修饰的寡核苷酸可以具有至少一个化学修饰的糖部分。
在一些实施方案中,可以修饰糖部分,从而用甲氧基乙基(MOE)、二甲基氨基氧基乙氧基(DMAOE)、二甲基氨基乙氧基乙基(DMAEOE)、甲基(OMe)、氨基丙氧基(AP)或氟(F)取代核苷酸中戊糖的2'位置处的-H基团,或者可以用F-ANA取代糖部分。
在一些实施方案中,糖部分可以以LNA(锁核酸)或PNA(肽核酸)的形式化学修饰。
在一些实施方案中,寡核苷酸可以是这样的状态,其中GalNAc(N-乙酰半乳糖胺)通过接头结合至3'或5'端。
在一些实施方案中,具有化学修饰的寡核苷酸可以具有两个或更多个选自以下的化学修饰:核苷间连接的化学修饰和糖部分的化学修饰。
在一些实施方案中,具有两个或更多个化学修饰的寡核苷酸可以具有化学修饰,其中用硫代磷酸、二硫代磷酸、磷酰胺或硼烷磷酸化学修饰核苷酸的磷酸基团,并且
可以进一步具有化学修饰,其中用甲氧基乙基(MOE)、二甲基氨基氧基乙氧基(DMAOE)、二甲基氨基乙氧基乙基(DMAEOE)、甲基(OMe)、氨基丙氧基(AP)或氟(F)取代核苷酸中戊糖的2'位置处的-H基团,或者可以用F-ANA取代核苷酸的糖部分。
在一些实施方案中,具有两个或更多个化学修饰的寡核苷酸可以具有化学修饰,其中用硫代磷酸、二硫代磷酸、磷酰胺或硼烷磷酸化学修饰核苷酸的磷酸基团,并且
可以进一步具有化学修饰,其中糖部分以LNA(锁核酸)或PNA(肽核酸)的形式化学修饰。
在一些实施方案中,具有两个或更多个化学修饰的寡核苷酸可以具有化学修饰,其中用硫代磷酸、二硫代磷酸、磷酰胺或硼烷磷酸化学修饰核苷酸的磷酸基团,并且
可以进一步是这样的状态,其中GalNAc(N-乙酰半乳糖胺)通过接头结合至3'或5'端。
在一些实施方案中,寡核苷酸可以与HBV形成G-四链体。
在一些实施方案中,用于治疗或预防乙型肝炎的药物组合物可以减少HBV的cccDNA(共价闭合环状DNA)。
在一些实施方案中,用于治疗或预防乙型肝炎的药物组合物可以进一步包括药学可接受的载体。
在一些实施方案中,药学可接受的载体可以包括壳聚糖纳米颗粒、胶体分散系统、大分子复合物、纳米胶囊、纳米颗粒、微球、珠和水包油乳剂、胶束、混合胶束或脂质体,但不限于此。
在一些实施方案中,药学可接受的载体可以是壳聚糖纳米颗粒,并且壳聚糖可以具有50kDa-190kDa的分子量。
在一些实施方案中,用于治疗或预防乙型肝炎的药物组合物可以口服或肠胃外给药至个体。
在一些实施方案中,用于治疗或预防乙型肝炎的药物组合物可以腹腔内、静脉内、经皮、舌下、肌肉内、鼻内或皮下给药至个体。
在另一方面,提供一种筛选乙型肝炎的治疗剂的方法,包括:使乙型肝炎病毒(HBV)与候选材料接触并证实HBV是否与候选材料形成G-四链体。
在一些实施方案中,所述方法可以包括如果HBV与候选材料形成G-四链体,则选择候选材料作为乙型肝炎的治疗剂。
在一些实施方案中,可以通过以下方法证实G-四链体的形成:电泳迁移率变动分析(EMSA)、圆二色性(CD)、核磁共振(NMR)和利用G-四链体特异性抗体的方法。
在一些实施方案中,候选材料可以包括4个或更多个鸟嘌呤(G)。
在一些实施方案中,候选材料可以通过与HBV的增强子II区结合并形成G-四链体来抑制HBV表达。
有益效果
用于治疗或预防乙型肝炎的药物组合物,作为活性成分,其包括SEQ ID NO:1、2或6的核酸序列或者其互补核酸序列表示的寡核苷酸以及在寡核苷酸上具有至少一个化学修饰的寡核苷酸,形成G-四链体,从而减少HBV的cccDNA(共价闭合环状DNA),因此可以用于治疗和预防乙型肝炎,或者用于筛选乙型肝炎的治疗剂。
附图说明
图1示意性地示出通过HBV基因组分析的序列筛选图以及HBV DNA区的D1-D9。
图2示出寡核苷酸(D1、D2和D6)对蛋白表达[(a)和(b)]和HBV复制[(c)]具有抑制效果。(a)和(b)分别示出HBV 1.2质粒的HBeAg和HBsAg的分泌率,而(c)示出HBVDNASouthern印迹的结果。
图3示出寡核苷酸抑制病毒mRNA的转录。pg/preC RNA代表前基因组和前核RNA;pre-S/S RNA代表表面RNA;而HBx RNA代表产生HBx蛋白的RNA。
图4示出寡核苷酸抑制病毒表面蛋白。β-肌动蛋白是装载对照,而L、M和S代表3类表面蛋白,其中L代表大,M代表中,而S代表小。
图5(a)和(b)示出萤光素酶报告测定,说明寡核苷酸抑制HBV增强子活性。
图6(a)和(b)示出寡核苷酸抑制HBV增强子。
图7示出寡核苷酸的电泳迁移率变动分析(EMSA)的结果,说明寡核苷酸结合至HBV增强子I和II序列以形成G-四链体。
图8示出寡核苷酸的电泳迁移率变动分析(EMSA)的结果,说明寡核苷酸与HBV增强子II区部分形成G-四链体。
图9示出寡核苷酸的电泳迁移率变动分析(EMSA)的结果,说明寡核苷酸识别它们自己的核苷酸序列并形成G-四链体结构。
图10示出研究在寡核苷酸中引起点突变的突变核苷酸是否形成G-四链体的EMSA结果。具有点突变的寡核苷酸不形成G-四链体。
图11示出通过萤光素酶活性测定测量寡核苷酸的HBV抑制活性的结果。PS代表用硫代磷酸修饰的D2,OMe代表用O-甲基修饰的D2,PNA代表用PNA修饰的D2,PS-OMe代表用硫代磷酸和O-甲基修饰的D2,而PS-LNA代表用硫代磷酸和LNA修饰的D2。
图12(a)示意性地示出HepG2-NTCP细胞的HBV转染和病毒蛋白分析。(b)和(c)示出用修饰的寡核苷酸处理时HBV蛋白表达的分析结果。PS代表用硫代磷酸修饰的D2,PS-OMe代表用硫代磷酸和O-甲基修饰的D2,而PS-LNA代表用硫代磷酸和LNA修饰的D2。D2的转染(D1,T.F)用作抗HBV效果的阳性对照。未修饰的D2处理(D2Tr)用作阴性对照。LMV是拉米夫定。
图13(a)示意性地示出PHH(原代人肝细胞)的HBV转染和病毒蛋白分析。(b)和(c)示出用修饰的寡核苷酸处理时HBV蛋白表达的分析结果。PS代表用硫代磷酸修饰的D2,PS-OMe代表用硫代磷酸和O-甲基修饰的D2,而PS-LNA代表用硫代磷酸和LNA修饰的D2。D2的转染(D1,T.F)用作抗HBV效果的阳性对照。未修饰的D2处理(D2Tr)用作阴性对照。
图14示出萤光素酶测定的结果,说明修饰的D2的抗HBV活性。PS代表其骨架用PS修饰的D2,PS-Ome(4,4)代表其骨架是PS并且其中用O-甲基修饰在5'和3'两端的4个核苷酸的D2,PS-Ome(5,5)代表其骨架是PS并且其中用O-甲基修饰在5'和3'两端的5个核苷酸的D2,PS-Ome(所有)代表其骨架是PS并且其中用O-甲基修饰所有核苷酸的D2,PS-LNA(2,2)代表其骨架是PS并且其中用LNA修饰在5'和3'两端的2个核苷酸的D2,PS-LNA(3,3)代表其骨架是PS并且其中用LNA修饰在5'和3'两端的3个核苷酸的D2,PS-LNA(4,4)代表其骨架是PS并且其中用LNA修饰在5'和3'两端的4个核苷酸的D2,PS-LNA(5,5)代表其骨架是PS并且其中用LNA修饰在5'和3'两端的5个核苷酸的D2,而PS-LNA(所有)代表其骨架是PS并且其中用LNA修饰所有核苷酸的D2。
图15示出HepG2细胞中58种修饰的寡核苷酸的HBeAg抑制活性。
图16示出HepG2细胞中58种修饰的寡核苷酸的HBsAg抑制活性。
图17示出HepG2-NTCP细胞中58种修饰的寡核苷酸的HBeAg抑制活性。
图18示出HepG2-NTCP细胞中58种修饰的寡核苷酸的HBsAg抑制活性。
图19示出PHH细胞中58种修饰的寡核苷酸的HBeAg抑制活性。
图20示出PHH细胞中58种修饰的寡核苷酸的HBsAg抑制活性。
图21示出在体内模型中寡核苷酸抑制HBV。(a)是体内实验计划的示意图,而(b)和(c)分别是病毒蛋白HBeAg和HBsAg的测量结果。(b)和(c)的第一条Mock代表对照小鼠,第二条代表包含具有空载体的HBV的实验组,而第三条代表包含HBV DNA和D2的实验组。
图22示出在静脉内注射入体内模型时修饰的寡核苷酸抑制HBV。(a)是体内静脉内(IV)注射实验计划的示意图,而(b)和(c)分别是病毒蛋白HBeAg和HBsAg的测量结果。(d)是通过Southern印迹证实的结果,并且每个数值表示实验中使用的小鼠上的数字。PS代表用PS修饰其骨架的D2,PS-OMe代表其骨架是PS并且其中用O-甲基修饰所有核苷酸的D2,而PS-LNA代表其骨架是PS并且其中用LNA修饰所有核苷酸的D2。
图23示出用纳米颗粒(壳聚糖)衣壳化修饰的寡核苷酸然后静脉内注射入体内模型时的HBV抑制。(a)是体内静脉内(IV)注射实验计划的示意图,(b)和(c)分别是病毒蛋白HBeAg和HBsAg的测量结果,而(d)是通过Southern印迹证实的结果。
图24示出从开始用修饰的寡核苷酸处理细胞时的HBV抑制。(a)是用HBV转染PHH的方法的示意图,(b)和(c)是证实修饰的寡核苷酸具有去除cccDNA的优越效果的结果,而(e)和(f)是通过实时PCR定量评价的结果。
图25示出在再转染条件下用修饰的寡核苷酸处理细胞时的HBV抑制。(a)是用HBV转染PHH的方法的示意图,(b)和(c)是处理不同浓度的修饰的寡核苷酸的结果,而(d)是普通PCR之后通过电泳证实DNA的量不同的结果。
图26示出证实在HepG2-NTCP中修饰的寡核苷酸是否有效识别cccDNA并形成G-四链体的结果。(a)通过识别G-四链体的BG4抗体证实当用NTCP中通过转染产生的HBV cccDNA和修饰的寡核苷酸处理细胞时D2和cccDNA形成G-四链体,而(b)示出BG4的集落数量。
具体实施方式
在一实施方案中,提供一种用于治疗或预防乙型肝炎的药物组合物,作为活性成分,其包括:至少一种选自以下的寡核苷酸:SEQ ID NO:1、2或6的核酸序列或者其互补核酸序列表示的寡核苷酸;以及在寡核苷酸上具有至少一个化学修饰的寡核苷酸。
根据下文的实施例,证实当具有SEQ ID NO:1、2或6的核酸序列的寡核苷酸处理HBV转染的肝癌细胞系并注射入HBV小鼠模型时抑制HBV蛋白的生成。因此,证实具有SEQ IDNO:1、2或6的核酸序列的寡核苷酸对HBV具有抗病毒作用。因此,与具有SEQ ID NO:1、2或6的核酸序列的寡核苷酸互补的核酸序列可能也对HBV具有抗病毒作用。
在另一实施方案中,为了促进寡核苷酸的细胞渗透,合成在SEQ ID NO:1、2或6的核酸序列表示的寡核苷酸上具有至少一个化学修饰的寡核苷酸并处理HBV转染的肝癌细胞系和HBV转染的小鼠模型。结果,证实抑制HBV蛋白的生成,从而证实尽管进行了化学修饰,具有至少一个化学修饰的寡核苷酸对HBV具有优秀的抗病毒作用。
因此,一种或多种选自以下的寡核苷酸:SEQ ID NO:1、2或6的核酸序列或者其互补核酸序列表示的寡核苷酸;以及在寡核苷酸上具有至少一个化学修饰的寡核苷酸可以用作治疗或预防乙型肝炎的药物的活性成分。
在所述药物组合物中,具有化学修饰的寡核苷酸可以具有至少一个化学修饰的核苷间连接,或者至少一个化学修饰的糖部分。
在一实施方案中,寡核苷酸是指但不限于由约5-40个,例如10-13个核苷酸组成的聚合物。
核苷酸由碱基、戊糖和磷酸基团(磷酸)组成。碱基可以是嘌呤(腺嘌呤或鸟嘌呤)或嘧啶(胞嘧啶、胸腺嘧啶或尿素)。此外,戊糖可以是核糖、脱氧核糖、阿拉伯糖、木糖、来苏糖、阿洛糖、altose、葡萄糖、甘露糖、古洛糖、艾杜糖、半乳糖、塔罗糖或者糖的稳定修饰形式。
例如,当戊糖是脱氧核糖时,核苷酸可以通过以下化学式1表示。
[化学式1]
在化学式1中,
B是碱基,并且R1是-H。
化学修饰会更详细地描述如下。与天然寡核苷酸相比,化学修饰的寡核苷酸可以包括各种化学修饰,包括核苷间连接、核糖单元和/或天然核苷碱基(腺嘌呤、鸟嘌呤、胞嘧啶、胸腺嘧啶等)。修饰可以在寡核苷酸合成期间或之后发生。在合成期间,修饰的碱基可以掺入内部或在其末端。合成之后,可以利用激活基团(通过氨基改性剂,通过3'或5'羟基,或者通过磷酸基团)进行修饰。修饰寡核苷酸的方法是本领域技术人员公知的。
在一些实施方案中,具有化学修饰的寡核苷酸可以具有化学修饰的核苷间连接。
核苷间连接的化学修饰表示核苷互相连接的磷酸基团中的氧被一个或多个其他取代基代替。
在一些实施方案中,其中不参与核苷间连接的磷酸基团上的氧被硫代替的核酸分子的稳定的糖磷酸骨架称作“硫代磷酸骨架”。除了硫代磷酸,还可以用二硫代磷酸、磷酰胺或硼烷磷酸取代核苷酸的磷酸基团,但不限于此。硫代磷酸、二硫代磷酸、磷酰胺或硼烷磷酸的骨架分别通过以下化学式2-5表示。
[化学式2]
[化学式3]
[化学式4]
[化学式5]
在化学式2-5中,B是碱基。
在一些实施方案中,具有化学修饰的寡核苷酸可以具有至少一个化学修饰的糖部分。
糖部分的化学修饰表示化学修饰核苷酸中的戊糖。
糖部分的化学修饰包括例如用另一取代基取代核苷酸中戊糖的2'位置处的-H基团,或者戊糖基本结构中的修饰。
其中用另一取代基取代核苷酸中戊糖的2'位置处的-H基团的糖部分的化学修饰表示用除-H以外的另一取代基取代下文化学式1的戊糖的2'位置处的R1。
[化学式1]
在化学式1中,
B是碱基,并且R1是-H。
在一些实施方案中,糖部分不限于核苷酸中戊糖的2'位置处的-H基团,而是可以通过用甲氧基乙氧基[2'-O-CH2CH2OCH3,2'-O-(2-甲氧基乙基);MOE],化学式6)、二甲基氨基氧基乙氧基([2'-O(CH2)2ON(CH3)2;DMAOE],化学式7)、二甲基氨基乙基氧基乙基([2'-OCH2CH2-O-CH2CH2-N(CH3)2;DMAEOE],化学式8)、甲氧基([2'-OCH3;OMe],化学式9)、氨基丙氧基([2'-OCH2CH2CH2NH2;AP],化学式10)或氟(2'-F,化学式11)取代修饰,或者糖部分可以通过用F-ANA(2'-F-β-D-阿拉伯呋喃糖基,化学式12)取代修饰。
[化学式6]
[化学式7]
[化学式8]
[化学式9]
[化学式10]
[化学式11]
[化学式12]
在化学式6-12中,B是碱基。
在一些实施方案中,核苷酸中戊糖基本结构的修饰可以包括LNA(锁核酸)或PNA(肽核酸)形式的核苷酸中戊糖的化学修饰。
LNA(锁核酸)也称作“锁核酸”或“双环核苷”,并且包括这样的核苷,其在核苷酸的戊糖的2'位置和4'位置之间包括共价桥。LNA通过化学式13表示。
[化学式13]
在化学式13中,B是碱基。
PNA(肽核酸)也称作“肽核酸”,其中核苷酸基本骨架中的碱基保留,并且直接或间接结合至基本骨架中的酰胺的氮杂-氮原子。PNA可以通过以下化学式14表示。
[化学式14]
在化学式14中,B是碱基。
在一些实施方案中,寡核苷酸可以是这样的状态,其中GalNAc(N-乙酰半乳糖胺)通过接头结合至3'或5'端。
按照需要可以将GalNAc引入连接至寡核苷酸末端的接头部分;例如,可以引入1、2或3个单元,但是GalNAc不限于此。
因为GalNAc结合至肝细胞的脱唾液酸糖蛋白受体,已开发通过结合作为肝靶向部分的GalNAc至寡核苷酸使得能够肝特异性递送的技术。因为寡核苷酸需要肝特异性递送,可以利用这类已知的GalNAc结合技术进一步化学修饰寡核苷酸。
在一些实施方案中,具有化学修饰的寡核苷酸可以具有至少两个选自以下的化学修饰:核苷间连接的化学修饰和糖部分的化学修饰。
糖部分的化学修饰可以相同或不同。
在一些实施方案中,寡核苷酸的一个或多个核苷酸可以包括糖部分的化学修饰,并且可以通过具有化学修饰的核苷间连接结合。一个核苷酸的化学修饰独立于相同寡核苷酸内存在的其他核苷酸的化学修饰。
在一些实施方案中,寡核苷酸的所有核苷酸可以包括糖部分的化学修饰。
在一些实施方案中,具有化学修饰的寡核苷酸可以修饰至少50%、至少60%、至少70%、至少80%、至少90%、至少95%或100%的核苷酸。
例如,具有两个或更多个化学修饰的寡核苷酸可以是这样的,其中用硫代磷酸、二硫代磷酸、磷酰胺或硼烷磷酸化学修饰核苷酸的磷酸基团,并且进一步地,用甲氧基乙基(MOE)、二甲基氨基氧基乙氧基(DMAOE)、二甲基氨基乙氧基乙基(DMAEOE)、甲基(OMe)、氨基丙氧基(AP)或氟(F)取代核苷酸中戊糖的2'位置处的-H基团,或者用F-ANA取代核苷酸的糖部分。
在另一实施方案中,具有两个或更多个化学修饰的寡核苷酸可以是这样的,其中用硫代磷酸、二硫代磷酸、磷酰胺或硼烷磷酸化学修饰核苷酸的磷酸基团,并且
进一步地,糖部分以LNA(锁核酸)或PNA(肽核酸)的形式化学修饰。
在一些实施方案中,寡核苷酸的5'端可以包括1、2、3、4或5个连续化学修饰。
在一些实施方案中,寡核苷酸的3'端可以包括1、2、3、4或5个连续化学修饰。
在一些实施方案中,寡核苷酸在5'和3'端可以包括1、2、3、4或5个连续化学修饰。
例如,如可以从以下实施方案看到的,寡核苷酸可以是PS-OMe(4,4),其中整个骨架修饰为硫代磷酸(PS)骨架,并且用O-甲基代替寡核苷酸的5'和3'两端的4个核苷酸;或者PS-OMe(5,5),其中用O-甲基修饰寡核苷酸的5'和3'两端的5个核苷酸。
在另一实施方案中,寡核苷酸可以是PS-LNA(2,2),其整个骨架是硫代磷酸(PS),并且用LNA修饰5'和3'两端的2个核苷酸;PS-LNA(3,3),其中用LNA修饰5'和3'两端的3个核苷酸;PS-LNA(4,4),其中用LNA修饰5'和3'两端的4个核苷酸;或者PS-LNA(5,5),其中用LNA修饰5'和3'两端的5个核苷酸。
在一些实施方案中,寡核苷酸可以包括SEQ ID NO:1的核酸序列表示的寡核苷酸,或者与其互补的寡核苷酸。
在一些实施方案中,寡核苷酸可以包括SEQ ID NO:2的核酸序列表示的寡核苷酸,或者与其互补的寡核苷酸。
在一些实施方案中,寡核苷酸可以包括SEQ ID NO:6的核酸序列表示的寡核苷酸,或者与其互补的寡核苷酸。
在一实施方案中,SEQ ID NO:20-57的核酸序列表示的寡核苷酸用作具有化学修饰的寡核苷酸,但不限于此。
在一些实施方案中,寡核苷酸可以与HBV形成G-四链体。
在根据Watson-Crick模型的双螺旋DNA中,腺嘌呤通过氢键与胸腺嘧啶配对,并且鸟嘌呤通过氢键与胞嘧啶配对。但是,根据Hoogsteen,鸟嘌呤富集区中的4个鸟嘌呤在一个平面上通过氢键连接在一起形成四聚体,并且3个四聚体垂直叠加形成一个结构,以提出其为G-四链体。一般来说,已报道鸟嘌呤作为G-四链体存在于具有很多鸟嘌呤富集区的基因中。
在一些实施方案中,证实了寡核苷酸结合至HBV以形成G-四链体,从而抑制HBV活性(见实施例3)。
寡核苷酸可以用来抑制个体中的HBV表达,如细胞、组织等。
在一些实施方案中,组合物,即,药物组合物可以配制用于给药至使用寡核苷酸的个体。制剂可以包括药学可接受的赋形剂。药学可接受的赋形剂是排除活性药物成分(例如,寡核苷酸、治疗剂等)的物质。赋形剂在添加的剂量不表现出效果。
在一实施方案中,提供一种用于治疗或预防乙型肝炎的药物组合物,其包括至少一种寡核苷酸。
寡核苷酸可以描述为“抗病毒寡核苷酸”或“抗HBV寡核苷酸”。
在一些实施方案中,用于治疗或预防乙型肝炎的药物组合物可以通过减少HBV的cccDNA(共价闭合环状DNA)来抑制HBV活性。
因此,提供一种包括“抗病毒寡核苷酸”的药物组合物。
药物组合物可以包括寡核苷酸或多种寡核苷酸,并且包含不干扰在体内用作抗病毒剂的其他物质。这类其他物质可以包括但不限于稀释剂、赋形剂、载体物质和/或其他抗病毒物质。
在一实施方案中,寡核苷酸可以配制为各种药物组合物。将药物组合物制备为适合预期用途的形式。一般来说,需要制备基本上不含热原以及可以对人或动物有害的其他杂质的组合物。示例性递送/制剂系统包括壳聚糖纳米颗粒、胶体分散系统、大分子复合物、纳米胶囊、纳米颗粒、微球、珠、和包括水包油乳剂的基于脂质的系统、胶束、混合胶束和脂质体。
在一些实施方案中,药学可接受的载体可以是壳聚糖纳米颗粒,并且壳聚糖可以具有50kDa-190kDa的分子量。
所述组合物或制剂可以采用多种治疗性寡核苷酸,包括本文描述的至少一种。例如,所述组合物或制剂可以采用本文描述的至少1、2或3种抗病毒寡核苷酸。
在一些实施方案中,寡核苷酸可以与其他治疗剂组合使用。组合还可以通过使细胞同时与一种或多种不同组合物或制剂接触来实现。或者,组合可以顺序给药。
在一些实施方案中,寡核苷酸可以配制用于常规皮下或静脉内给药,例如,通过用适当的水性稀释剂配制,包括无菌水和生理盐水。
所述药物组合物和制剂可以采用适当的盐和缓冲剂以使递送媒介物稳定并允许被靶细胞摄取。在一实施方案中,所述药物组合物包括有效量的递送媒介物,包括抑制剂寡核苷酸(例如,脂质体、纳米颗粒或其他复合物),并且溶解或分散于药学可接受的载体或水性介质中。“药学可接受的”或“药理学可接受的”是指当给药至动物或人时不产生有害、过敏或其他不良反应的分子实体或组合物。
如本文所用,“药学可接受的载体”可以包括一种或多种溶剂、缓冲剂、溶液、分散介质、包衣、抗细菌和抗真菌剂、等渗和吸收延迟剂等,可接受用于配制药物,如适合给药至人的药物。这类介质和物质用于药学活性物质的使用是本领域公知的。还可以将补充活性成分掺入所述组合物。
药物组合物的给药或递送可以通过任何途径,只要靶组织通过相同途径可用。例如,给药可以是局部或者通过皮内、皮下、肌肉内、腹腔内、动脉内、冠状动脉内、鞘内或静脉内注射,或者通过直接注射入靶组织(例如,心脏组织)。本文公开的寡核苷酸的稳定性和/或效力允许方便的给药途径,包括皮下、皮内、静脉内和肌肉内途径。此外,包括本文所述寡核苷酸的药物组合物可以通过导管系统或隔离冠状动脉循环用于递送治疗剂至心脏的系统给药。递送治疗剂至心脏和冠状动脉血管的各种导管系统是本领域已知的。
寡核苷酸和药物组合物可以包含在试剂盒、容器、包装或分配器中。
在一些实施方案中,包括至少一种寡核苷酸的药物组合物在细胞、组织或个体中有效抑制HBV表达。此外,所述组合物或制剂还可以腹腔内、静脉内、经皮、舌下、肌肉内、鼻内或皮下给药。例如,可以在与表面活性剂如羟丙基纤维素适当混合的水中制备作为游离碱或药理学可接受的盐的缀合物溶液。还可以在甘油、液体聚乙二醇和它们的混合物中以及在油中制备分散液。
在普通储存和使用条件下,这些制品一般包含防腐剂以防止微生物生长。适合注射使用或导管递送的药物形式包括例如无菌水性溶液或分散液以及用于临时制备无菌注射溶液或分散液的无菌粉末。一般来说,这些制品是无菌的,并且是容易注射的流体。制品应当在制备和储存条件下稳定,并且应当受到保护抗微生物如细菌和真菌的污染作用。合适的溶剂或分散介质可以包含例如水、乙醇、多元醇(例如甘油、丙二醇、液体聚乙二醇等)、它们的合适混合物以及植物油。例如,可以通过使用包衣如卵磷脂,在分散液的情况下通过保持所需的粒径以及通过使用表面活性剂来保持适当的流动性。防止微生物的作用可以通过各种抗细菌和抗真菌剂实现,例如,对羟基苯甲酸酯、氯代丁醇、酚、山梨酸、硫柳汞等。在许多情况下,可能优选包括等渗剂,例如糖或氯化钠。注射组合物延长的吸收可以通过在组合物中使用吸收延迟剂来实现,例如单硬脂酸铝和明胶。
可以通过将适当量的缀合物加入溶剂连同期望的任何其他成分(例如,如上文列举的)来制备无菌注射溶液。一般来说,通过将各种无菌活性成分加入包含基本分散介质和其他期望成分(例如,如上文列举的)的无菌媒介物来制备分散剂。在用于制备无菌注射溶液的无菌粉末的情况下,优选的制备方法包括真空干燥和冷冻干燥技术,其获得来自其先前无菌过滤的溶液的活性成分加上任何额外的期望成分的粉末。
一旦配制,溶液优选以与剂量制剂相容的方式和治疗有效的量给药。制剂可以以各种剂型如注射溶液、药物释放胶囊等容易地给药。对于在水性溶液中肠胃外给药,例如,溶液一般是适当缓冲的,并且首先使液体稀释剂是等渗的,例如,用足够的盐水或葡萄糖。例如,这类水性溶液可以用于静脉内、肌肉内、皮下和腹腔内给药。优选地,如本领域技术人员已知地采用无菌水性介质,特别是根据本公开。例如,可以将单一剂量溶于1ml等渗NaCl溶液中,并且添加至1000ml皮下灌注术液体或在建议的输注部位注射(参见例如,“Remington's Pharmaceutical Sciences”15th Edition,1035-1038和1570-1580页)。剂量的一些变化必然会发生,取决于治疗的个体的状况。无论如何,负责给药的人会确定单独个体的适当剂量。此外,为了给药至人,制品应当满足FDA生物制品标准办公室要求的无菌性、产热原性、一般安全性和纯度。
在一实施方案中,提供一种递送寡核苷酸至细胞的方法(例如,作为本文所述组合物或制剂的部分),以及一种治疗、改善或预防个体中的疾病状况发展的方法。如本文所用,术语“个体”或“患者”是指任何脊椎动物,包括但不限于人和其他灵长类(例如,黑猩猩以及其他猿和猴物种)、农场动物(例如,牛、绵羊、猪、山羊和马)、家养哺乳动物(例如,狗和猫)、实验室动物(例如,啮齿动物如小鼠、大鼠和豚鼠)和鸟(例如,家养、野生和捕猎的鸟如鸡、火鸡和其他家禽、鸭、鹅等)。在一些实施方案中,所述个体是哺乳动物。
在其他实施方案中,所述哺乳动物是人。
在一实施方案中,可以使寡核苷酸或药物组合物与靶细胞在体外或体内接触(例如,哺乳动物细胞)。在一些实施方案中,所述细胞可以是肝细胞。
此外,在一实施方案中,提供一种筛选乙型肝炎的治疗剂的方法,包括使乙型肝炎病毒(HBV)与候选材料接触,以及证实HBV是否与候选材料形成G-四链体。
在筛选方法中,当HBV与候选材料形成G-四链体时可以判断候选材料有效治疗乙型肝炎。
在一实施方案中,可以通过以下方法证实HBV和候选材料形成G-四链体:电泳迁移率变动分析(EMSA)、圆二色性(CD)、核磁共振(NMR)或利用G-四链体特异性抗体的方法,但是可以使用能够证实DNA和蛋白之间结合的任何方法而没有限制。
候选材料可以包括4个或更多个鸟嘌呤(G)。因为候选材料包括4个或更多个鸟嘌呤,可以形成G-四链体,其中4个螺旋基于4个鸟嘌呤键形成一个单一结构。
可以通过结合HBV的增强子II区与候选材料形成G-四链体。增强子II可以具有SEQID NO:19的核酸序列。
在以下实施例中,证实具有SEQ ID NO:2的核酸序列的寡核苷酸物理结合至HBV的增强子II区以部分形成G-四链体,从而抑制HBV增强子活性。
在一些实施方案中,候选材料可以是SEQ ID NO:1、2或6的核酸序列或者其互补核酸序列表示的寡核苷酸,或者在寡核苷酸上具有至少一个化学修饰的寡核苷酸,或者可以包括两种寡核苷酸。
寡核苷酸的上述描述可以直接适用。
在一些实施方案中,提供一种治疗或预防乙型肝炎的方法,包括向个体口服或肠胃外给药用于治疗或预防乙型肝炎的药物组合物。
对于临床使用,寡核苷酸可以通过有效达到期望治疗结果的任何合适的给药途径单独给药,或者可以配制于药物组合物中。寡核苷酸的给药“途径”可以包括肠内、肠胃外以及局部给药或吸入。寡核苷酸的肠内给药途径包括口服、胃肠道、肠道和直肠途径。肠胃外途径包括静脉内、腹腔内、肌肉内、椎管内、皮下、局部注射、阴道、局部、鼻部、粘膜和肺部给药。寡核苷酸的局部给药途径是指寡核苷酸外部施用至表皮、口和耳、眼和鼻。
如本文所用,术语“预防”是指通过向个体给药药物组合物来抑制或延缓炎性疾病或免疫疾病的所有行动。
如本文所用,术语“治疗”是指通过向怀疑患有乙型肝炎的个体给药药物组合物来减轻或有益地改变肝炎症状的所有行动。
如本文所用,术语“改善”是指至少减少与待治疗的疾病状况相关的参数如症状程度的所有行动。
通过以下额外的实施例进一步说明本发明,其不应当理解为限制性的。鉴于本发明,本领域技术人员应当理解可以对本文公开的具体实施方案进行许多变化,并且仍获得类似或相似的结果而不背离本发明的精神和范围。
除非另有定义,本文所用的所有技术和科学术语均具有与本发明所属技术领域的技术人员通常理解的相同的含义。一般来说,本文使用的命名法是本领域公知和常用的。
下文通过实施例详细描述本发明。但是,这些实施例仅为了说明目的给出,并且本发明的范围不受这些实施例限制。
实施例
实施例1:材料和方法
1-1:细胞培养和转染
人肝癌细胞系(HepG2和Huh7细胞)获得自美国典型培养物保藏中心(Manassas,VA,USA)。利用Lipofectamine 2000(Invitrogen),根据制造商的说明书将具有NCBI号hNTCP[NM_003049.3]的能够表达智人(Homo sapiens)溶质载体家族10(钠/胆汁酸协同转运蛋白)或成员(SLC10A1)的质粒转染至HepG2细胞,以便建立HepG2-hNTCP细胞系。将细胞系在DMEM中培养。DMEM补充了10%(v/v)FBS(Gibco BRL)并添加1%青霉素和1%链霉素待用。将HepG2和Huh7细胞在37℃下于产生5%CO2的培养箱中培养。经IRB批准从来自天主教大学医院(Uijeongbu,Gyeonggi-do,Korea)的患者的肝组织分离原代人肝细胞(PHH),并且使用。原代维持培养基(Gibco BRL,Oregon,USA)补充了CM4000(Thermo,Rockford,USA)并添加1%青霉素和1%链霉素,并且在其中培养PHH。当根据指导利用Lipofectamine 2000培养80%细胞时进行转染。转染15小时之后,用新鲜培养基替换细胞。在转染之后2或3天收获细胞。
1-2.HBV转染研究
为了收集可接种的HBV,将约100-倍浓缩的HepAD38细胞的培养上清液用6%PEG8000沉淀。在包含25%FBS的PBS中制备HBV颗粒。将传染性HBV储备储存在-80℃下。利用斑点印迹测定计算HBV定量。对于HBV转染,将HepG2-NTCP细胞和PHH细胞与包含4%PEG和2.5%DMSO的PMM一起使用。转染15小时之后,用新鲜PMM替换培养基。在转染之后7天收获转染细胞。
1-3.Southern印迹
通过Southern印迹检测病毒DNA。简单地说,在转染之后3天通过刮擦收获细胞团。然后,将收获的细胞溶于100μl的冷HEPES(10mM HEPES pH 7.5,100mM NaCl,1mM EDTA,0.5%NP-40)缓冲液中,并且用26%PEG8000缓冲液沉淀裂解物中的HBV核心衣壳。随后,在37℃下用0.5%SDS缓冲液(具有250mg蛋白酶K)降解HBV核心衣壳3小时。用酚-氯仿提取HBVDNA并用NaOAc和乙醇沉淀。通过在0.8%琼脂糖凝胶中于90V下电泳3小时分离总DNA并转移至XL硝化纤维素膜(GE Healthcare)。然后,用高纯随机HBV探针检测HBV DNA,并且利用phospho-imager定量相对HBV DNA复制水平。
1-4.Northern印迹
通过Northern印迹检测HBV mRNA。简单地说,利用TRIzol试剂(Invitrogen)根据制造商的方案提取总细胞RNA。通过在1%甲醛琼脂糖凝胶中于120V下电泳3小时分离20μl总RNA并转移至XL硝化纤维素膜(GE Healthcare)16-18小时。为了检测HBV特异性mRNA,将膜用高纯随机引物HBV探针杂交,并且利用phospho-imager定量相对HBV DNA复制水平。
1-5:蛋白印迹
转染2天之后,收获细胞并在4℃下于RIPA缓冲液[20mM Tris/HCl,1%NP-40,0.5%蛋白酶抑制剂混合物(Sigma,St.Louis,MO),150mM NaCl,2mM KCl,pH 7.4]中裂解30分钟。通过SDS-PAGE分离蛋白裂解物。SDS-PAGE之后,将聚丙烯酰胺凝胶的蛋白转移至PVDF膜。以1:2000的比例使用抗体。抗肌动蛋白(Sigma)、HBsAg(Abcam)和HBcAg(DAKO,USA)用作一抗(第一抗体)。
1-6:利用实时PCR的rcDNA和cccDNA的定量分析
为了定量HBV rcDNA,利用QIAamp DNAMini试剂盒(Qiagen)从用HBV转染的PHH提取全细胞DNA。在扩增cccDNA之前,用T5外切核酸酶(NEB)处理DNA。利用20μl包含20ng的DNA、0.5μmol/L的正向和反向引物、0.2μmol/L的3'-荧光素(FL)标记的探针以及0.4μmol/L的5'-Red640(R640)标记的探针的LightCycler(roche)进行实时PCR。用于cccDNA扩增的正向和反向引物分别具有5'-CTCCCCGTCTGTGCCTTCT-3'(SEQ ID NO:10)和5'-GCCCCAAAGCCACCCAAG-3'(SEQ ID NO:11)的结构,并且分别使用5'-CTCGTGGTGGACTTCTCTC-3'(SEQ ID NO:12)和5'-CTGCAGGATGAAGAGGAA-3'(SEQ ID NO:13)的正向和反向引物,用于肝中的rcDNA扩增。
对于FRET杂交探针,分别使用5'-GTTCACGGTGGTCTCCATGCAACGT-FL-3'(SEQ IDNO:14)和5'-R640-AGGTGAAGCGAAGTGCACACGGACC-3'(SEQ ID NO:15)的正向和反向引物,用于cccDNA的扩增,并且分别使用5'-CACTCACCAACCTCCTGTCCTCCAA-FL-3'(SEQ ID NO:16)和5'-R640TGTCCTGGTTATCGCTGGATGTGTCT-3'(SEQ ID NO:17)的正向和反向引物,用于rcDNA的扩增。HBV DNA总量的扩增进行如下:将DNA在95℃下处理10分钟,然后在95℃下处理10秒,在58℃下处理10秒,并且在72℃下处理15秒,这个循环重复45次。cccDNA的扩增进行如下:将cccDNA在95℃下处理10分钟,然后在95℃下处理10秒,在58℃下处理5秒,并且在72℃下处理20秒,这个循环重复45次。为了归一化,利用LightCyclerβ-珠蛋白对照试剂盒(Roche)扩增β-珠蛋白基因。将包含HBV单体的质粒(pHBVEcoRI)逐步稀释并用作定量标准品。
1-7:萤光素酶报告基因的分析
进行增强子萤光素酶测定以证实HBV增强子活性。在12-孔板中制备2×105个HepG2细胞系并用0.5μg的增强子-Luc(pEnhI.II,pEnhI.ΔII,pEnhIXp,pXp.EnhII,pNRE.EnhII,pEnhII/cp,pEnhI△Xp-D2,pEnhIXp-D6,pEnhI△Xp-D7,and pEnhIXp-D8;见图5和6)和50nM的D2转染。转染48小时之后,收获细胞并在Promega裂解缓冲液中裂解,然后利用萤光素酶试剂(Promega,Madison,WI)测量增强子萤光素酶活性。
表1
1-8:寡核苷酸的构建
1-8-1:未修饰的寡核苷酸的构建
图1示出通过HBV的基因组分析的序列筛选图,其能够通过形成特定结构如G-四链体表现出抗病毒作用。
通过Cosmogenetech(Seoul,Korea)或Bio Basic(Canada)合成本发明中使用的低聚化合物D1-D9。每种化合物的详细描述在下文表2中示出。
表2
D1-D9是未修饰的寡核苷酸。在它们中,用待用的PS(硫代磷酸)、OMe(O-甲基)、PNA(肽核酸)、LNA(锁核酸)、PS-OMe和PS-LNA修饰D2。
用PS修饰的寡核苷酸可以容易地渗透入细胞并防止被外切核酸酶降解。用OMe修饰的寡核苷酸具有与RNA相似的特征,但是其特征在于针对细胞中的核酸酶和水解增加的稳定性。此外,双结构的Tm增加约1℃-4℃。PNA是人工产生的聚合物,其具有与DNA或RNA相似的结构,并且其骨架具有通过肽键重复连接的N-(2-氨基乙基)-甘氨酸。用LNA修饰的寡核苷酸具有这样的结构,其中2'-氧和4'-碳连接并锁定,其Tm在杂交期间增加,并且针对降解稳定。部分修饰的D2在5'和3'端序列部分修饰。例如,PS-LNA(4,4)表示其整个骨架是PS并且其中用LNA修饰5'和3'两端的4个核苷酸的寡核苷酸。通过这类部分修饰构建的D2的实例包括PS-OMe(4,4)、PS-OMe(5,5)、PS-LNA(2,2)PS-LNA(3,3)(SEQ ID NO:76)、PS-LNA(4,4)(SEQ ID NO:77)和PS-LNA(5,5)。
1-8-2:修饰的寡核苷酸的构建
为了优化抗病毒作用,利用1-8-1的D2通过以下命名法制备各种类型的寡核苷酸。利用大写字母A、G、C和T命名DNA,并且利用小写字母a、g、c和t命名RNA。当用O-甲基修饰核苷酸中戊糖的2'-位置时在核酸前面加上小写字母m,当用LNA修饰寡核苷酸时在核酸前面加上字母l。当骨架是硫代磷酸(PS)时,通过方括号([])表示DNA骨架。正常DNA没有方括号。以上命名法在表3中示出。
根据以上命名法则合成总计58种寡核苷酸并在表4和5中示出。在本发明中,表4和5中示出的寡核苷酸通过SEQ ID NO:20-SEQ ID NO:77顺序表示,并且表4和5中分配给核酸序列的编号是oligo修饰#。
表3
[表4]
1 | [mT | mG | mC | mT | mG | IG | IG | mG | mG | mG | mA | mA | mT | mT | mG | mA] |
2 | [mT | mG | mC | mT | IG | IG | IG | IG | mG | mG | mA | mA | mT | mT | mG | mA] |
3 | [mT | mG | mC | mT | IG | IG | IG | IG | IG | IG | mA | mA | mT | mT | mG | mA] |
4 | [mT | mG | mC | IT | IG | IG | IG | IG | IG | IG | IA | mA | mT | mT | mG | mA] |
5 | [mT | mG | mC | IT | IG | IG | IG | IG | IG | IG | IA | IA | IT | mT | mG | mA] |
6 | [mT | mG | IC | IT | IG | IG | IG | IG | IG | IG | IA | IA | IT | IT | mG | mA] |
7 | [IT | IG | mC | mT | mG | mG | mG | mG | mG | mG | mA | mA | IT | IT | IG | IA] |
8 | [IT | IG | IC | mT | mG | mG | mG | mG | mG | mG | mA | IA | IT | IT | IG | IA] |
9 | [IT | IG | IC | IT | mG | mG | mG | mG | mG | mG | IA | IA | IT | IT | IG | IA] |
10 | [IT | IG | IC | IT | IG | mG | mG | mG | mG | IG | IA | IA | IT | IT | IG | IA] |
11 | [IT | IG | IC | IT | IG | IG | mG | mG | IG | IG | IA | IA | IT | IT | IG | IA] |
12 | [T | G | C | T | G | G | IG | IG | G | G | A | A | T | T | G | A] |
13 | [T | G | C | T | G | IG | IG | IG | IG | G | A | A | T | T | G | A] |
14 | [T | G | C | T | IG | IG | IG | IG | IG | IG | A | A | T | T | G | A] |
15 | [T | G | C | IT | IG | IG | IG | IG | IG | IG | IA | A | T | T | G | A] |
16 | [T | G | C | IT | IG | IG | IG | IG | IG | IG | IA | IA | IT | T | G | A] |
17 | [T | G | IC | IT | IG | IG | IG | IG | IG | IG | IA | IA | IT | IT | G | A] |
18 | [IT | IG | C | T | G | G | G | G | G | G | A | A | IT | IT | IG | IA] |
19 | [IT | IG | IC | T | G | G | G | G | G | G | A | IA | IT | IT | IG | IA] |
20 | [IT | IG | IC | IT | G | G | G | G | G | G | IA | IA | IT | IT | IG | IA] |
21 | [IT | IG | IC | IT | IG | G | G | G | G | IG | IA | IA | IT | IT | IG | IA] |
22 | [IT | IG | IC | IT | IG | IG | G | G | IG | IG | IA | IA | IT | IT | IG | IA] |
23 | [IT | mG | IC | mT | IG | mG | IG | mG | IG | mG | IA | mA | IT | mT | IG | mA] |
24 | [mT | IG | mC | IT | mG | IG | mG | IG | mG | IG | mA | IA | mT | IT | mG | IA] |
25 | [IT | G | IC | T | IG | G | IG | G | IG | G | IA | A | IT | T | IG | A] |
26 | [T | IG | C | IT | G | IG | G | IG | G | IG | A | IA | T | IT | G | IA] |
27 | [mT | mG | mC | mT | mG | mG | mG | mG | mG | mG | IA | IA | IT | IT | IG | IA] |
28 | [IT | IG | IC | IT | IG | IG | IG | IG | IG | IG | mA | mA | mT | mT | mG | mA] |
29 | [T | G | C | T | G | G | G | G | G | G | IA | IA | IT | IT | IG | IA] |
30 | [IT | IG | IC | IT | IG | IG | IG | IG | IG | IG | A | A | T | T | G | A] |
[表5]
31 | [mT | mG | mC | mT | mG | mG | mG | mG | mG | mG | A | A | T | T | G | A] |
32 | [T | G | C | T | G | G | G | G | G | G | mA | mA | mT | mT | mG | mA] |
33 | [mT | mG | mC | mT | IG | IG | IG | IG | IG | IG | A | A | T | T | G | A] |
34 | [mT | mG | mC | mT | G | G | G | G | G | G | IA | IA | IT | IT | IG | IA] |
35 | [T | G | C | T | IG | IG | IG | IG | IG | IG | mA | mA | mT | mT | mG | mA] |
36 | [T | G | C | T | mG | mG | mG | mG | mG | mG | IA | IA | IT | IT | IG | IA] |
37 | [IT | IG | IC | IT | mG | mG | mG | mG | mG | mG | A | A | T | T | G | A] |
38 | [IT | IG | IC | IT | G | G | G | G | G | G | mA | mA | mT | mT | mG | mA] |
39 | [IT | IG | IC | IT | IG | IG | IG | IG | IG | IG | IA | IA | IT | IT | IG | IA] |
40 | IT | IG | IC | IT | IG | IG | IG | IG | IG | IG | IA | IA | IT | IT | IG | IA |
41 | [mT | mG | mC | mT] | IG | IG | IG | IG | IG | IG | [mA | mA | mT | mT | mG | mA] |
42 | [IT | IG | IC | IT] | mG | mG | mG | mG | mG | mG | [IA | IA | IT | IT | IG | IA] |
43 | [T | G | C | T] | IG | IG | IG | IG | IG | IG | [A | A | T | T | G | A] |
44 | [IT | IG | IC | IT] | G | G | G | G | G | G | [IA | IA | IT | IT | IG | IA] |
45 | [mT | mG | mC | mT] | IG | IG | IG | IG | IG | IG | [A | A | T | T | G | A] |
46 | [mT | mG | mC | mT] | G | G | G | G | G | G | [IA | IA | IT | IT | IG | IA] |
47 | [T | G | C | T] | IG | IG | IG | IG | IG | IG | [mA | mA | mT | mT | mG | mA] |
48 | [T | G | C | T] | mG | mG | mG | mG | mG | mG | [IA | IA | IT | IT | IG | IA] |
49 | [IT | IG | IC | IT] | mG | mG | mG | mG | mG | mG | [A | A | T | T | G | A] |
50 | [IT | IG | IC | IT] | G | G | G | G | G | G | [mA | mA | mT | mT | mG | mA] |
51 | T | G | C | T | [IG | IG | IG | IG | IG | IG] | A | A | T | T | G | A |
52 | [mu | mG | mC | mu | IG | IG | IG | IG | IG | IG | mA | mA | mu | mu | mG | mA] |
53 | [u | G | C | u | G | G | IG | IG | G | G | A | A | u | u | G | A] |
54 | [lu | mG | IC | mu | IG | mG | IG | mG | IG | mG | IA | mA | Iu | mu | IG | IA] |
55 | [Iu | IG | IC | Iu | IG | IG | IG | IG | IG | IG | IA | IA | Iu | Iu | IG | IA] |
56 | [mu | mG | mC | mu] | IG | IG | IG | IG | IG | IG | [A | A | u | u | G | A] |
(3,3) | [IT | IG | IC] | T | G | G | G | G | G | G | A | A | T | [IT | IG | IA] |
(4,4) | [IT | IG | IC | IT] | G | G | G | G | G | G | A | A | [IT | IT | IG | IA] |
1-9:电泳迁移率变动分析(EMSA)
使用30ng的HBV增强子DNA并用[32P]-γ同位素标记。500ng的D2用来形成G-四链体。将DNA(D2、pEnhIΔXp、pEnhIΔXp-D2以及增强子I和II)与缓冲溶液(10mM Tris-HCl pH7.5、0.1 M KCl、1mM DTT和10mM MgCl2)混合并加热,然后冷却,从而DNA可以折叠。与DNA混合物反应之后,添加BG4抗体(Absolute Antibody,United Kingdom)以通过DNA-蛋白结合鉴定特异性G-四链体DNA。在室温下结合反应之后,利用6%聚丙烯酰胺凝胶使DNA-DNA复合物在低温下进行电泳。电泳之后,将凝胶在70℃干燥30分钟。利用phospho-imager分析结果。
1-10:小鼠中利用水动力注射的实验
利用水动力注射方法将质粒DNA(25μg的HBV 1.2,25μg的D2和5μg的b-gal)递送至6周龄小鼠(BALB/C)。制备体积对应于小鼠体重10%的PBS并静脉内注射入小鼠的尾巴。还将修饰的D2(50μg)静脉内注射至小鼠的尾巴。利用注射器快速静脉内注射包含DNA的PBS4-6秒。所有动物实验均由Konkuk大学动物护理委员会配准。
1-11:利用显微镜的细胞中G-四链体的分析方法
将盖玻片放在6-孔板的底上并在其中培养细胞。用HBV转染细胞并用500nM修饰的D2处理。将细胞用丙酮固定,用PBS洗涤3次,并且用包含3%BSA的PBS封闭。用PBS洗涤3次之后,将BG4(Absolute Antibody,Ab00174-1.1)抗体以1:300的比例混合并在冷室中反应过夜。用PBS洗涤3次之后,将盖玻片放在6-孔板的底上并在其中用抗小鼠Alexa 568培养细胞1小时。用HBV转染细胞并用500nM修饰的D2处理。将细胞用丙酮固定,用PBS洗涤三次,并且用包含3%BSA的PBS封闭。用PBS洗涤三次后,将BG4(Absolute Antibody,Ab00174-1.1)抗体以1:300的比例混合,并在冷室中反应过夜。用PBS洗涤3次之后,将细胞与抗小鼠Alexa568反应1小时。用PBS洗涤3次之后,将核用DAPI染色30分钟。用PBS洗涤3次之后,将盖玻片安装在载玻片上并干燥。
1-12:利用壳聚糖纳米颗粒的体内实验
利用水动力注射方法将质粒DNA(25μg的HBV 1.2和5μg的b-gal)递送至6周龄小鼠(BALB/C)。制备体积对应于小鼠体重10%的PBS并静脉内注射入其尾巴。利用注射器快速静脉内注射包含DNA的PBS 4-6秒。第二天,将8μg壳聚糖纳米颗粒D2也静脉内注射入小鼠的尾巴。壳聚糖纳米颗粒是具有低细胞毒性和免疫原性以及高效生物相容性的分子,并且具有高效递送寡核苷酸如siRNA的特征(Targeted Gene Silencing Using RGD-LabeledChitosan Nanoparticles,Hee Dong Han,Clin Cancer Res.2010)。
基于壳聚糖(MW 50kDa-190kDa)和D2之间的离子凝胶化制备上述实验中使用的壳聚糖纳米颗粒。将TPP(0.25%w/v)和D2(1μg/μl)添加至1%(w/v)壳聚糖溶液。连续反应在室温下发生,并且在温育反应之后,通过在4℃下以13,000RPM离心40分钟获得沉淀。将由此获得的沉淀用DW洗涤3次并储存在4℃下直至使用。所有动物实验均由Konkuk大学动物护理委员会批准。
实施例2:抗病毒作用的证实
2-1:证实寡核苷酸表现出抗病毒作用
用D1-D9寡核苷酸作为HBV转染肝癌细胞系,并且通过病毒蛋白(HBsAg和HBeAg)形成的抑制和复制的抑制判断抗病毒作用。
具体地,将HBV 1.2质粒和寡核苷酸(D1-D9,分别为SEQ ID NO:1-9)转染至HepG2。在转染之后将细胞和上清液培养3天。测量分泌的HBeAg和HBsAg以确定HBV蛋白表达。利用HBeAg和HBsAg ELSIA试剂盒(Wantai Pharm Inc.,Beijing,China)分析培养基中的HBeAg和HBsAg。通过Southern印迹测量病毒HBV DNA。
结果,如图2所示,D1、D2和D6表现出抗病毒作用。
2-2:HBV RNA表达的抑制
进行实验以证实利用D2寡核苷酸抑制HBV RNA表达,这显示D1、D2和D6寡核苷酸中最优越的抗病毒效力,如实施例2-1中证实的。具体地,为了确定D2寡核苷酸抑制HBV生命周期的哪个阶段,用HBV 1.2-聚体转染Huh7细胞,并且通过Northern印迹分析HBV mRNA水平。
结果,如图3所示,证实D2寡核苷酸以剂量依赖性方式抑制HBV RNA。因此,证实D2寡核苷酸还抑制HBV RNA表达,从而证实D2寡核苷酸通过在病毒的RNA转录阶段作用抑制表达。
2-3:证实HBV蛋白表达的抑制
为了证实D2寡核苷酸是否抑制HBV蛋白表达,用HBV 1.2-聚体和D2寡核苷酸转染Huh7细胞,然后通过蛋白印迹分析测量表面蛋白表达水平。
结果,如图4所示,证实D2寡核苷酸以浓度依赖性方式抑制HBV蛋白之一表面蛋白的表达。
2-4:HBV增强子/启动子活性的抑制
为了研究D2寡核苷酸怎样降低HBV mRNA水平,利用HBV增强子进行萤光素酶报道测定。
结果,如图5(a)和(b)所示,证实通过D2寡核苷酸转染抑制HBV增强子I和II的约80%活性。这些结果证实D2寡核苷酸抑制增强子I和II的活性。但是,在增强子I(EnhI)和pEnhIΔXp的上游未观察到影响。通过D2寡核苷酸转染抑制HBV增强子II(EnhII)约48%。基于这些结果,证实HBV增强子II的1742G-富集区(1742-1747的区域)对于通过D2寡核苷酸抑制增强子活性很重要。
因此,图5的结果证实D2寡核苷酸减少增强子I和II的活性,从而在转录阶段表现出抗病毒作用。
为了研究D2寡核苷酸怎样减少HBV增强子的活性,构建上述报告质粒,并且测量报告物活性。如图6(a)所示,将表2中示出的D2、D6、D7和D8的碱基G-丰富的HBV基序引入报告质粒启动子区。
结果,如图6(b)所示,证实pEnhI△Xp萤光素酶克隆没有效果,但是包含D2或D6基序的pEnhI△Xp萤光素酶克隆强烈抑制萤光素酶活性。
因此,证实虽然D2寡核苷酸虽然在pEnhI△Xp报告物中不发挥功能,其是增强子I(EnhI)的上游,但是当通过将相同核苷酸序列添加至D2构建报告质粒时其表现出强抑制作用,并且进一步证实包含具有与D2寡核苷酸相似的核苷酸序列的D6寡核苷酸的报告物也被抑制。这些结果表明D2寡核苷酸识别并抑制其核苷酸序列。
实施例3:G-四链体结构的形成
3-1:证实D2寡核苷酸通过识别HBV增强子I和II区形成G-四链体。
利用D2寡核苷酸和P32标记的HBV增强子序列进行体外电泳迁移率变动分析(EMSA),以便证实D2寡核苷酸是否与增强子I和II的序列形成G-四链体(图7(a))。
结果,通过EMSA证实D2寡核苷酸与增强子I和II的序列部分形成G-四链体。如图7(b)所示,利用G-四链体特异性BG4抗体通过条带超级迁移证实G-四链体的形成。这是通过用磷成像可视化凝胶获得的结果。即,通过图7证实D2寡核苷酸物理结合至HBV增强子区以形成G-四链体,从而抑制HBV增强子活性。
3-2:证实D2寡核苷酸与HBV增强子II区形成G-四链体
利用D2和HBV进行体外EMSA以证实D2寡核苷酸是否与增强子II区形成G-四链体。
结果,通过EMSA证实D2寡核苷酸与增强子II序列部分形成G-四链体。利用G-四链体特异性BG4抗体通过条带超级迁移证实G-四链体的形成。此外,通过图8证实D2寡核苷酸通过HBV增强子II区形成G-四链体。
3-3:证实D2寡核苷酸与具有其自身核苷酸序列的区域形成完整G-四链体结构
进行体外EMSA以证实D2寡核苷酸是否通过其自身序列与HBV基因组形成完整G-四链体。
结果,如图9所示,证实D2寡核苷酸通过其自身序列与HBV基因组形成完整G-四链体。利用G-四链体特异性BG4抗体通过条带超级迁移证实G-四链体的形成,并且用磷成像使凝胶可视化。
根据图9,D2寡核苷酸不结合至增强子I区(EnhI△Xp),但是与引入其自身序列的区域(EnhI△Xp-D2)形成完整G-四链体结构。这些结果表明D2寡核苷酸识别其自身核苷酸序列并形成G-四链体结构,因此与病毒抑制相关。
3-4:证实在D2寡核苷酸的核苷酸序列中引入点突变的D3寡核苷酸不形成G-四链结构。
如图10所示,通过体外EMSA证实其中在D2寡核苷酸的核苷酸序列中引入点突变的D3寡核苷酸不形成G-四链体结构。具体地,其中在D2寡核苷酸序列的中间区域中将保守GGGGGG点突变为GGGTGG的D3寡核苷酸不与HBV基因组形成G-四链体。从这些结果,可以看到D2寡核苷酸的G-富集区对于G-四链体的形成非常重要。
此外,如图2中可以看到的,D3寡核苷酸完全不表现出病毒抑制活性。参考通过图10中示出的EMSA证实的表明D3寡核苷酸不形成G-四链体的结果,G-四链体结构的形成对于抗病毒作用至关重要。
实施例4:HBV活性的抑制
4-1:证实修饰的D2寡核苷酸通过渗透入细胞抑制HBV增强子活性
用HBV增强子I和II的质粒转染HepG2细胞。转染之前,用多种修饰的D2寡核苷酸(PS,OMe,PNA,LNA,PS-OMe,PS-LNA)预处理HepG2细胞(以500nM的终浓度)。第二天,用包含500nM修饰的D2寡核苷酸的新鲜培养基(DMEM)替换细胞。然后,转染之后将细胞培养24小时,并且利用稳定Glo-萤光素酶系统分析萤光素活性。
结果,如图11所示,证实具有PS修饰的修饰的寡核苷酸表现出优越的细胞渗透和HBV抑制活性。根据上述结果,在寡核苷酸的骨架中用硫代磷酸(PS)或锁核酸(LNA)修饰提高寡核苷酸的渗透性,结果增加寡核苷酸的抗病毒作用。
4-2:证实修饰的D2寡核苷酸在HBV转染模型中抑制HBV
为了研究修饰的D2寡核苷酸是否在HBV转染模型中也表现出抑制作用,用HBV转染HepG2-NTCP细胞系,其是HBV感染细胞系,然后用PS(PS,PS-OMe,PS-LNA)修饰的D2寡核苷酸处理。具体地,图12(a)的HepG2-NTCP细胞的HBV转染和病毒蛋白分析在示意图中示出,实验方法如下:用2000HBV基因组当量/细胞(Geq/细胞)转染HepG2-NTCP细胞,所述细胞在包含2%DMSO和4%PEG8000的PMM(PHH维持培养基,Gibco)中培养16-20小时。然后,将细胞用500μl的PBS洗涤3次,维持在PMM(2%DMSO)中,并且在转染之后培养7天。为了分析HBV蛋白表达,测量分泌的HBeAg和HBsA。利用HBeAg和HBsAg ELISA试剂盒(Wantai Pharm Inc.,Beijing,China)分析培养基中的HBeAg和HBsAg。D2寡核苷酸的转染(D1,T.F)用作抗HBV作用的阳性对照。未修饰的D2寡核苷酸处理(D2Tr)用作阴性对照。LMV是拉米夫定。
作为HBV蛋白表达的结果,证实在HepG2-NTCP(HBV感染细胞系)中用PS(PS,PS-OMe,PS-LNA)修饰的D2寡核苷酸也抑制HBV,从而证实在用修饰的D2寡核苷酸处理时表现出抗病毒作用,如图12(b)和(c)所示。
4-3:证实修饰的D2寡核苷酸在原代人肝细胞(PHH)中抑制HBV
为了证实用PS修饰的D2寡核苷酸是否在原代人肝细胞(PHH)中抑制HBV,在肝脏手术之后从患者的肝组织分离PHH,然后用HBV转染细胞以研究修饰的D2寡核苷酸的抗病毒作用。具体地,图13(a)的PHH的HBV转染和病毒蛋白分析在示意图中示出,实验方法如下:用5000HBV基因组当量/细胞(Geq/细胞)转染PHH,所述细胞在包含2%DMSO和4%PEG8000的PMM(PHH维持培养基,Gibco)中培养16-20小时。然后,将细胞用500μl的PBS洗涤3次,维持在PMM(2%DMSO)中,并且在转染之后培养7天。为了分析HBV蛋白表达,测量分泌的HBeAg和HBsA。利用HBeAg和HBsAg ELISA试剂盒(Wantai Pharm Inc.,Beijing,China)分析培养基中的HBeAg和HBsAg。未修饰的D2寡核苷酸用作阴性对照。LMV是拉米夫定。
作为HBV蛋白表达分析的结果,证实修饰的D2寡核苷酸具有很好的抗病毒作用,特别地,用PS-LNA修饰的D2寡核苷酸抑制病毒超过90%,表现出最强的抑制作用,如图13所示。这些结果证实当用修饰的D2寡核苷酸处理人细胞时表现出抗病毒作用。
4-4:D2寡核苷酸的分析
为了找到表现出最佳效果的修饰形式,修饰D2寡核苷酸末端的3个(3,3)、4个(4,4)或5个(5,5)核苷酸。为了分析,用HBV增强子I和II的结构转染HepG2细胞。转染之前,用多种修饰的D2(PS,PS-OMe(4,4),PS-OMe(5,5),PS-OMe(所有),PS-LNA(2,2),PS-LNA(3,3),PS-LNA(4,4),PS-LNA(5,5),PS-LNA(所有))预处理HepG2细胞(终浓度500nM)。第二天,用包含500nM修饰的D2寡核苷酸的新鲜培养基(DMEM)替换细胞。转染两天之后,利用萤光素酶测定系统(Promega;Madison,WI)根据方案测定HBV增强子的萤光素酶活性。
结果,如图14所示,证实修饰的D2寡核苷酸表现出优越的抗病毒作用。在它们中,其中用LNA修饰5'和3'两端4个核苷酸的PS-LNA(4,4)表现出最强的抗病毒作用。
4-5:证实修饰的寡核苷酸在HepG2细胞中的抗病毒作用
将1-8-2中制备的寡核苷酸与HBV一起插入HepG2细胞。以50nM的浓度使用58种寡核苷酸,并且与1μg的HBV一起添加至2ml培养基以转染细胞。第二天,用新鲜培养基(DMEM)替换培养基并将细胞培养72小时。随后,利用HBeAg和HBsAg ELISA试剂盒(Wantai PharmInc.,Beijing,China)分析培养基中的HBeAg和HBsAg。HBeAg和HBsAg的结果分别在图15和图16中示出。
结果,如图15所示,证实许多寡核苷酸抑制HBeAg。特别地,有效减少HepG2细胞中的HBeAg的物质是9、17、18、20、21、34、37、40、41、42、43、44、46、47、50、51、54、55、(3,3)和(4,4)。
此外,如图16所示,证实许多寡核苷酸抑制HBsAg。特别地,有效减少HepG2细胞中的HBsAg的物质是9、10、18、20、21、24、28、34、37、40、41、44、48和(3,3)。
4-6:证实修饰的寡核苷酸在HepG2-NTCP细胞中的作用
为了证实1-8-2中制备的寡核苷酸在寡核苷酸转染模型中的作用,使用HBV感染细胞HepG2-NTCP细胞。具体地,用2000HBV基因组当量/细胞(Geq/细胞)转染HBV,所述细胞在包含2%DMSO和4%PEG8000的PMM(PHH维持培养基,Gibco)中培养16-20小时。然后,将细胞用500μl的PBS洗涤3次,维持在PMM(2%DMSO)中,然后培养7天。从转染之后3天,将细胞用58种修饰的寡核苷酸每天处理。处理浓度是500nM。在转染之后第7天,通过测量分泌的HBeAg和HBsAg分析HBV蛋白表达。利用HBeAg和HBsAg ELISA试剂盒分析培养基中的HBeAg和HBsAg。HBeAg和HBsAg的结果分别在图17和图18中示出。
结果,如图17所示,证实许多寡核苷酸抑制HBeAg。特别地,有效减少HepG2-NTCP细胞中的HBeAg的物质是8、17、18、19、20、21、27、40、44、47、55、(3,3)和(4,4)。此外,如图18所示,证实许多寡核苷酸还抑制HBsAg。特别地,有效减少HepG2-NTCP细胞中的HBsAg的物质是7、8、9、18、19、20、40、42、44、45、(3,3)和(4,4)。
4-7:证实修饰的寡核苷酸在PHH(原代人肝细胞)细胞中的作用
为了证实1-8-2中制备的寡核苷酸是否在原代人肝细胞(PHH)中抑制HBV,在肝脏手术之后从患者的肝组织分离PHH并用HBV转染以证实58种修饰的寡核苷酸的抗病毒作用。具体地,PHH的HBV转染和病毒蛋白分析如下:用2000HBV基因组当量/细胞(Geq/细胞)转染HBV,所述细胞在包含2%DMSO和4%PEG8000的PMM(PHH维持培养基,Gibco)中培养16-20小时。然后,如HepG2-NTCP细胞,将PHH细胞用500μl的PBS洗涤3次,维持在PMM(2%DMSO)中,然后培养11天。从转染之后5天,将细胞用58种修饰的寡核苷酸每天处理。处理浓度是500nM。在转染之后第11天,通过测量分泌的HBeAg和HBsAg分析HBV蛋白表达。HBeAg和HBsAg的结果分别在图19和图20中示出。
结果,证实许多寡核苷酸抑制HBeAg。特别地,如图19所示,有效减少PHH细胞中的HBeAg的物质是7、8、18、19、20、52、(3,3)和(4,4)。此外,如图20所示,证实许多寡核苷酸还抑制HBsAg。特别地,有效减少PHH细胞中的HBsAg的物质是6、7、8、15、16、18、19、42、(3,3)和(4,4)。
作为HBV蛋白表达分析的结果,如各个结果所示证实修饰的D2寡核苷酸具有很好的抗病毒作用,特别地,在各种类型的gapmer中,用PS-LNA部分修饰的D2表现出最强的抑制作用。HBeAg和HBsAg抑制作用很好,特别是在未修饰的重复出现G的区域中,如(3,3)或(4,4)。这表明可以降低寡核苷酸合成所需要的成本,并且进一步地,证实当用部分或完全修饰的D2寡核苷酸处理人细胞时表现出抗病毒作用。
实施例5:体内模型
5-1:证实D2寡核苷酸在体内小鼠模型中抑制HBV
为了研究D2寡核苷酸是否在体内也发挥功能,利用HBV小鼠模型进行实验。根据图21(a)进行实验。将雄性6周龄小鼠用于每个组。注射PBS作为对照(Mock)。通过水动力注射方法注射(HI)的DNA如下:25μg的HBV-1.2mer、25μg的空载体或D2寡核苷酸以及5μg的b-gal。b-gal用作注射对照。将小鼠处死以获得血液样品。将小鼠血清用PBS稀释(对于HBeAg以1:50,而对于HBsAg以1:2000)。用ELISA试剂盒测量病毒蛋白(HBeAg和HBsAg)。
结果,如图21(b)和(c)所示,证实在用D2寡核苷酸注射的小鼠中表现出强抗病毒作用。此外,如图21(d)所示,利用Southern印迹证实在用D2寡核苷酸注射的小鼠中HBV DNA大大减少。
5-2:证实修饰的D2寡核苷酸在静脉内注射入体内小鼠模型时抑制HBV
为了研究修饰的D2寡核苷酸是否在注射时在体内发挥功能,利用HBV小鼠模型进行实验。根据图22(a)进行体内实验。将雄性6周龄小鼠用于每个组。仅用HBV注射的小鼠用作对照。通过水动力注射方法注射包含25μg的HBV-1.2mer和5μg的b-gal的质粒。然后,将50μg修饰的DNA(PS、PS-OMe和PS-LNA)静脉内注射3天。注射4天之后,将小鼠处死以获得血液样品。b-gal用作注射对照。将小鼠血清用PBS稀释(对于HBeAg以1:50,而对于HBsAg以1:2000)。用ELISA试剂盒测量病毒蛋白(HBeAg和HBsAg)。
结果,如图22(b)和(c)所示,在用修饰的D2寡核苷酸注射的小鼠中表现出强抗病毒作用。此外,如图22(d)所示,证实在HBV DNA水平也表现出抗病毒作用。这些结果证实当注射修饰的D2寡核苷酸时,它们递送至小鼠的肝并表现出病毒抑制作用。
5-3:证实D2寡核苷酸在用纳米颗粒(壳聚糖)包裹并静脉内注射入体内小鼠模型时抑制HBV
为了研究D2寡核苷酸是否在用纳米颗粒(壳聚糖)包裹并注射时在体内发挥功能,利用HBV小鼠模型进行实验。当D2寡核苷酸被纳米颗粒包裹时,它们有效递送至肝。根据图23(a)进行体内实验。将雄性6周龄小鼠用于每个组。通过水动力注射方法注射包含25μg的HBV-1.2mer和5μg的b-gal的质粒。然后,用HBV转染8μg纳米颗粒D2并静脉内注射一次。注射4天之后,将小鼠处死以获得血液样品。b-gal用作注射对照。将小鼠血清用PBS稀释(对于HBeAg以1:50,而对于HBsAg以1:2000)。用ELISA试剂盒测量病毒蛋白(HBeAg和HBsAg)。
结果,如图23(b)和(c)所示,在用纳米颗粒D2注射的小鼠中表现出抗病毒作用。在这里,第一条代表Mock,第二条代表HBV,第三条代表HBV和壳聚糖纳米颗粒D2,而第四条代表HBV和壳聚糖纳米颗粒D4。壳聚糖纳米颗粒D4用作完全不抑制HBV的阴性对照。如图23(d)所示,在HBV DNA水平也表现出抗病毒作用。这些结果证实当注射壳聚糖纳米颗粒D2时,其递送至小鼠的肝并强烈抑制病毒活性。
实施例6:PHH(原代人肝细胞)中HBV cccDNA的抑制
6-1:证实修饰的D2在从开始处理时抑制HBV
为了研究D2寡核苷酸是否从PHH去除HBV cccDNA,通过用HBV转染PHH进行实验。在这个实验中,从HBV转染之后第二天用修饰的D2寡核苷酸处理细胞,并且这个方法作为用HBV转染PHH的程序在图24(a)中计划。这时,IFN-α用作阳性对照,并且未修饰的D2寡核苷酸用作阴性对照,因为当用未修饰的D2寡核苷酸处理时它们完全不可以抑制HBV。
利用实时PCR定量进行实验,并且结果在图24(d)和(e)中示出。
结果,在图24(b)和(c)中证实当用PS-LNA(3,3)、PS-LNA(4,4)和PS-LNA(所有)部分修饰的D2寡核苷酸处理细胞时,HBeAg和HBsAg减少。此外,如图24(d)所示,在用PS-LNA(3,3)、PS-LNA(4,4)和PS-LNA(所有)部分修饰的D2寡核苷酸中有效减少HBV rcDNA。为了从根本上治疗HBV,必须去除cccDNA。在这方面,如图24(e)所示,当用PS-LNA(3,3)、PS-LNA(4,4)和PS-LNA(所有)部分修饰的D2寡核苷酸处理细胞时减少HBV cccDNA。
6-2:证实修饰的D2寡核苷酸甚至在产生足够的cccDNA时抑制HBV
为了研究D2寡核苷酸是否甚至在HBV转染之后5天的足够重新转染条件下从PHH去除HBV cccDNA,进行实验。在这个实验中,从HBV转染之后5天用修饰的D2寡核苷酸(0.5μM的PS-LNA(所有),1μM的PS-LNA(所有))处理细胞,并且这个方法作为用HBV转染PHH的程序在图25(a)中计划。这时,IFN-α用作抑制HBeAg、HBsAg、rcDNA和cccDNA的阳性对照,并且未修饰的D2寡核苷酸用作阴性对照,因为当用未修饰的D2寡核苷酸处理细胞时它们完全不可以抑制HBV。
结果,图25(b)和(c)显示当用不同浓度的修饰的D2寡核苷酸处理细胞时HBeAg和HBsAg减少。图25(d)通过在进行普通PCR之后进行DNA电泳证实HBV DNA和cccDNA量的差异。此外,如图25(d)所示,在部分修饰的D2寡核苷酸中HBV rcDNA和cccDNA以浓度依赖性方式减少。
实施例7:证实HepG2-NTCP中通过D2寡核苷酸和HBV cccDNA形成的G-四链体
为了研究用PS-LNA修饰的D2寡核苷酸是否有效识别HepG2-NTCP中的HBV cccDNA并形成G-四链体,用HBV转染NTCP细胞并用D2寡核苷酸处理,然后在显微镜下观察。在这个实验中,从HBV转染之后5天用修饰的D2寡核苷酸处理细胞,并且在第7天将细胞固定。然后,制备载玻片以显示红色信号,从而可以利用BG4抗体观察G-四链体。
结果,当用产生自NTCP中转染的HBV cccDNA和修饰的D2寡核苷酸处理细胞时,图26(a)通过识别G-四链体的BG4抗体证实D2寡核苷酸和cccDNA形成G-四链体。此外,通过证实HBeAg在HBV中正常表达,但是当用修饰D2处理时HBeAg水平降低,还检测到修饰的D2寡核苷酸的抗病毒作用。当检查图26(b)中示出的图和图26(a)底部总结的BG4的集落数量时,在Mock情况下,在正常细胞条件下鉴定了约5种内源形式的G-四链体信号。当用修饰的D2寡核苷酸单独处理细胞时鉴定了约6种信号。当用HBV单独处理细胞时鉴定了约7种信号。特别地,当用HBV和修饰的D2寡核苷酸处理细胞时鉴定了约16种信号。这些结果表明由于修饰的D2寡核苷酸,HBV cccDNA形成G-四链体。
序列表
<110> 阿姆科学有限公司
<120> 用于预防或治疗乙型肝炎的药物组合物
<130> X17E10C0283
<150> KR 10-2016-0169681
<151> 2016-12-13
<160> 77
<170> PatentIn version 3.2
<210> 1
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> D1
<400> 1
aagcctccaa gctgtgcctt gggtggct 28
<210> 2
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> D2
<400> 2
tgctgggggg aattga 16
<210> 3
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> D3
<400> 3
tgctgggtgg aattga 16
<210> 4
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> D4
<400> 4
actagacact atttaa 16
<210> 5
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> D5
<400> 5
cgttgatgcc tttgta 16
<210> 6
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> D6
<400> 6
ttctaggggg aactac 16
<210> 7
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> D7
<400> 7
gatgtggtat tggggg 16
<210> 8
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> D8
<400> 8
aggagttggg ggagga 16
<210> 9
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> D9
<400> 9
cataaggtgg ggaact 16
<210> 10
<211> 19
<212> DNA
<213> Artificial Sequence
<220>
<223> cccDNA primer_F
<400> 10
ctccccgtct gtgccttct 19
<210> 11
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> cccDNA primer_R
<400> 11
gccccaaagc cacccaag 18
<210> 12
<211> 19
<212> DNA
<213> Artificial sequence
<220>
<223> RC DNA primer_F
<400> 12
ctcgtggtgg acttctctc 19
<210> 13
<211> 18
<212> DNA
<213> Artificial sequence
<220>
<223> RC DNA primer_R
<400> 13
ctgcaggatg aagaggaa 18
<210> 14
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223> hybridization probe with cccDNA prime_F
<400> 14
gttcacggtg gtctccatgc aacgt 25
<210> 15
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223> hybridization probe with cccDNA primer_R
<400> 15
aggtgaagcg aagtgcacac ggacc 25
<210> 16
<211> 25
<212> DNA
<213> Artificial sequence
<220>
<223> hybridization probe with RC DNA primer_F
<400> 16
cactcaccaa cctcctgtcc tccaa 25
<210> 17
<211> 26
<212> DNA
<213> Artificial sequence
<220>
<223> hybridization probe with RC DNA primer_R
<400> 17
tgtcctggtt atcgctggat gtgtct 26
<210> 18
<211> 398
<212> DNA
<213> Artificial sequence
<220>
<223> enhancer I
<400> 18
aaattgcctg taaatagacc tattgattgg aaagtatgtc aaagaattgt gggtcttttg 60
ggctttgctg ccccttttac acaatgtggc tatcctgctt tgatgccttt atatgcatgt 120
atacaatcta agcaggcttt cactttctcg ccaacttaca aggcctttct gtgtaaacaa 180
tatctgcacc tttaccccgt tgcccggcaa cggtcaggtc tctgccaagt gtttgctgac 240
gcaaccccca ctggatgggg cttggccatt ggccatcggc gcatgcgtgg aacctttgtg 300
gctcctctgc cgatccatac cgcggaactc ctagcggctt gttttgctcg cagccggtct 360
ggagcgaaac ttatcgggac tgacaactct gttgtcct 398
<210> 19
<211> 212
<212> DNA
<213> Artificial sequence
<220>
<223> enhancer II
<400> 19
cgcttcacct ctgcacgtcg catggagacc accgtgaacg cccaccaggt cttgcccaag 60
gtcttacata agaggactct tggactctca gcaatgtcaa cgaccgacct tgaggcatac 120
ttcaaagact gtttgtttaa agactgggag gagttggggg aggagattag gttaaaggtc 180
tttgtattag gaggctgtag gcataaattg gt 212
<210> 20
<211> 16
<212> DNA
<213> Artificial Sequence
<220>
<223> Oligo modification#1
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a 2'OMe thymidine wherein phosphate of T is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a 2'OMe cytidine.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (5)..(5)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (6)..(7)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (8)..(10)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a 2'OMe adenosine.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a 2'OMe adenosine wherein phosphate of A is a
modified as phosphorothioate.
<400> 20
tgctgggggg aattga 16
<210> 21
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#2
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a 2'OMe thymidine wherein phosphate of T is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a 2'OMe cytidine.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (5)..(8)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (9)..(10)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a 2'OMe adenosine.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a 2'OMe adenosine wherein phosphate of A is a
modified as phosphorothioate.
<400> 21
tgctgggggg aattga 16
<210> 22
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#3
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a 2'OMe thymidine wherein phosphate of T is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a 2'OMe cytidine.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a 2'OMe adenosine.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a 2'OMe adenosine wherein phosphate of A is a
modified as phosphorothioate.
<400> 22
tgctgggggg aattga 16
<210> 23
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#4
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a 2'OMe thymidine wherein phosphate of T is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a 2'OMe cytidine.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(11)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (12)..(12)
<223> A represents a 2'OMe adenosin.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a 2'OMe adenosine wherein phosphate of A is a
modified as phosphorothioate.
<400> 23
tgctgggggg aattga 16
<210> 24
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#5
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a 2'OMe thymidine wherein phosphate of T is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a 2'OMe cytidine.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(13)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (14)..(14)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a 2'OMe adenosine wherein phosphate of A is a
modified as phosphorothioate.
<400> 24
tgctgggggg aattga 16
<210> 25
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#6
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a 2'OMe thymidine wherein phosphate of T is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a 2'OMe adenosine wherein phosphate of A is a
modified as phosphorothioate.
<400> 25
tgctgggggg aattga 16
<210> 26
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#7
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a 2'OMe cytidine.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a 2'OMe adenosine.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 26
tgctgggggg aattga 16
<210> 27
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#8
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (11)..(11)
<223> A represents a 2'OMe adenosine.
<220>
<221> misc_feature
<222> (12)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 27
tgctgggggg aattga 16
<210> 28
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#9
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 28
tgctgggggg aattga 16
<210> 29
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#10
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(5)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (6)..(9)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (10)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 29
tgctgggggg aattga 16
<210> 30
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#11
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic aci.
<220>
<221> misc_feature
<222> (5)..(6)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (7)..(8)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (9)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 30
tgctgggggg aattga 16
<210> 31
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#12
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine wherein phosphate of T is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (7)..(8)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<400> 31
tgctgggggg aattga 16
<210> 32
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#13
<220>
<221> misc_feature
<222> (6)..(9)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(1)
<223> T represents a thymidine wherein phosphate of T is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<400> 32
tgctgggggg aattga 16
<210> 33
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#14
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine wherein phosphate of T is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<400> 33
tgctgggggg aattga 16
<210> 34
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#15
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine wherein phosphate of T is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(11)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<400> 34
tgctgggggg aattga 16
<210> 35
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#16
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine wherein phosphate of T is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(13)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<400> 35
tgctgggggg aattga 16
<210> 36
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#17
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine wherein phosphate of T is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<400> 36
tgctgggggg aattga 16
<210> 37
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#18
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 37
tgctgggggg aattga 16
<210> 38
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#19
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (12)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 38
tgctgggggg aattga 16
<210> 39
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#20
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 39
tgctgggggg aattga 16
<210> 40
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#21
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(5)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (10)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 40
tgctgggggg aattga 16
<210> 41
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#22
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(6)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (9)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 41
tgctgggggg aattga 16
<210> 42
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#23
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (5)..(5)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (6)..(6)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (7)..(7)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (8)..(8)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (9)..(9)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (10)..(10)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (11)..(11)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (12)..(12)
<223> A represents a 2'OMe adenosine.
<220>
<221> misc_feature
<222> (13)..(13)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (14)..(14)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a 2'OMe adenosine wherein phosphate of A is modified
as phosphorothioate.
<400> 42
tgctgggggg aattga 16
<210> 43
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#24
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a 2'OMe thymidine wherein phosphate of T is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a 2'OMe cytidine.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(5)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (6)..(6)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (7)..(7)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (8)..(8)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (9)..(9)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (10)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(11)
<223> A represents a 2'OMe adenosine.
<220>
<221> misc_feature
<222> (12)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(13)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (14)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 43
tgctgggggg aattga 16
<210> 44
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#25
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(5)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (7)..(7)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (9)..(9)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(11)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(13)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<400> 44
tgctgggggg aattga 16
<210> 45
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#26
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine wherein phosphate of T is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (6)..(6)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (8)..(8)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (10)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (12)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (14)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 45
tgctgggggg aattga 16
<210> 46
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#27
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a 2'OMe thymidine wherein phosphate of T is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a 2'OMe cytidine.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 46
tgctgggggg aattga 16
<210> 47
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#28
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a 2'OMe adenosine.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a 2'OMe adenosine wherein phosphate of A is modified
as phosphorothioate.
<400> 47
tgctgggggg aattga 16
<210> 48
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#29
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine wherein phosphate of T is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 48
tgctgggggg aattga 16
<210> 49
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#30
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<400> 49
tgctgggggg aattga 16
<210> 50
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#31
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a 2'OMe thymidine wherein phosphate of T is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a 2'OMe cytidine.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<400> 50
tgctgggggg aattga 16
<210> 51
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#32
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine wherein phosphate of T is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a 2'OMe adenosine.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a 2'OMe adenosine wherein phosphate of A is modified
as phosphorothioate.
<400> 51
tgctgggggg aattga 16
<210> 52
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#33
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a 2'OMe thymidine wherein phosphate of T is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a 2'OMe cytidine.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<400> 52
tgctgggggg aattga 16
<210> 53
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#34
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a 2'OMe thymidine wherein phosphate of T is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a 2'OMe cytidine.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 53
tgctgggggg aattga 16
<210> 54
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#35
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine wherein phosphate of T is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a 2'OMe adenosine.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a 2'OMe adenosine wherein phosphate of A is modified
as phosphorothioate.
<400> 54
tgctgggggg aattga 16
<210> 55
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#36
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine wherein phosphate of T is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 55
tgctgggggg aattga 16
<210> 56
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#37
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<400> 56
tgctgggggg aattga 16
<210> 57
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#38
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a 2'OMe adenosine.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a 2'OMe adenosine wherein phosphate of A is modified
as phosphorothioate.
<400> 57
tgctgggggg aattga 16
<210> 58
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#39
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 58
tgctgggggg aattga 16
<210> 59
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#40
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 59
tgctgggggg aattga 16
<210> 60
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#41
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a 2'OMe thymidine wherein phosphate of T is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a 2'OMe cytidine.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a 2'OMe adenosine.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a 2'OMe adenosine wherein phosphate of A is modified
as phosphorothioate.
<400> 60
tgctgggggg aattga 16
<210> 61
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#42
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 61
tgctgggggg aattga 16
<210> 62
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#43
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine wherein phosphate of T is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine wherein phosphate of T is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(11)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<400> 62
tgctgggggg aattga 16
<210> 63
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#44
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (11)..(11)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (12)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 63
tgctgggggg aattga 16
<210> 64
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#45
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a 2'OMe thymidine wherein phosphate of T is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a 2'OMe cytidine.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a 2'OMe thymidine wherein phosphate of T is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(11)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<400> 64
tgctgggggg aattga 16
<210> 65
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#16
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a 2'OMe thymidine wherein phosphate of T is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a 2'OMe cytidine.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a 2'OMe thymidine wherein phosphate of T is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (11)..(11)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (12)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 65
tgctgggggg aattga 16
<210> 66
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#47
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine wherein phosphate of T is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine wherein phosphate of T is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(11)
<223> A represents a 2'OMe adenosine wherein phosphate of A is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (12)..(12)
<223> A represents a 2'OMe adenosine.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a 2'OMe adenosine wherein phosphate of A is modified
as phosphorothioate.
<400> 66
tgctgggggg aattga 16
<210> 67
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#48
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine wherein phosphate of T is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine wherein phosphate of T is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (11)..(11)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (12)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 67
tgctgggggg aattga 16
<210> 68
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#49
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (11)..(11)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<400> 68
tgctgggggg aattga 16
<210> 69
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#50
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a 2'OMe adenosine wherein phosphate of A is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (13)..(14)
<223> T represents a 2'OMe thymidine.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a 2'OMe adenosine wherein phosphate of A is modified
as phosphorothioate.
<400> 69
tgctgggggg aattga 16
<210> 70
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#51
<220>
<221> misc_feature
<222> (5)..(5)
<223> G represents a guanosine of locked nucleic acid wherein phosphate
of G is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (6)..(9)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (10)..(10)
<223> G represents a guanosine of locked nucleic acid wherein phosphate
of G is modified as phosphorothioate.
<400> 70
tgctgggggg aattga 16
<210> 71
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#52
<220>
<221> misc_feature
<222> (1)..(1)
<223> u represents a 2'OMe uridine wherein phosphate of u is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a 2'OMe cytidine.
<220>
<221> misc_feature
<222> (4)..(4)
<223> u represents a 2'OMe uridine.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a 2'OMe adenosine.
<220>
<221> misc_feature
<222> (14)..(14)
<223> u represents a 2'OMe uridine.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a 2'OMe adenosine wherein phosphate of A is modified
as phosphorothioate.
<400> 71
ugcugggggg aauuga 16
<210> 72
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#53
<220>
<221> misc_feature
<222> (1)..(1)
<223> u represents a uridine wherein phosphate of u is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (7)..(8)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 72
ugcugggggg aauuga 16
<210> 73
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#54
<220>
<221> misc_feature
<222> (1)..(1)
<223> u represents a uridine of locked nucleic acid wherein phosphate
of u is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> u represents a 2'OMe uridine.
<220>
<221> misc_feature
<222> (5)..(5)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (6)..(6)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (7)..(7)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (8)..(8)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (9)..(9)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (10)..(10)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (11)..(11)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (12)..(12)
<223> A represents a 2'OMe adenosine.
<220>
<221> misc_feature
<222> (13)..(13)
<223> u represents a uridine of locked nucleic acid.
<220>
<221> misc_feature
<222> (14)..(14)
<223> u represents a 2'OMe uridine.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 73
ugcugggggg aauuga 16
<210> 74
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#55
<220>
<221> misc_feature
<222> (1)..(1)
<223> u represents a uridine of locked nucleic acid wherein phosphate
of u is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> u represents a uridine of locked nucleic acid.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(12)
<223> A represents a adenosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (13)..(14)
<223> u represents a uridine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 74
ugcugggggg aauuga 16
<210> 75
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#56
<220>
<221> misc_feature
<222> (1)..(1)
<223> u represents a 2'OMe uridine wherein phosphate of u is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a 2'OMe guanosine.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a 2'OMe cytidine.
<220>
<221> misc_feature
<222> (4)..(4)
<223> u represents a 2'OMe uridine wherein phosphate of u is modified
as phosphorothioate.
<220>
<221> misc_feature
<222> (5)..(10)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (11)..(11)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine wherein phosphate of A is modified as
phosphorothioate.
<400> 75
ugcugggggg aauuga 16
<210> 76
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#(3,3)
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid wherein phosphate
of C is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (14)..(14)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 76
tgctgggggg aattga 16
<210> 77
<211> 16
<212> DNA
<213> Artificial sequence
<220>
<223> Oligo modification#(4,4)
<220>
<221> misc_feature
<222> (1)..(1)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (2)..(2)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (3)..(3)
<223> C represents a cytidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (4)..(4)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (13)..(13)
<223> T represents a thymidine of locked nucleic acid wherein phosphate
of T is modified as phosphorothioate.
<220>
<221> misc_feature
<222> (14)..(14)
<223> T represents a thymidine of locked nucleic acid.
<220>
<221> misc_feature
<222> (15)..(15)
<223> G represents a guanosine of locked nucleic acid.
<220>
<221> misc_feature
<222> (16)..(16)
<223> A represents a adenosine of locked nucleic acid wherein phosphate
of A is modified as phosphorothioate.
<400> 77
tgctgggggg aattga 16
Claims (39)
1.一种用于治疗或预防乙型肝炎的药物组合物,作为活性成分,其包含:
至少一种选自以下的寡核苷酸:SEQ ID NO:1、2或6的核酸序列或者其互补核酸序列表示的寡核苷酸;以及
在寡核苷酸上具有至少一个化学修饰的寡核苷酸。
2.权利要求1的组合物,其中具有化学修饰的寡核苷酸具有至少一个化学修饰的核苷间连接。
3.权利要求2的组合物,其中具有化学修饰的核苷间连接的寡核苷酸具有这样的化学修饰,其中用硫代磷酸、二硫代磷酸、磷酰胺或硼烷磷酸化学修饰核苷酸的磷酸基团。
4.权利要求1的组合物,其中具有化学修饰的寡核苷酸具有至少一个化学修饰的糖部分。
5.权利要求4的组合物,其中修饰所述糖部分,从而用甲氧基乙基(MOE)、二甲基氨基氧基乙氧基(DMAOE)、二甲基氨基乙氧基乙基(DMAEOE)、甲基(OMe)、氨基丙氧基(AP)或氟(F)取代核苷酸中戊糖的2'位置处的-H基团,或者
用F-ANA取代所述糖部分。
6.权利要求4的组合物,其中所述糖部分以LNA(锁核酸)或PNA(肽核酸)的形式化学修饰。
7.权利要求1的组合物,其中所述寡核苷酸是这样的状态,其中GalNAc(N-乙酰半乳糖胺)通过接头结合至3'或5'端。
8.权利要求1的组合物,其中具有化学修饰的寡核苷酸具有两个或更多个选自以下的化学修饰:核苷间连接的化学修饰和糖部分的化学修饰。
9.权利要求8的组合物,其中具有两个或更多个化学修饰的寡核苷酸具有这样的化学修饰,其中用硫代磷酸、二硫代磷酸、磷酰胺或硼烷磷酸化学修饰核苷酸的磷酸基团,并且
进一步具有这样的化学修饰,其中用甲氧基乙基(MOE)、二甲基氨基氧基乙氧基(DMAOE)、二甲基氨基乙氧基乙基(DMAEOE)、甲基(OMe)、氨基丙氧基(AP)或氟(F)取代核苷酸中戊糖的2'位置处的-H基团,或者用F-ANA取代核苷酸的糖部分。
10.权利要求8的组合物,其中具有两个或更多个化学修饰的寡核苷酸具有这样的化学修饰,其中用硫代磷酸、二硫代磷酸、磷酰胺或硼烷磷酸化学修饰核苷酸的磷酸基团,并且
进一步具有这样的化学修饰,其中所述糖部分以LNA(锁核酸)或PNA(肽核酸)的形式化学修饰。
11.权利要求1的组合物,其中所述寡核苷酸与HBV形成G-四链体。
12.权利要求1的组合物,其减少HBV的cccDNA(共价闭合环状DNA)。
13.权利要求1的组合物,其进一步包含药学可接受的载体。
14.权利要求13的组合物,其中所述药学可接受的载体包含壳聚糖纳米颗粒、胶体分散系统、大分子复合物、纳米胶囊、纳米颗粒、微球、珠和水包油乳剂、胶束、混合胶束或脂质体。
15.权利要求14的组合物,其中所述药学可接受的载体是壳聚糖纳米颗粒,并且所述壳聚糖具有50kDa-190kDa的分子量。
16.权利要求1的组合物,其经由口服或肠胃外给药至个体。
17.权利要求1的组合物,其经由腹腔内、静脉内、经皮、舌下、肌肉内、鼻内或皮下给药至个体。
18.一种用于治疗或预防乙型肝炎的方法,包括:
向个体给药有效剂量的用于治疗或预防乙型肝炎的药物组合物,所述药物组合物,作为活性成分,包含至少一种选自以下的寡核苷酸:SEQ ID NO:1、2或6的核酸序列或者其互补核酸序列表示的寡核苷酸;以及在寡核苷酸上具有至少一个化学修饰的寡核苷酸。
19.权利要求18的方法,其中具有化学修饰的寡核苷酸具有至少一个化学修饰的核苷间连接。
20.权利要求19的方法,其中具有化学修饰的核苷间连接的寡核苷酸具有这样的化学修饰,其中用硫代磷酸、二硫代磷酸、磷酰胺或硼烷磷酸化学修饰核苷酸的磷酸基团。
21.权利要求18的方法,其中具有化学修饰的寡核苷酸具有至少一个化学修饰的糖部分。
22.权利要求21的方法,其中修饰所述糖部分,从而用甲氧基乙基(MOE)、二甲基氨基氧基乙氧基(DMAOE)、二甲基氨基乙氧基乙基(DMAEOE)、甲基(OMe)、氨基丙氧基(AP)或氟(F)取代核苷酸中戊糖的2'位置处的-H基团,或者
用F-ANA取代所述糖部分。
23.权利要求21的方法,其中所述糖部分以LNA(锁核酸)或PNA(肽核酸)的形式化学修饰。
24.权利要求18的方法,其中所述寡核苷酸是这样的状态,其中GalNAc(N-乙酰半乳糖胺)通过接头结合至3'或5'端。
25.权利要求18的方法,其中具有化学修饰的寡核苷酸具有两个或更多个选自以下的化学修饰:核苷间连接的化学修饰和糖部分的化学修饰。
26.权利要求25的方法,其中具有两个或更多个化学修饰的寡核苷酸具有这样的化学修饰,其中用硫代磷酸、二硫代磷酸、磷酰胺或硼烷磷酸化学修饰核苷酸的磷酸基团,并且
进一步具有这样的化学修饰,其中用甲氧基乙基(MOE)、二甲基氨基氧基乙氧基(DMAOE)、二甲基氨基乙氧基乙基(DMAEOE)、甲基(OMe)、氨基丙氧基(AP)或氟(F)取代核苷酸中戊糖的2'位置处的-H基团,或者用F-ANA取代核苷酸的糖部分。
27.权利要求25的方法,其中具有两个或更多个化学修饰的寡核苷酸具有这样的化学修饰,其中用硫代磷酸、二硫代磷酸、磷酰胺或硼烷磷酸化学修饰核苷酸的磷酸基团,并且
进一步具有这样的化学修饰,其中所述糖部分以LNA(锁核酸)或PNA(肽核酸)的形式化学修饰。
28.权利要求18的方法,其中所述寡核苷酸与HBV形成G-四链体。
29.权利要求18的方法,用于治疗或预防乙型肝炎的组合物减少HBV的cccDNA(共价闭合环状DNA)。
30.权利要求18的方法,其中用于治疗或预防乙型肝炎的组合物进一步包含药学可接受的载体。
31.权利要求30的方法,其中所述药学可接受的载体包含壳聚糖纳米颗粒、胶体分散系统、聚合物复合物、纳米胶囊、纳米颗粒、微球、珠、水包油乳剂、胶束、混合胶束或脂质体。
32.权利要求31的方法,其中所述药学可接受的载体是壳聚糖纳米颗粒,并且所述壳聚糖具有50kDa-190kDa的分子量。
33.权利要求18的方法,包括向个体口服或肠胃外给药用于治疗或预防乙型肝炎的组合物。
34.权利要求18的方法,向个体腹腔内、静脉内、经皮、舌下、肌肉内、鼻内或皮下给药用于治疗或预防乙型肝炎的组合物。
35.一种筛选乙型肝炎的治疗剂的方法,包括:
使乙型肝炎病毒(HBV)与候选材料接触并证实HBV是否与候选材料形成G-四链体。
36.权利要求35的方法,进一步包括:如果HBV与候选材料形成G-四链体,则选择所述候选材料作为乙型肝炎的治疗剂。
37.权利要求35的方法,其中通过选自以下的方法证实G-四链体的形成:电泳迁移率变动分析(EMSA)、圆二色性(CD)、核磁共振(NMR)和利用G-四链体特异性抗体的方法。
38.权利要求35的方法,其中所述候选材料包含4个或更多个鸟嘌呤(G)。
39.权利要求35的方法,其中通过使HBV的具有SEQ ID NO:19的核酸序列的增强子II区与候选材料结合形成G-四链体。
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WO2019240504A1 (en) * | 2018-06-12 | 2019-12-19 | Am Sciences Co., Ltd. | Modified oligonucleotides for inhibition of target gene expression |
CN114829599A (zh) * | 2019-12-19 | 2022-07-29 | 豪夫迈·罗氏有限公司 | Scamp3抑制剂用于治疗乙型肝炎病毒感染的用途 |
TW202223089A (zh) * | 2020-07-27 | 2022-06-16 | 美商艾利格斯醫療公司 | 與hbv結合之寡核苷酸及使用方法 |
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CN1580070A (zh) * | 2003-08-06 | 2005-02-16 | 北京三诺佳邑生物技术有限责任公司 | 一组抗hbv感染及防治乙型肝炎的核苷酸序列及其应用 |
CN103582648A (zh) * | 2011-04-21 | 2014-02-12 | Isis制药公司 | 乙型肝炎病毒(hbv)表达的调节 |
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US20030206887A1 (en) | 1992-05-14 | 2003-11-06 | David Morrissey | RNA interference mediated inhibition of hepatitis B virus (HBV) using short interfering nucleic acid (siNA) |
US20040127446A1 (en) * | 1992-05-14 | 2004-07-01 | Lawrence Blatt | Oligonucleotide mediated inhibition of hepatitis B virus and hepatitis C virus replication |
AU6056799A (en) * | 1998-09-21 | 2000-04-10 | University Of Massachusetts | Hepatitis b core antigen nucleic acid vaccine |
WO2011145885A2 (ko) | 2010-05-18 | 2011-11-24 | 연세대학교 산학협력단 | B형 간염 치료활성 물질의 스크리닝용 조성물 및 스크리닝 방법 |
EP2468866A1 (en) | 2010-12-21 | 2012-06-27 | Index Pharmaceuticals AB | Biologically active oligonucleotides capable of modulating the immune system |
CN103403158B (zh) | 2011-08-11 | 2015-01-28 | 松下电器产业株式会社 | 检测g-四联体螺旋形成的方法 |
ITMI20120275A1 (it) | 2012-02-24 | 2013-08-25 | Biogenera Societa A Responsabilita Limitata | Oligonucleotidi per la modulazione dell'espressione genica e loro usi |
JP6431478B2 (ja) | 2012-06-01 | 2018-11-28 | ドレクセル ユニバーシティ | B型肝炎ウイルスのcccdnaの転写の調節 |
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- 2017-12-13 EP EP17881424.0A patent/EP3556402A4/en active Pending
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- 2017-12-13 CA CA3047076A patent/CA3047076A1/en not_active Abandoned
- 2017-12-13 WO PCT/KR2017/014662 patent/WO2018110980A1/ko unknown
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Patent Citations (3)
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CN1580070A (zh) * | 2003-08-06 | 2005-02-16 | 北京三诺佳邑生物技术有限责任公司 | 一组抗hbv感染及防治乙型肝炎的核苷酸序列及其应用 |
CN103582648A (zh) * | 2011-04-21 | 2014-02-12 | Isis制药公司 | 乙型肝炎病毒(hbv)表达的调节 |
US20150176007A1 (en) * | 2013-05-01 | 2015-06-25 | Isis Pharmaceuticals, Inc. | Compositions and methods for modulating hbv expression |
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EP3556402A1 (en) | 2019-10-23 |
RU2019120163A3 (zh) | 2021-01-15 |
WO2018110980A1 (ko) | 2018-06-21 |
AU2017375819A1 (en) | 2019-07-25 |
AU2017375819B2 (en) | 2021-01-28 |
US11033571B2 (en) | 2021-06-15 |
JP6811338B2 (ja) | 2021-01-13 |
KR20180068320A (ko) | 2018-06-21 |
US20190343864A1 (en) | 2019-11-14 |
JP2020513421A (ja) | 2020-05-14 |
CA3047076A1 (en) | 2018-06-21 |
EP3556402A4 (en) | 2020-08-05 |
KR101954130B1 (ko) | 2019-03-05 |
MX2019006803A (es) | 2019-11-18 |
RU2019120163A (ru) | 2021-01-15 |
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