CN117535287A - 乙型肝炎病毒基因的小干扰核酸及其用途 - Google Patents
乙型肝炎病毒基因的小干扰核酸及其用途 Download PDFInfo
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Abstract
本发明涉及一种靶向乙型肝炎病毒基因的小干扰核酸(siRNA)及其用途。此外,本发明涉及一种包含所述小干扰核酸的缀合物,特别是GalNAc‑siRNA缀合物。本发明所合成的siRNA和缀合物(GalNAc‑siRNA)对HBV mRNA有明显的沉默作用。本发明根据HBV序列信息,通过设计特异性siRNA序列,靶向HBV mRNA,有效的“破坏”病毒复制环节中产生的mRNA,阻断病毒生命周期,大幅度降低机体病毒抗原的水平,给机体免疫力恢复以足够的空间,有望治疗HBV感染者。
Description
技术领域
本发明属于生物工程领域,具体涉及一种乙型肝炎病毒(HBV)基因的小干扰核酸及其用途。
技术背景
乙型病毒性肝炎简称乙型肝炎,是由乙型肝炎病毒(Hepatitis B virus,HBV)引起的传染性疾病。HBV是一种嗜肝病毒,主要存在于肝细胞内并损害肝细胞,引起肝细胞炎症、坏死、纤维化。据2016年世界卫生组织报道,全球约有3.5亿人为慢性HBV感染者,每年约有65万人死于慢性HBV感染导致的肝硬化或肝癌。我国是感染HBV人数最多的国家,约有1.2亿乙肝病毒携带者,慢性患者达3000万,每年新发病人约50万,有近25万人死于乙型肝炎相关疾病。每年因乙型肝炎造成的直接医疗费用约为300亿人民币,造成的国民经济直接损失约为3600亿。因此,乙型肝炎不仅危害我国人民的身体健康,而且还为国家带来严重的社会经济负担。
HBV病毒属于DNA病毒,由3200bp组成,为部分双链环状DNA。电子显微镜下可呈3种形态的颗粒结构:直径约42nm的大球形颗粒、直径约22nm的小球形颗粒及管型颗粒。大球形颗粒为完整的病毒颗粒,有感染性,由包膜和核衣壳组成。包膜含表面抗原(HBsAg)、糖蛋白和细胞脂肪,核心颗粒含核心蛋白(HBcAg)、环状双股HBV-DNA和HBV-DNA多聚酶。小球形颗粒及管型颗粒均由与病毒包膜相同的脂蛋白组成,前者主要由HBsAg形成中空颗粒,不含DNA和DNA多聚酶,不具传染性;后者是小球形颗粒串联聚合而成,成分与小球形颗粒相同,也不具有传染性。
HBV通过低亲和力受体(如硫酸乙酰肝素、蛋白多糖等),黏附到肝细胞表面,再通过包膜蛋白与病毒受体(NTCP受体)结合介导细胞对病毒的内吞。在内吞体,病毒包膜和内吞体膜融合将核衣壳释放入细胞质,核衣壳被运送至细胞核的核孔复合体,核衣壳内部的病毒基因组rcDNA释放入细胞核,在细胞核内,rcDNA转化成共价闭合环状DNA(cccDNA)。cccDNA有高度的稳定性,在细胞核内可以维持数月至数年,这是抗病毒治疗结束后病毒反弹的根本原因,因此清除cccDNA对根治乙型肝炎具有决定性意义。
在细胞内HBV复制过程可分6个环节,①病毒进入肝细胞浆,其核衣壳被裂解而形成松弛型环状DNA(rcDNA),后者进入肝细胞核,并在病毒DNA聚合酶和宿主酶的作用下,修复rcDNA成为cccDNA;②以cccDNA为模板,在宿主细胞RNA聚合酶作用下,转录成4种不同长度的病毒信使RNA(mRNA)和前基因组RNA(pgRNA),翻译HBV的各种蛋白;③病毒聚合酶和衣壳化信号共同作用于pgRNA,启动逆转录和核衣壳组装;④通过逆转录,合成病毒负链DNA,同时病毒聚合酶消化病毒RNA模板;⑤病毒负链DNA合成后,剩余的RNA作为引物,启动正链DNA合成;⑥完成正链DNA合成,形成rcDNA,同时核衣壳被包被上外膜并作为感染性病毒体分泌至细胞外;或再回到同一个肝细胞核内,扩增或维持cccDNA库。
针对HBV进入细胞以及自身生命活动的各个环节,抗乙肝病毒药物研发可分为两个方向:一是直接干扰病毒复制的某一环节(DAAs),二是通过修饰宿主细胞功能以抑制病毒复制,即宿主靶向制剂。目前临床上常用的抗病毒药物有干扰素类和核苷(酸)类似物两大类。干扰素并不直接杀伤或抑制病毒,主要通过细胞表面受体作用使细胞产生抗病毒蛋白,从而抑制乙肝病毒的复制;同时还可增强自然杀伤细胞(NK细胞)、巨噬细胞和T淋巴细胞的活力,从而起到免疫调节作用,并增强抗病毒能力。代表药物有聚乙二醇干扰素α-2a(派罗欣)、聚乙二醇干扰素α-2b(佩乐能)和普通干扰素;核苷(酸)类似物通过抑制病毒DNA多聚酶和逆转录酶的活性,同时竞争性抑制核苷酸进入病毒DNA链,终止病毒DNA链的延长,干扰病毒DNA的合成,从而发挥抗病毒作用。代表药物有拉米夫定、阿德福韦酯、恩替卡韦、替比夫定、Levovir(Clevudine)、Besivo(besifovir)、富马酸替诺福韦二吡呋酯(TDF)、替诺福韦艾拉酚胺(TAF)。
然而上述两大类药物均只能通过抑制病毒复制来延缓病情,治愈率非常有限,无法实现大范围的功能治愈,且多数使用口服药物的患者需要终生用药。部分患者在现有药物治疗过程中出现耐药。研究人员认为新型作用机制药物或成为唯一选择,因此,开发新型治疗方法毫无疑义成为当前迫切需求。
发明内容
RNA干扰是生物界普遍存在的一种现象,是由双链RNA(dsRNA)分子在mRNA水平关闭相应基因的表达或使该基因沉默的过程。该现象广泛存在于动物、植物等真核生物细胞内。外源性或内源性的sdRNA被RNAseIII核酶家族的Dicer剪切成siRNA,随后siRNA与诱导复合体RISC结合,解旋成单链,引导链特异性的结合到靶标mRNA上,切割mRNA,引发其被Argonaute蛋白特异性分解,从而达到基因沉默。从理论上讲,其可以通过序列特异性的方式抑制或阻断任何感兴趣的目的基因的表达,从而达到治疗疾病的目的。
为了解决上述至少一种现有技术存在的问题,本发明在于根据HBV序列信息,设计合适的小干扰RNA(siRNA)序列在细胞内HBV复制过程的6个环节中,有效的“破坏”复制环节中产生的mRNA,阻止其继续表达为病毒蛋白,则能大幅度降低机体病毒抗原的水平,给机体免疫力恢复以足够的空间。
本发明的目的在于提供包含siRNA的核酸分子以抑制与乙型肝炎病毒相关的基因的表达。
具体的,本发明的技术方案如下:
本发明的第一个方面提供了一种乙型肝炎病毒基因的小干扰核酸(siRNA),其特征在于,所述小干扰核酸的序列为9组序列中的至少一组,所述9组序列分别如SEQ ID NO:1-2、SEQ ID NO:3-4、SEQ ID NO:5-6、SEQ ID NO:7-8、SEQ ID NO:9-10、SEQ ID NO:11-12、SEQ ID NO:13-14、SEQ ID NO:15-16、SEQ ID NO:17-18所示。其中,每一组序列分别为正义链和反义链。
进一步的,本发明的小干扰核酸的序列与上述所示的9组序列中的其中一组具有85%以上同源性。
本发明的第二个方面提供了一种缀合物,该缀合物包含本发明所述的乙型肝炎病毒基因的小干扰核酸(siRNA)。
优选地,缀合物中的靶向性配体通过连接基团与siRNA正义链5’末端连接。
更优选地,所述缀合物中的靶向性配体通过硫代磷酸酯键与siRNA正义链5’末端连接。
在一个实施方案中,所述缀合物的为GalNAc-siRNA。
进一步的实施方式中,所述缀合物为下述中的一种:
本发明的第三个方面提供了本发明所述的小干扰核酸siRNA或缀合物在制备治疗乙型肝炎药物中的应用。
本发明的第四个方面提供了一种用于乙型肝炎的药物组合物,该药物组合物包括本发明所述乙型肝炎病毒基因的小干扰核酸。
本发明的第四个方面提供了一种用于乙型肝炎的药物组合物,其特征在于,该药物组合物包括本发明所述的缀合物。
与现有技术相比,本发明至少具有以下区别技术特征:
本发明于根据HBV序列信息,通过设计特异性小干扰RNA序列(siRNA),靶向HBV基因组,有效的“破坏”复制环节中产生的mRNA,阻止其继续表达为病毒蛋白,则能大幅度降低机体病毒抗原的水平,给机体免疫力恢复以足够的空间,有望治疗HBV感染者。
附图说明
图1显示合成反应1-17;
图2显示NPD007s-1在人血浆及大鼠溶酶体中稳定性测试结果;
图3显示NPD007s-1在Hep3B细胞上对HBV mRNA具有明显的抑制作用,其中,EC50为0.44nM;
图4显示NPD007s-1在靶效应显著,且没有脱靶现象;
图5显示NPD007s-1在Hep2.2.15细胞上对HBsAg有较高的抑制效果,EC50为0.12nM,且没有观察到细胞毒性;
图6显示在转基因小鼠上,NPD007s-1在5mpk时,显著抑制HBsAg的表达,且具有长效性;
图7显示在转基因小鼠上,NPD007s-1在5mpk时,显著抑制HBeAg的表达,且具有长效性;
图8显示在转基因小鼠上,NPD007s-1在5mpk时,显著沉默HBV DNA的表达,且具有长效性。
具体实施方式
下面通过详细的实施例对本申请进行进一步的阐述,应该理解,下述实施例仅是为了用于说明本申请,并不对发明内容进行限定。
实施例中所用仪器、设备、试剂均可通过各种渠道获得,例如购买得到,或可以制备而得。
实施例1:HBV基因小核酸序列的设计
根据HBV的基因组成,查找能够影响HBV复制的基因靶点(S/C/P/X基因区),以X和S区为目的区域,进行小核酸序列设计。
利用设计软件,针对HBV靶基因S/C/P/X四个基因区序列信息,按照siRNA设计的指导原则进行siRNA设计,每个区域设计10条siRNA序列。设计原则为:(1)GC含量在35%—55%之间,(2)避免处于重复序列或低复杂性序列区域内(3)避免出现4个以上连续碱基序列(4)避免含有单核苷酸多态性位点(5)避免处于读码框起始密码和终止密码的50—100bp内。除此之外,还要分析核苷酸序列的组成和热力学性质。通过BLAST分析,设计的HBVsiRNA不能与人类基因序列有很大的同源性(16个以上碱基)。
实施例2:在Hep2.2.15细胞上测试小干扰核酸(siRNA)的活性
使用HepG2.2.15细胞,用含有10%胎牛血清、380μg/ml G418DMEM完全培养基培养。第1天种细胞到96孔板,种细胞同时用RNAiMax将不同浓度的siRNA转入HepG2.2.15细胞;第4天,更换新鲜培养基,再次转染siRNA。第7天,收集细胞培养上清,ELISA检测HBsAg(剩余上清冻存备用检测DNA)。最后收集细胞,提取细胞内RNA,RT-PCR分别检测总HBV RNA,同时检测GAPDH基因RNA作为内参。化合物均3个浓度点,平行测定2复孔。
抑制百分比计算公式如下:
%HBsAg抑制率=(1-样品中HBsAg含量/对照组中HbsAg含量)×100
%HBV RNA抑制率=(1-样品中HBV RNA含量/对照组中HBV RNA含量)×100
本发明设计了9条小干扰核酸(siRNA),并检测这些序列对HBV基因mRNA和HBsAg的抑制效果,小干扰核酸序列如下表1所示。
表1.小干扰核酸序列
细胞实验结果如下表2和表3所示,从表2和表3可以得出,除了NPD-S2之外,另外的的8条序列均能够抑制HBsAg的表达。
表2.HepG2.2.15细胞中mRNA的沉默效果
表3.HepG2.2.15细胞中HBV表面抗原抑制效果
实施例3:siRNA修饰与缀合
一、GalNAc靶头1043的合成
按照以下方法,合成了TO-23(1043靶头)及TP-23(1043靶头连接siRNA的前体)的一种非对映异构体。
1、中间体GN-17-01的合成(图1合成反应1)
(1)在N2氛围下,将GC-1(12g,25.89mmol)溶于DCM(200mL)中,冰水浴降温至0~5℃,加入HBTU(11.78g,31mmol)和DIEA(10g,77.67mmol),搅拌10分钟,
(2)随后加入N-叔丁氧羰基-1,4-丁二胺(4.87g,25.89mmol),升温至25℃搅拌反应16小时,TLC显示原料基本消失。
(3)加入饱和的氯化铵溶液(100mL)淬灭,分液,DCM(100mL×2)萃取,
(4)合并有机相并用饱和食盐水(100mL)洗涤,无水Na2SO4干燥,过滤并浓缩。经柱层析纯化(DCM/MeOH=20/1)得白色固体化合物GN-17-01(15g,收率91%)。
2、中间体GN-17的合成(图1合成反应2)
(1)将GN-17-01(15g,23.67mmol)溶于DCM(150mL)中,加入TFA(50mL),25℃搅拌1小时,TLC显示原料基本消失,浓缩,
(2)用乙腈(100mL×3)与TFA共沸除去多余TFA,得泡沫状固体GN-17(TFA盐,12.6g)。
3、中间体TO-23-01的合成(图1合成反应3)
(1)在N2氛围下,将NC-4(2.6g,4.7mmol)溶于DCM(200mL)中,冰水浴降温至0~5℃,加入HATU(5.6g,14.83mmol)和DIEA(4.85g,37.6mmol)搅拌20分钟,
(2)随后加入GN-17-01(8.45g,15.5mmol),升温至25℃搅拌反应4小时。TLC检测,原料基本消失,
(3)加入饱和氯化铵溶液(50mL)淬灭,分液,DCM(100mL×2)萃取,
(4)合并有机相并用饱和食盐水(100mL)洗涤,无水Na2SO4干燥,
(5)过滤并浓缩得粗制品。经柱层析纯化(DCM/MeOH=10/1)得白色固体TO-23-01(6.3g,收率63.1%)。
4、化合物TO-23(1043靶头)的合成(图1合成反应4)
(1)向TO-23-01(6.3g,3.0mmol)的MeOH(100mL)溶液中加入10%Pd/C(600mg)和Pd(OH)2/C(600mg),H2置换3次,25℃搅拌反应3小时,TLC(DCM/MeOH=8/1)检测原料基本消失,
(2)过滤,浓缩得粗制品。经柱层析纯化(DCM/MeOH/TEA=10/1/0.1)得白色固体TO-23(4.5g,收率75%)。
1H NMR(400MHz,DMSO-d6)δ7.88-7.81(m,9H),7.14(s,1H),5.21(d,J=3.4Hz,3H),4.95(dd,J=11.2,3.4Hz,3H),4.53(d,J=8.5Hz,3H),4.07-3.97(m,9H),3.88(dt,J=11.0,9.0Hz,3H),3.77-3.71(m,3H),3.63-3.50(m,24H),3.49-3.41(m,8H),3.38-3.35(m,2H),3.08-2.98(m,12H),2.35–2.25(m,14H),2.10(s,9H),2.00(s,9H),1.89(s,9H),1.78(s,9H),1.40-1.33(s,12H).
MS(ESI):m/z[1/2M+H]+理论值1000.5,实测值1000.3。
5、化合物TP-23(1043靶头连接siRNA的前体)的合成(图1合成反应5)
(1)N2氛围下,将TO-23(2.3g,1.15mmol)溶于干燥DCM(40mL)中,加入DIEA(0.86mL,5.2mmol),利用注射器缓慢滴加2-氰乙基-N,N-二异丙基氯代亚磷酰胺(0.46mL,2.1mmol)的干燥DCM(2mL)溶液。25℃反应1小时。TLC检测,原料基本消失,(2)加入饱和NaHCO3(20mL)淬灭,分液,有机相用饱和NaHCO3(20mL)溶液,饱和食盐水(20mL)洗涤,无水MgSO4干燥,过滤浓缩得粗制品。经柱层析纯化(硅胶柱预先经1.5%TEA/DCM碱化,DCM/MeOH/TEA=15/1/0.1)得白色固体TP-23(1.8g,收率71.1%)。
1H NMR(400MHz,DMSO-d6)δ7.91-7.79(m,9H),7.15(s,1H),5.21(d,J=3.4Hz,3H),4.95(dd,J=11.2,3.4Hz,3H),4.53(d,J=8.5Hz,3H),4.06-3.97(m,9H),3.88(dt,J=11.1,8.9Hz,3H),3.78-3.66(m,6H),3.63-3.41(m,36H),3.07-2.98(m,12H),2.76(t,J=5.9Hz,2H),2.35-2.24(m,14H),2.10(s,9H),2.00(s,9H),1.89(s,9H),1.78(s,9H),1.40-1.33(m,12H),1.13(dd,J=6.7,4.1Hz,12H);
31P NMR(162MHz,DMSO-d6)δ147.81;
MS(ESI):m/z[1/2M+Na]+理论值1122.5,实测值1122.4。
二、GalNAc靶头1046的合成
按照以下方法,合成了TO25(1046靶头)及TP-25(1046靶头连接siRNA的前体)的一种非对映异构体。
1、中间体NC-6-01的合成(图1合成反应6)
(1)N2氛围下,向1000mL三口瓶中加入干燥的THF(300mL),冰浴降温至0-5℃搅拌,分批加入60%NaH(14g,354.8mmol),随后缓慢滴加2-氯乙氧基乙醇(40g,322.5mmol)的THF溶液(200mL),保温反应30分钟,再向反应瓶中滴加溴化苄(60.3g,354.8mmol),升至25℃搅拌16小时,TLC监测原料基本消耗完毕。
(2)缓慢滴加饱和氯化铵溶液(150mL)淬灭,分液,水相用EtOAc(100mL×2)萃取,合并有机相并用饱和食盐水(300mL)洗涤、无水Na2SO4干燥,过滤浓缩获得粗制产品。粗产品用硅胶柱层析纯化(石油醚/EtOAc=5/1)得淡黄色油状物化合物NC-6-01(53g,收率78%)。
MS(ESI):m/z[M+H]+理论值215.1,实测值215.1。
2、中间体NC-6-02的合成(图1合成反应7)
(1)将乙二胺(196g,3.26mol)置于2000mL三口瓶中,加入乙腈(1000mL)、碳酸钾(90g,0.65mol)和碘化钠(60.6g,0.33mol)搅拌。随后将NC-6-01(70g,0.33mol)的乙腈(100mL)溶液缓慢滴加至反应瓶中,升温至60℃搅拌16小时,TLC检测原料基本消耗完毕。
(2)停止反应,浓缩,加入纯化水(300mL),浓盐酸调节pH至4-5,EtOAc(200mL×3)萃取三次,水相加入氢氧化钠固体调节pH至13-14,经DCM(200mL×3)萃取三次,合并有机相并用饱和食盐水(300mL)洗涤、无水Na2SO4干燥,过滤浓缩得淡黄色油状物NC-6-02(69.5g,87%)。
MS(ESI):m/z[M+H]+理论值239.2,实测值239.1。
3、中间体NC-6-03的合成(图1合成反应8)
(1)将NC-6-02(69.5g,0.29mol)和溴乙酸叔丁酯(187g,0.96mol)加入到四氢呋喃(700mL)和纯化水(350mL)中,搅拌,冰水浴降温至5℃以下,加入碳酸钾(322g,2.34mol)。25℃搅拌反应14小时,TLC检测原料转化完全。
(2)反应液加入纯化水(300mL),静置分层,分出有机相,水相经EtOAc(200mL×2)萃取两次,合并有机相,加入饱和食盐水(500mL)洗涤、无水Na2SO4干燥,过滤浓缩得淡黄色油状物NC-6-03(201g)。
MS(ESI):m/z[M+H]+理论值581.4,实测值581.3。
4、中间体NC-6的合成(图1合成反应9)
(1)将NC-6-03(23g,39.6mmol)溶于1,4-二氧六环(200mL)中,加入浓盐酸(40mL),升温至60℃反应2小时,TLC检测原料基本消耗完毕。
(2)浓缩,再次加入1,4-二氧六环(200mL)浓缩,得白色固体粗品,将粗品加入到乙酸乙酯(200mL)中打浆2小时,抽滤,收集滤饼,50℃真空烘干得得白色固体化合物NC-6(22.6g,96.9%)。
(3)MS(ESI):m/z[M+H]+理论值413.2,实测值413.1。
5、中间体TO-25-01的合成(图1合成反应10)
(1)N2氛围下,将NC-6(1.5g,3.6mmol)、HBTU(4.5g,12.0mmol)和DIEA(4.75g,36mmol)加入到DCM(50mL)中搅拌30分钟,随后滴加GN-17(6.4g,12.0mmol)与DIEA(4.75g,36mmol)的DCM(50mL)溶液,25℃搅拌16小时,LCMS检测,原料基本消耗完毕。
(2)加入DCM(100mL)稀释,向反应液中加入1N盐酸溶液(80mL×2)洗涤,合并有机相,经饱和碳酸氢钠(100mL)洗涤、饱和食盐水(100mL)洗涤、无水Na2SO4干燥过滤浓缩获得粗制产品。粗产品经硅胶柱层析纯化(DCM/MeOH=7/1)得白色固体化合物TO-25-01(4.3g,收率60%)。
(3)MS(ESI):m/z[M/2+H]+理论值980.0,实测值979.9。
6、中间体TO-25(1046靶头)的合成(图1合成反应11)
(1)将TO-25-01(4.3g,2.2mmol)溶于甲醇(80mL)中,加入10%钯碳(1.0g),H2置换三次,25℃搅拌2小时,LCMS检测,原料基本消失。
(2)过滤,浓缩,加入DCM(20mL)溶解,缓慢滴加至MTBE(300mL)中,搅拌析晶30分钟,抽滤,得到白色固体化合物TO-25(3.7g,收率90%)。
1H NMR(400MHz,DMSO-d6)δ8.48(d,J=5.6Hz,1H),8.06(t,J=5.7Hz,2H),7.85(dd,J=11.7,6.8Hz,6H),5.21(d,J=3.3Hz,3H),4.95(dd,J=11.2,3.3Hz,3H),4.53(d,J=8.5Hz,3H),4.08-3.83(m,14H),3.75(p,J=4.8Hz,5H),3.68-3.26(m,28H),3.21-2.95(m,14H),2.30(q,J=7.9,6.7Hz,6H),1.94-1.78(m,,36H),1.41-1.38(m,12H);
MS(ESI):m/z[1/2M+H]+理论值934.9,实测值934.8。
7、TP-25的(1046靶头连接siRNA的前体)合成(图1合成反应12)
(1)N2氛围下,将TO-25(700mg,0.37mmol)溶于干燥DCM(10mL)中,加入DIEA(0.31mL,1.9mmol),利用注射器缓慢滴加2-氰乙基-N,N-二异丙基氯代亚磷酰胺(0.19mL,0.74mmol)的干燥DCM(1mL)溶液,25℃反应30分钟,TLC检测,原料基本消失。
(2)加入饱和NaHCO3(10mL)淬灭,DCM(10mL)稀释,分液,有机相用饱和NaHCO3(10mL)溶液,饱和食盐水(10mL)洗涤,无水NaSO4干燥,过滤浓缩得粗制品。经柱层析纯化(硅胶柱预先经1.5%TEA/DCM碱化,DCM/MeOH/TEA=15/1/0.1)得白色固体TP-25(405mg,收率53%)。
1H NMR(400MHz,DMSO-d6)δ8.12(t,J=6.0Hz,2H),7.98-7.75(m,7H),5.21(d,J=3.4Hz,3H),4.96(dd,J=11.2,3.4Hz,2H),4.54(d,J=8.4Hz,2H),4.02(q,J=5.3,4.5Hz,9H),3.95-3.83(m,3H),3.82-3.50(m,23H),3.40-3.26(m,4H),3.12-2.94(m,27H),2.76-2.59(m,7H),2.29(t,J=6.7Hz,5H),2.11-1.78(m,38H),1.38(s,12H),1.16(d,J=7.5Hz,12H);
31P NMR(162MHz,DMSO-d6)δ147.97;
MS(ESI):m/z[1/2M+Na]+理论值1057.0,实测值1057.4。
三、GalNAc靶头1048的合成
按照以下方法,合成了TO-26(1048靶头)及TP-26(1048靶头连接siRNA的前体)的一种非对映异构体。
1、中间体GN-18-01的合成(图1合成反应13)
(1)N2氛围下,将GC-2(20.1g,39.7mmol)溶于DCM(200mL)中,分批加入CDI(7.09g,73.7mmol),25℃搅拌3小时,随后将N-Boc-乙二胺(7.0g,43.7mmol)和三乙胺(12.05g,119.1mmol)加入反应液中,反应16小时,LCMS检测显示原料消失。
(2)加入饱和碳酸氢钠溶液(200mL)淬灭,分液,水相经DCM(100mL×3)萃取,合并有机相并使用饱和氯化铵溶液(200mL)、饱和氯化钠溶液(200mL)洗涤,无水Na2SO4干燥,过滤浓缩获得粗制产品。粗产品经甲基叔丁基醚(100mL)洗涤,油状产物浓缩得白色固体化合物GN-18-01(24.43g,收率95.1%)。
MS(ESI):m/z[M+H]+理论值650.3,实测值650.5。
2、中间体GN-18的合成(图1合成反应14)
(1)将GN-18-01(45.52g,70mmol)分批加入到HCl/EtOAc溶液(2N,500mL)中,25℃搅拌2小时,LCMS检测显示原料消失。
(2)倾倒出溶剂,固体浓缩得粗制产品。粗产品经甲基叔丁基醚(200mL)打浆纯化,过滤,滤饼40℃真空干燥得白色固体GN-18(49.6g)。
MS(ESI):m/z[M+H]+理论值550.3,实测值550.5。
3、中间体TO-26-01的合成(图1合成反应15)
(1)N2氛围下,将NC-6(1.5g,3.6mmol)、PyBOP(6.2g,12.0mmol)和DIEA(4.75g,36mmol)加入到DCM(50mL)中搅拌30分钟,随后滴加GN-18(6.6g,12.0mmol)与DIEA(4.75g,36mmol)的DCM(50mL)溶液,25℃搅拌16小时,LCMS检测,原料基本消耗完毕。
(2)加入DCM(100mL)稀释,向反应液中加入1N盐酸溶液(80mL×2)洗涤,合并有机相,经饱和碳酸氢钠(100mL)、饱和食盐水(100mL)洗涤、无水Na2SO4干燥过滤浓缩获得粗制产品。粗产品经硅胶柱层析纯化(DCM/MeOH=7/1)得白色固体化合物TO-26-01(4.7g,收率65%)。
MS(ESI):m/z[M/2+H]+理论值1004.0,实测值1004.2。
4、中间体TO-26(1048靶头)的合成(图1合成反应16)
(1)将TO-26-01(4.0g,2.0mmol)溶于甲醇(80mL)中,加入10%钯碳(1.0g),H2置换三次,25℃搅拌2小时,LCMS检测,原料基本消失。
(2)过滤,浓缩,加入DCM(20mL)溶解,缓慢滴加至MTBE(200mL)中,搅拌析晶30分钟,抽滤,得到白色固体化合物TO-26(3.5g,收率91%)。
1H NMR(400MHz,DMSO-d6)δ8.54(s,1H),8.14(s,2H),7.95-7.92(m,3H),7.84(d,J=7.8Hz,3H),5.21(d,J=3.4Hz,3H),4.97(dd,J=11.2,3.4Hz,3H),4.54(d,J=8.5Hz,3H),4.13-3.66(m,21H),3.60-3.44(m,37H),3.14(d,J=13.8Hz,15H),2.31(t,J=6.4Hz,6H),2.10(s,9H),2.00(s,9H),1.89(s,9H),1.77(s,9H).
MS(ESI):m/z[1/2M+H]+理论值958.9,实测值959.1。
5、TP-26(1048靶头连接siRNA的前体)的合成(图1合成反应17)
(1)N2氛围下,将TO-26(900mg,0.47mmol)溶于干燥DCM(12mL)中,加入DIEA(0.39mL,0.44mmol),利用注射器缓慢滴加2-氰乙基-N,N-二异丙基氯代亚磷酰胺(277mg,1.17mmol)的干燥DCM(1mL)溶液,25℃反应30分钟,TLC检测,原料基本消失。
(2)加入饱和NaHCO3(10mL)淬灭,DCM(10mL)稀释,分液,有机相用饱和NaHCO3(10mL)溶液,饱和食盐水(10mL)洗涤,无水NaSO4干燥,过滤浓缩得粗制品。经柱层析纯化(硅胶柱预先经1.5%TEA/DCM碱化,DCM/MeOH/TEA=15/1/0.1)得白色固体TP-26(600mg,收率60%)。
1H NMR(400MHz,DMSO-d6)1H NMR(400MHz,DMSO-d6)δ8.15(s,2H),7.94-7.81(m,7H),5.22(d,J=3.4Hz,3H),4.97(dd,J=11.2,3.4Hz,3H),4.55(d,J=8.5Hz,3H),4.03(s,8H),3.88(dt,J=11.2,8.9Hz,3H),3.81-3.67(m,7H),3.64-3.46(m,30H),3.11(d,J=13.1Hz,19H),2.76(t,J=5.9Hz,3H),2.65-2.54(m,7H),2.31(t,J=6.6Hz,7H),2.11(s,9H),2.00(s,9H),1.89(s,9H),1.77(s,9H),1.13(d,J=6.8,12H).
31P NMR(162MHz,DMSO-d6)δ147.89;
MS(ESI):m/z 1/2[M-i-Pr2N]理论值1007.9,实测值1008.2。
以下RNAi剂双链体的反义链和正义链的寡核苷酸序列部分,以及靶向性配体与RNA的连接,均按照J.Org.Chem.2012,77,4566-4577;Curr.Protoc.Nucleic Acid Chem.,81,e107报道的亚磷酰胺偶联技术在用于寡核苷酸合成的固相上合成。靶向性配体1046、1048、1043均通过硫代磷酸酯键与siRNA正义链5’末端连接。
合成的GalNAc-siRNA缀合物(NPD007s-1)如表4所述,表4中第二列的缀合物结构包括三部分,例如,G1043-S2A2-A265的结构为:1043靶头通过硫代磷酸酯键与编号为A265的siRNA正义链5’末端连接,S2A2为对A265的siRNA的修饰类型,具体的修饰基团和修饰方式见表4。
核酸序列中,Ao表示腺嘌呤核苷,Uo表示尿嘧啶核苷,Go表示鸟嘌呤核苷,Co表示胞嘧啶核苷,直接相邻的核苷酸之间没有符号,表示以正常的磷酸酯键连接。
DNA:A G C T(A表示2’-脱氧腺嘌呤核苷,T表示2’-脱氧胸腺嘧啶核苷,G表示2’-脱氧鸟嘌呤核苷,C表示2’-脱氧胞嘧啶核苷);
2'-F:aFgFcFuF(aF表示2’-氟代腺嘌呤核苷,uF表示2’-氟代尿嘧啶核苷,gF表示2’-氟代鸟嘌呤核苷,cF表示2’-氟代胞嘧啶核苷);
2'-OMe:aMgMcMuM(aM表示2’-O-甲基腺嘌呤核苷,uM表示2’-O-甲基尿嘧啶核苷,gM表示2’-O-甲基鸟嘌呤核苷,cM表示2’-O-甲基胞嘧啶核苷);
*:表示以硫代磷酸酯键连接;
序列中的y、z代表靶头的位置。
表4.缀合物核苷酸序列
实施例4:缀合物在体外稳定性测试
4.1人血浆反应体系
取Human plasma(肝素抗凝)700ul加300ul无菌1XPBS得到70%的血浆工作液。取0.1mg/ml受试样品5ul(0.5ug),加入70%的血浆工作液15ul混匀(终浓度50%),37℃恒温孵育。分别在倒计时0h、4h、24h、72h每个时间放置一支样品。反应结束,加入4μLDNA上样缓冲液(6Xloading buffer)。0h表示将待测样品与血浆混匀后,立即加loading buffer。取10ul样品(0.25ug)进行后续跑胶检测。
4.2大鼠溶酶体反应体系
取2.5mg/ml Rat Liver Tritosome(R0610.LT;Sekisui Xenotech,KansasCity,MO,USA)母液10μL,加入90uL 20mM PH5的NaOAc,得到0.25mg/ml的酸化工作液。取0.1mg/ml受试样品5ul(0.5ug),加入Tritosome酸化工作液15ul(3.75ug)混匀,在37℃恒温孵育。分别于倒计时0h、4h、24h、72h每个时间放置一支样品。反应结束,加入4μL DNA上样缓冲液(6Xloading buffer)。0h表示将待测样品与溶酶体裂解液混匀后,立即加loading buffer。取10ul样品(0.25ug)进行后续跑胶检测。
NPD007s-1在人血浆及大鼠溶酶体中稳定性测试如图2,结果显示,缀合物样品在经过人血浆处理72小时后,电泳均未观察到条带迁移(左)。经Tritosome处理4小时电泳均出现条带迁移(右);Tritosome处理72小时后受试样品未观察到明显的强度降低。
实施例5:缀合物在Hep3B细胞上活性测试
人肝癌Hep3B细胞(中国科学院上海细胞库),培养于添加10%胎牛血清(FBS)(Gibco,US)的DMEM(Gibco,US)中,于37℃,5%CO2条件下培养(il60,Thermo Fisher)。转染实验进行当天,使用0.25%Trypsin(Gibco,US)消化细胞,计数并以450μL/孔、5万/孔的密度接种于24孔板。随后,以lipofectmine2000(Thermo Fisher)转染方式加入受试样品。按RNAiMAX试剂说明说书标准流程转染,siRNA终浓度为10nM/1nM/0.5nM/0.25nM/0.1nM/0.05nM/0.01nM。转染组以siNC为阴性对照,其序列为:
正义链(sense):5’-UUCUCCGAACGUGUCACGUTT-3’
反义链(antisense):5’-ACGUGACACGUUCGGAGAATT-3’。
24h后,提取细胞的总RNA,通过实时定量PCR(Quantitative Real-Time PCR)检测细胞中HBV mRNA序列的表达情况,其中用于扩增内参基因PPIB、HBV的PCR引物如下所示:QPCR引物序列信息:HBV forword:5‘-CCTTTGTTTACGTCCCGTCG-3’,reverse:5‘-GGAGAAGGGGACGAGAGAGT-3’;内参基因human cyclophilin B forword:5‘-GGTGATCTTTGGTCTCTTCGG-3’,human cyclophilin B reverse:5‘-TAGATGCTCTTTCCTCCTGTG-3’。图3结果显示,NPD007s-1在Hep3B细胞上对HBV mRNA具有明显的抑制作用,EC50为0.44nM。
实施例6:缀合物在体外细胞毒性测试
人肝癌Hep3B细胞(中国科学院上海细胞库),培养于添加10%胎牛血清(FBS)(Gibco,US)的DMEM(Gibco,US)中,于37℃,5%CO2条件下培养(il60,Thermo Fisher)。转染实验进行当天,使用0.25%Trypsin(Gibco,US)消化细胞,计数并以3000cells/孔的密度,体积90μL/孔接种于96孔板。24h后,以RNAiMAX(Thermo Fisher)为载体按标准配置方法转染,RNAiMAX 0.2μl/孔。siRNA终浓度为0.25nM、0.5nM、1nM、10nM、100nM、300nM。转染72小时后,移除上清液,加入10μL CCK8(索莱宝),孵育1-2h后使用全波长酶标仪(SYNERGY H1,BioTek)于450nm波长下检测吸光度。数据以细胞活率mean±SD(%)的形式展示。表5结果显示,NPD007s-1的加入对细胞活力几乎没有影响。
表5.缀合物在体外细胞毒性测试
实施例7:缀合物在体外脱靶效应评估测试
7.1针对序列构建4个重组质粒,其中GCM表示在靶质粒,PCM、GSM、PSM表示脱靶质粒。质粒设计依据如下:
表6.缀合物在体外脱靶效应评估
将目标序列克隆到psiCHECKTM-2质粒Xho 1/Not 1位点。
7.2 HEK293(ATCC)细胞培养于添加10%FBS(Gibco)的DMEM(Gibco)培养基中。转染前一天以每孔10000个细胞的密度接种于96孔白色酶标板,贴壁后转染。转染过程参照lipofectemine2000说明书标准流程。每孔分别配置两只混合溶液:a.0.2ullipofectemine2000混合10ng对应质粒、b.0.2ul lipofectemine2000混合选定剂量的siRNA受试样品。
将混合物a、b分别加入细胞中,孵育24小时。每个转染条件设置3个平行孔。转染24h后,裂解细胞检测双荧光素酶活性,按照双荧光素酶报告基因检测试剂盒(E2940,Promega)标准步骤检测。检测前裂解液置于4℃溶解。待试剂稳定到室温,1:1混合Luciferase Reagent与细胞完全培养基。将混合液加入移去培养基的孔板中,室温孵育10分钟后检测发光,读数为萤火虫荧光发光值(firefly luciferase)发光值。按1:100稀释/>Stop&/>Substrate到/>Stop&Glo缓冲液中,配制1X工作液,向96孔板中加入40μL工作液。室温孵育10分钟后检测发光,读数为海肾荧光发光值(renilla luciferase)发光值。未添加siRNA作为对照组,该组海肾荧光发光值/萤火虫荧光发光值的比值设定为100%。其他各浓度组海肾荧光发光值/萤火虫荧光发光值与该组数据比较得到相对的发光值读值。数据以mean±SEM的形式展示。
图4结果显示,NPD007s-1在靶效应明显,且没有脱靶现象。
实施例8:缀合物在细胞水平药效测试
取人肝癌Hep2.2.15细胞(中国科学院上海细胞库),培养于添加10%胎牛血清(FBS)(Gibco,US)的DMEM(Gibco,US)中,于37℃,5%CO2条件下培养(il60,ThermoFisher)。第一天,使用0.25%Trypsin(Gibco,US)消化细胞,计数并以20000cells/孔的密度,体积90μL/孔接种于96孔板。24h后,以RNAiMAX(Thermo Fisher)为载体按标准配置方法转染,RNAiMAX 0.2μl/孔。siRNA终浓度为0.0001nM、0.001nM、0.01nM、0.1nM、1nM、2.5nM、10nM、50nM。转染72h后,收集上清,检测HBsAg的表达。同时,测试细胞活性的变化。
图5结果显示,NPD007s-1在Hep2.2.15细胞上对HBsAg均有较高的抑制效果,EC50为0.12nM,且没有观察到细胞毒性。
实施例9:缀合物对HBV转基因小鼠的药效研究
取8—10周龄乙肝转基因小鼠,雄性。在给药前两天检测HBV DNA及HBV HBsAg,选取符合要求的小鼠入组。每组5只。按照5mpk的剂量,皮下单次给药,给药体积为100—200ul。分别在给药后7,14,21,28天采外周血检测HBV DNA,HBsAg,HBeAg的表达。
图6、7、8的结果显示,在转基因小鼠上,NPD007s-1在5mpk时,能够沉默HBV DNA、抑制HBsAg和HBeAg表达,且具有长效性。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种乙型肝炎病毒基因的小干扰核酸(siRNA),其特征在于,所述小干扰核酸的序列为9组序列中的至少一组,所述9组序列分别如SEQ ID NO:1-2、SEQ ID NO:3-4、SEQ ID NO:5-6、SEQ ID NO:7-8、SEQ ID NO:9-10、SEQ ID NO:11-12、SEQ ID NO:13-14、SEQ ID NO:15-16、SEQ ID NO:17-18所示。
2.根据权利要求1所述的乙型肝炎病毒基因的小干扰核酸,其特征在于,所述小干扰核酸的序列与权利要求1中所示的9组序列中的其中一组具有85%以上同源性。
3.一种缀合物,其特征在于,所述缀合物包含如权利要求1或2所述的乙型肝炎病毒基因的小干扰核酸(siRNA)。
4.根据权利要求3所述的缀合物,其特征在于,所述缀合物中的靶向性配体通过连接基团与siRNA正义链5’末端连接。
5.根据权利要求4所述的缀合物,其特征在于,所述缀合物中的靶向性配体通过硫代磷酸酯键与siRNA正义链5’末端连接。
6.根据权利要求3所述的缀合物,其特征在于,所述缀合物为GalNAc-siRNA。
7.根据权利要求3所述的缀合物,其特征在于,所述缀合物具有下述中的一种的序列:
8.根据权利要求1-2中任一项所述的小干扰核酸siRNA或权利要求3-7中任一项所述的缀合物在制备治疗乙型肝炎药物中的应用。
9.一种用于治疗乙型肝炎的药物组合物,其特征在于,所述药物组合物包括权利要求1或2所述乙型肝炎病毒基因的小干扰核酸。
10.一种用于治疗乙型肝炎的药物组合物,其特征在于,所述药物组合物包括权利要求3-7中任一项所述的缀合物。
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