CN117305301A - 一种抑制asgpr-1基因的寡核苷酸或其药学上可接受的盐及应用 - Google Patents
一种抑制asgpr-1基因的寡核苷酸或其药学上可接受的盐及应用 Download PDFInfo
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- CN117305301A CN117305301A CN202310673495.3A CN202310673495A CN117305301A CN 117305301 A CN117305301 A CN 117305301A CN 202310673495 A CN202310673495 A CN 202310673495A CN 117305301 A CN117305301 A CN 117305301A
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Abstract
本发明属于生物医药技术领域，涉及一种抑制ASGPR‑1基因的寡核苷酸或其药学上可接受的盐及应用，本发明公开的抑制ASGPR‑1基因的寡核苷酸或其药学上可接受的盐、缀合物和组合物，能够显著抑制ASGPR‑1基因的表达水平，抑制SREBP1和脂肪生成，从而促进胆固醇排泄和降低血脂水平，疗效确切、副作用小、依从性高。
Description
技术领域
本发明属于生物医药技术领域，尤其涉及一种用于抑制ASGPR-1基因表达的寡核苷酸或其药学上可接受的盐及应用。
背景技术
心血管疾病是全球导致死亡的首要原因之一。非高密度脂蛋白(non-HDL)胆固醇和低密度脂蛋白(LDL)胆固醇水平与冠状动脉疾病和心肌梗死的风险之间存在因果关系。非HDL胆固醇已被证明比LDL胆固醇更能预测心血管风险，因为它包含所有含胆固醇的促动脉粥样硬化脂蛋白，包括LDL胆固醇、极低密度脂蛋白、中密度脂蛋白、脂蛋白(a)和乳糜微粒。目前的降脂药物主要通过三种机制降低血脂水平，他汀类药物主要降低胆固醇的合成和增加低密度脂蛋白(LDL)的重吸收，依折麦布(ezetimibe)抑制肠道对胆固醇吸收，PCSK9抑制剂增加肝脏对LDL的重吸收。目前，尚没有通过靶向胆固醇降解或排泄的降脂药物上市。虽然有多种降脂药物上市，但仍有大部分患者存在复发性心血管疾病的风险，其胆固醇水平控制在目标范围内。因此，新型降胆固醇药物亟待开发。
肝去唾液酸糖蛋白受体(Asialoglycoprotein Receptor，ASGPR)，在啮齿动物和人类中高度表达和保守，由高度同源的主要(ASGPR-1)和次要(ASGPR-2)亚基组成。ASGPR-1主要表达在肝实质细胞膜上，具有识别和调控细胞的内吞作用、介导多种去唾液酸糖蛋白的内吞和溶酶体降解，通过介导具有暴露的末端半乳糖或N-乙酰半乳糖胺残基的糖蛋白的内吞作用和溶酶体降解，在血清糖蛋白稳态中起关键作用。
2016年人类遗传学研究已经确定，与非携带者相比，去唾液酸糖蛋白受体ASGPR-1亚基功能丧失变异等位基因的人类携带者血清中非高密度脂蛋白(HDL)胆固醇水平较低，冠状动脉疾病和心肌梗死的风险较低。因此，靶向ASGPR-1功能的疗法代表了一种降低非HDL胆固醇水平和治疗心血管疾病，特别是冠状动脉疾病的新方法。
2022年宋保亮研究团队通过敲除细胞中ASGPR-1，并进行RNA测序、多种动物实验等多方面证实了ASGPR-1通过mTOR/AMPK→BRCA1/BARD1→LXR→ABCG5/G8通路调控脂代谢的详细机制。去唾液酸糖蛋白与位于肝细胞膜上的ASGPR-1结合，通过网格蛋白介导的内吞作用进入到溶酶体中，在溶酶体酸性条件下被降解成游离氨基酸等营养物质激活溶酶体表面的mTORC1及抑制AMPK来调节胆固醇代谢。增加下游泛素连接酶BRCA1/BARD1的蛋白水平，促进LXR蛋白降解。而当ASGPR-1缺失或被抑制时，糖蛋白内吞和溶酶体降解减少，溶酶体内的氨基酸水平降低，从而抑制了mTORC1并激活了AMPK。一方面，AMPK通过降低E3泛素连接酶复合物BRCA1/BARD1来引起LXR增多，使ABCG5/G8表达增加，胆固醇外排增多；另一方面AMPK抑制胆固醇调节元件结合蛋白(SREBP)通路，防止脂肪酸合成增加。总之，靶向ASGPR-1上调LxRα、ABCA1和ABCG5/G8，抑制SREBP1和脂肪生成，从而促进胆固醇排泄和降低血脂水平。综上所述，ASGPR-1缺失会抑制胆固醇生成，是降血脂潜在的治疗靶点。
发明内容
本发明针对上述现有技术存在的不足，提供一种用于抑制ASGPR-1基因表达的寡核苷酸或其药学上可接受的盐、缀合物、组合物及应用，具体的技术方案如下：
本发明第一方面提供一种抑制ASGPR-1基因的寡核苷酸或其药学上可接受的盐，包含正义链和反义链，所述正义链如SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7或SEQ ID NO:8所示的不超过3个核苷酸差异的核苷酸序列；所述反义链如SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17或SEQ ID NO:18所示的不超过3个核苷酸差异的核苷酸序列；所述正义链与所述反义链至少80％互补。
进一步地，所述正义链为长度为19-25个核苷酸(碱基)。
进一步地，互补的正义链和反义链至少包含1个修饰的核苷酸，所述修饰的核苷酸独立地选自2’-甲基修饰的核苷酸、2’甲氧基修饰的核苷酸、2’-氨基乙基修饰的核苷酸、2’-O-甲氧基乙基修饰的核苷酸、2’-氟修饰的核苷酸、5’-硫代磷酸酯基团的核苷酸、连接于胆固醇基衍生物或月桂酸二癸酰胺基团或VP的末端核苷酸。
进一步地，修饰的核苷酸选自2’-脱氧-2’-氟修饰的核苷酸、2’-脱氧-修饰的核苷酸、锁核苷酸、未锁核苷酸(unlocked nucleotide)、构象限制核苷酸、约束乙基核苷酸(constrained ethyl nucleotide)、无碱基核苷酸、2’-氨基-修饰的核苷酸、2’-烷基-修饰的核苷酸、2’-O-烯丙基修饰的核苷酸、2’-C-烯丙基修饰的核苷酸、2’-羟基修饰的核苷酸、吗啉代核苷酸、氨基磷酸酯和包含非天然碱基的核苷酸等。
进一步地，所述正义链与所述反义链包含硫代磷酸酯基修饰和乙烯基磷酸酯基修饰。
进一步地，所述硫代磷酸酯基修饰位于正义链末端，包含至少1个，且不超过3个硫代磷酸酯基修饰；所述硫代磷酸酯基修饰位于反义链末端，包含至少1个，且不超过3个硫代磷酸酯基修饰；所述乙烯基磷酸酯基修饰位于反义链第1位。
进一步地，所述寡核苷酸或其药学上可接受的盐的正义链如SEQ IDNO:23、SEQ IDNO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ IDNO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ IDNO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41或SEQID NO:42所示的不超过3个核苷酸差异的修饰核苷酸序列；其反义链的核苷酸序列如SEQID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50或SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54或SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ IDNO:63或SEQ ID NO:64所示的不超过3个核苷酸差异的修饰核苷酸序列。
在一个实施方案中，所述寡核苷酸或其药学上可接受的盐，不包含3’突出端端。另一个实施方案中，至少一条链包含至少1个核苷酸的3’突出端。在另一个实施方案中，至少一条链包含至少2个核苷酸的3’突出端。
进一步地，所述寡核苷酸包括siRNA、shRNA、miRNA或ASO等。
本发明第二方面提供了一种缀合物，包括配体和通过配体缀合的如上所述寡核苷酸或其药学上可接受的盐，所述配体缀合在正义链3’末端或/和5’末端；更进一步地，所述配体缀合在正义链3’末端。
进一步地，所述配体为去唾液酸糖蛋白受体的配体。
进一步地，所述配体为半乳糖或N-乙酰半乳糖胺，优选3价或4价态N-乙酰半乳糖胺，更优选4价态N-乙酰半乳糖胺。
进一步地，所述缀合物的结构如式(I)下所示：
其中，R1结构如式(Ⅱ)所示，R2为氧、硫或碳等原子，优选碳原子；Q为磷酸酯、碳酸脂或硫酸酯等，优选磷酸酯；n为4-10，优选4；Nu为功能性寡核苷酸；
其中R3为H离子、乙酰基、苄基或叔丁氧羰基等，优选乙酰基或氢离子；n1为1-6，优选1。
进一步地，所述缀合物的结构如式(Ⅲ)下所示：
即R1结构如式(Ⅱ)所示，R2为碳原子；Q为磷酸酯；n4；Nu为功能性寡核苷酸；R3为乙酰基或氢离子；n1为1。
本发明第三方面提供了一种组合物，含有如上所述寡核苷酸或其药学上可接受的盐和药学上可接受的载体；所述寡核苷酸或其药学上可接受的盐与药学上可接受的载体重量比为1：1-500，优选的重量比为1:1-50。
其中，所述药学上可接受的载体可以为本领域常规采用的各种载体，包括但不限于脂质体、聚合物、多肽、蛋白、单抗、外泌体、纳米材料等。所述脂质体为可离子化脂质，优选阳离子脂质体。
进一步地，所述药学上可接受的载体还含有辅助脂质和聚乙二醇化脂质。所述辅助脂质为胆固醇、胆固醇的类似物和/或胆固醇的衍生物；所述聚乙二醇化脂质为1,2-二棕榈酰胺-sn-甘油-3-磷脂酰乙醇胺-N-[甲氧基(聚乙二醇)]-2000。
进一步地，所述药学上可接受的载体含有有机胺。
进一步地，所述药学上可接受的载体含有去唾液酸糖蛋白受体的配体，优先半乳糖或N-乙酰半乳糖胺，优选3价或4价态N-乙酰半乳糖胺，更优选4价态N-乙酰半乳糖胺。
进一步地，所述寡核苷酸或其药学上可接受的盐、缀合物或组合物，包括但不限于药学上可接受的赋形剂、稀释剂、缓冲剂或稳定剂等。
本发明第四方面提供一种如上所述寡核苷酸或其药学上可接受的盐、缀合物或组合物在制备用于预防、治疗或缓解ASGPR-1相关疾病和症状的药物中的用途。
本发明第五方面提供了一种用于预防、治疗或缓解ASGPR-1相关疾病和症状的方法，该方法包括将如第一方面所述寡核苷酸或其药学上可接受的盐和/或如第二方面所述缀合物和/或如第三方面所述组合物给予有需要的患者。
本发明第六方面提供一种用于抑制肝细胞中ASGPR-1基因表达的方法，该方法包括将如第一方面所述寡核苷酸或其药学上可接受的盐和/或如第二方面所述缀合物和/或如第三方面所述组合物导入所述肝细胞。
本发明第一方面所述寡核苷酸或其药学上可接受的盐和/或如第二方面所述缀合物和/或如第三方面所述组合物可有效的预防、治疗或缓解ASGPR-1相关疾病和症状。具体表现为，在细胞水平上，所述寡核苷酸或其药学上可接受的盐对ASGPR-1基因的抑制效果可达到80％以上；当皮下给药时，缀合物的配体可以有效的将RNAi递送到肝脏，血浆中ASGPR-1含量明显下降。
通过上述技术方案，本发明提供的寡核苷酸或其药学上可接受的盐、缀合物和组合物，能够显著抑制ASGPR-1基因的表达水平，可有效的预防、治疗或缓解ASGPR-1相关疾病和症状，疗效确切、副作用小、依从性高，实现对患者血压的有效、长期控制。siRNA在Hep 3B细胞的ASGPR-1基因表达的抑制有显著效果，抑制效果能达到85％；修饰后siRNA在Hep 3B细胞的ASGPR-1基因表达的抑制效果能达到85％；siRNA缀合物采用皮下给药后，可以显著降低小鼠血液中ASGPR-1蛋白水平。
具体实施方式
以下结合实例对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。
实施例：
1.化合物10的制备：
化合物7与苄胺8反应得到二取代化合物9，随后水解得到化合物10。化合物10作为中间体用于化合物(H01)的合成。
称取化合物7(6-溴-己酸甲酯，7.33g，35.0mmol)，化合物8(苄胺，1.5g，14.0mmol)，无水碳酸钾(5.8g，42.0mmol)，碘化钾(1.16g，7.0mmol)置于反应瓶中，加入无水乙醇50mL搅拌混悬，将反应体系置于82℃条件下搅拌加热回流，反应12h。停止加热，降至室温，减压脱除溶剂乙醇，加入水30mL，二氯甲烷30mL搅拌静置分层，分出有机相，水相二氯甲烷萃取2次，合并有机相，干燥脱溶剂后得到粗品黄色油状液体。柱层析纯化，以石油醚:乙酸乙酯＝10:1～3:1梯度洗脱，得化合物9浅黄色油状液体3.71g，收率为73％。
称取化合物9(3.7g，10.17mmol)置于反应瓶中，加入乙醇10mL搅拌溶解，称取氢氧化钠(1.63g，40.68mmol)加入到上述反应液中，将反应体系置于40℃条件下加热搅拌2h，停止加热降至室温，减压脱除溶剂乙醇，加水溶解后，相二氯甲烷洗涤2次，水相用1N盐酸调pH为2-3，加入固体氯化钠至饱和，二氯甲烷萃取产品3次，合并有机相，干燥后脱除溶剂得到产品中间体10黄色油状液体2.63g，收率为77％。
MS m/z[M+H]+(ESI):336.05。
2.化合物6的制备
化合物2与乙酸酐反应得到乙酰基保护的化合物3，环合得到化合物4，化合物4与4-(N-叔丁氧羰基氨基)-1-丁醇反应开环得到化合物5，盐酸托Boc保护基得到化合物6。化合物6作为中间体用于化合物(H01)的合成。
化合物2(50.0g，232mmol)加入反应瓶中，继续加入乙酸酐(165mL)，冰浴条件下加入吡啶(220mL)，DMAP(2.4g，19.7mmol)，三乙胺(23.5g，232mmol)。加毕室温搅拌过夜。过滤，滤饼用甲苯淋洗，水淋洗，45℃减压干燥，得到白色固体化合物3，73.2g，收率81％。
化合物3(20.0g，51.4mmol)加入反应瓶，加入4A分子筛干燥的二氯甲烷(100mL)。氩气保护下，加入三氟甲磺酸三甲基硅酯(13.7g，61.7mmol)，室温搅拌过夜。加入三乙胺(15.6g，154.2mmol)，搅拌。减压脱除溶剂和三乙胺，得到化合物4，油状物32.1g，不经纯化直接投入下步反应。
上步化合物4溶于经4A分子筛干燥的1，2-二氯乙烷(120mL)，加入4-(N-叔丁氧羰基氨基)-1-丁醇(10.2g，54mmol)，室温下，继续加入三氟甲磺酸三甲基硅酯(2.3g，10.3mmol)，搅拌过夜。反应液中加入饱和碳酸氢钠溶液(60mL)，水80mL，分液，有机相水洗(80mL×1)，10％柠檬酸水溶液洗(100g×2)，无水硫酸钠干燥，减压脱除溶剂得到粗品5，棕黄色油状物16.5g。两步收率62％。
化合物5(16.0g，30.8mmol)加入反应瓶，加入盐酸二氧六环溶液(4.0mol/L，65mL)，室温搅拌3h。减压脱除溶剂，得到琥珀色泡沫状物化合物6，12.3g，收率95％。该化合物可不经纯化直接用于下一步反应。
MS m/z[M+H]+(ESI):418.06。
3.化合物H01的制备
化合物10与化合物6在酰胺缩合剂作用下制备化合物11，化合物11催化氢化脱除苄基得到化合物12，化合物12再次与化合物11在缩合剂作用下得到带有四个靶头的化合物13，化合物13氢化脱除苄基得到化合物14，最后与化合物12反应得到化合物21，化合物21与丁二酸酐反应在仲醇位置引入酯基得到化合物(H01)。
反应瓶中加入化合物10(6.0g，17.9mmol)，加入二氯甲烷(90mL)溶解。加入N，N-二异丙基乙胺(9.2g，71.1mmol)，继续加入HATU(2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯，14.3g，37.6mmol)，室温搅拌3h。化合物6(15.7g，37.6mmol)，用二氯甲烷(60mL)溶解，将此溶液滴加进反应体系，室温搅拌反应4h。加入饱和氯化钠溶液100mL，分液，二氯甲烷相无水硫酸钠干燥，减压脱除溶剂得到红色油状物粗品。加入二氯甲烷30mL，甲基叔丁基醚120mL，搅拌1h，过滤得到黄色固体。重复以上结晶操作两次得到化合物11，9.1g，收率45％。
化合物11(9.0g，7.9mmol)加入反应瓶中，加入水100mL，搅拌溶解。加入5％Pd-C(1.0g)，室温通入氢气3h。过滤除去Pd-C，水中加入氯化钠固体至饱和，二氯甲烷萃取(70mL×4)。合并有机相，无水硫酸钠干燥，减压脱除溶剂得到灰白色固体8.0g，即化合物12，收率96％。
化合物10(1.27g，3.8mmol)加入反应瓶中，加入二氯甲烷10mL搅拌。加入HATU(3.03g，8mmol)，室温搅拌3h。加入化合物12(7.95g，7.6mmol，溶于20mL二氯甲烷)，室温反应2h。加入10％氯化钠溶液100g，分液，分出二氯甲烷相，10％氯化钠溶液洗涤(50g×4)，二氯甲烷相无水硫酸钠干燥，减压脱除溶剂得到棕色固体粗品，加入10mL二氯甲烷，100mL乙酸乙酯，搅拌，倾倒出上清液，瓶底得到胶状固体。加入25mL乙腈，超声溶解胶状固体，静置过夜，过滤得到胶状固体。重复以上结晶操作三次，得到淡黄色胶状固体2.81g，收率31％，即化合物13。
化合物13(2.75g，1.15mmol)加入反应瓶中，加入5％Pd-C(0.7g)，室温通入氢气3h。过滤除去Pd-C，水中加入氯化钠固体至饱和，二氯甲烷萃取(20mL×4)。合并有机相，无水硫酸钠干燥，减压脱除溶剂，得到棕黄色固体2.51g，收率95％，即化合物14。
化合物20(672mg，1.1mmol)加入反应瓶，加入二氯甲烷5mL，继续加入HATU(543mg，1.43mmol)，室温搅拌3h。化合物14(2.50g，1.09mmol，溶于5mL二氯甲烷)加入反应体系，室温搅拌3h。反应也中加入10％氯化钠溶液10g，搅拌分液，有机相10％氯化钠溶液洗涤(10g×3)，无水硫酸钠干燥，减压脱除溶剂得到棕色油状物。加入二氯甲烷4mL，乙酸乙酯30mL，有固体析出，过滤得到灰白色固体725mg，收率23％，即化合物21。
化合物21(620mg，0.21mmol)加入反应瓶中，加入二氯甲烷5mL。继续加入丁二酸酐(210mg，2.1mol)，DMAP(4-二甲氨基吡啶，256mg，2.1mmol)，三乙胺(323mg，3.2mmol)，室温反应24h，LC-MS监测反应进行完全。反应液中加入10％氯化钠溶液10g，分液取有机相，饱和氯化钠洗涤有机相(8g×3)。有机相无水硫酸钠干燥，减压脱除溶剂得到粗品。加入8mL二氯甲烷溶解，再加入乙酸乙酯100mL，有固体析出，减压干燥得到固体580mg，214nm下，纯度71％。
制备液相进行分离纯化，型号Gilson GX281，色谱柱：waters X-bridge C18,19*250mm，10μm。流动相A：碳酸氢铵水溶液，pH＝8-9；流动性B：乙腈。流速20mL/min，监测波长214nm，每针进样量6.0μl，梯度洗脱，得到白色固体粉末180mg，即化合物H01的HPLC纯度98.84％。
HRMS m/z[M-H]-(ESI):2988.3838。
1H NMR(400MHz,Chloroform-d)δδ7.90-7.79(m,9H),7.36-7.25(m,4H),7.25-7.14(m,5H),6.91-6.82(m,4H),5.24(t,J＝7.0Hz,4H),5.18(t,J＝6.6Hz,4H),5.03(d,J＝5.5Hz,4H),4.99(p,J＝4.4Hz,1H),4.25(qd,J＝12.1,4.3Hz,8H),4.11(ddd,J＝9.1,7.1,5.5Hz,4H),3.96(dt,J＝6.4,4.4Hz,4H),3.90-3.78(m,8H),3.71-3.58(m,8H),3.52(qdd,J＝13.6,6.1,4.4Hz,2H),3.28-3.16(m,20H),2.77-2.63(m,4H),2.43(tt,J＝8.1,1.3Hz,6H),2.21(dt,J＝16.9,8.4Hz,10H),2.09(d,J＝15.6Hz,36H),2.02(s,12H),1.72-1.50(m,45H),1.46-1.28(m,24H).
4.化合物(H01)的固相载体的合成
化合物(H01)以酰胺缩合剂连接可控微孔玻璃珠(CPG)固相载体上，得到(H01)的固相载体，用于固相合成制备式缀合物。
将化合物(H01)(50mg，16.73μM)加入DMF 10ml，再加入HBTU(19mg、50μM)、DIEA(16.2mg、125μM)，加入氨基修饰的固相载体(CPG-NH2)0.4g，25℃振荡反应24h。反应完成后，依次用乙腈、二氯甲烷洗涤。继续加入20％乙酸酐/80％乙腈，25℃振荡反应24h。反应完成后，依次用乙腈、二氯甲烷洗涤，得到固相载体目标产物H01-CPG，测得GalNAc载量为24.38μM/g。
5.双链RNA的制备
siRNA合成所需核苷单体原料2'-O-甲基、2'-F、2'-O-TBDMS等核苷亚磷酰胺单体购自上海兆维科技发展有限公司。3％二氯乙酸作为脱保护剂，0.25M 5-乙硫基-1H-四唑乙腈溶液作为活化剂，N,N-二甲基-N'-(3-硫代-3H-1,2,4-二硫唑-5-基)甲脒的吡啶溶液作为硫化试剂，0.05M碘/吡啶/水溶液作为氧化剂，20％乙酸酐乙腈溶液为盖帽剂A，20％乙腈/N-甲基咪唑/吡啶溶液为盖帽剂B，以上相关合成试剂均购自苏州柯乐玛生物技术有限公司。每条RNA单链采用亚磷酰胺固相合成，以Universal CPG载体为起始，使用DNA合成仪根据合成程序连接核苷亚磷酰胺单体。每个核苷单体连接时包括脱保护、偶联、氧化或硫化、盖帽四步反应。含有H01缀合物的RNA单链以H01-CPG载体固相合成，H01-CPG载体按照上述方法合成。
固相合成完成后，寡核苷酸采用28％氨水在55℃条件下氨解16h。将上清液浓缩蒸干，使用Resource 15Q色谱柱纯化，通过溴化钠溶液梯度洗脱，并使用3％三氟乙酸溶液脱除DMTr，纯化获得寡核苷酸链。收集洗脱液，采用葡聚糖凝胶G25凝胶柱进行脱盐，获得的寡核苷酸链收集后冻干，离子对色谱检测纯度，质谱分析目标产物分子量。紫外定量所得到的单链寡核苷酸，按等摩尔比互补配对，溶于水中，按常规退火方法形成双链siRNA，并调整至实验所需浓度备用。
表1为合成的siRNA序列表；表2为合成修饰的siRNA序列表；表3为siRNA-H01缀合物。
其中，A、U、G、C代表腺嘌呤、尿嘧啶、鸟嘌呤、胞嘧啶核苷酸；m代表2'-OMe；f代表2'-F；s代表硫代磷酸酯；H01代表半乳糖胺配体。
表1合成的siRNA序列
表2修饰的siRNA序列
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表3 siRNA-H01缀合物
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6.siRNA脂质递送载体的制备
称取4-(N,N-二甲基氨基)丁酸(二亚油基)甲酯(Dlin-MC3-DMA)80mg、二硬脂酰基磷脂酰胆碱(DSPC)20mg、3β-[N-(N',N'-二甲基胺乙基)氨基甲酰基]胆固醇盐酸盐(DC-CHOL)4mg、1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000(PEG-DMG)10mg，加入8ml乙醇，摇匀，配成脂质溶液作为有机相；配制含2mg siRNA的水相溶液，按照siRNA与脂质的质量比为1:50，微量注射器通过Ignite芯片，超滤去除未包载的siRNA和乙醇，浓缩。
检测：
1、Hep 3B细胞抑制活性
检测合成的表2所示的siRNA在Hep 3B细胞上对ASGPR-1mRNA表达水平的抑制率，抑制率结果如表5所示：
将Hep 3B(购自中国科学院上海生科院细胞库)以2×104个细胞接种至96孔细胞培养板中，用含有10％胎牛血清的DMEM完全培养基培养，在37℃、5％CO2条件下培养至细胞密度约90％，每孔中加入0.25μl的Lipofectamin2000(购自赛默飞世尔科技(中国)有限公司集团)，用opti-MEM稀释为25μl，siRNA储备液用opti-MEM稀释为25μL，将稀释好的siRNA与稀释的Lipofectamin2000轻柔混匀，室温孵育30min后，加至96孔板中，并补加DMEM完全培养基至siRNA浓度为25nM,在37℃、5％CO2条件下继续孵育24h。使用磁珠法细胞总RNA提取试剂盒(购自天根生化科技(北京)有限公司)提取各组总RNA并逆转录cDNA，逆转录试剂盒使用Ⅱ1st Strand cDNA Synthesis SuperMix(购自翌圣生物科技股份有限公司)，将获得的cDNA按照Hieff/>Universal TaqMan multiplex qPCR master mix(UDG plus)试剂盒(购自翌圣生物科技股份有限公司)的说明书进行qPCR，按ΔCt法计算各组ASGPR-1mRNA相对含量水平。
用于扩增的目的基因、内参基因的引物和探针序列表4所示：
表4引物序列
表5各组siRNA对Hep 3B细胞人ASGPR-1抑制结果
由表5可见，各组对Hep 3B细胞的ASGPR-1基因表达的抑制有显著效果，且HLR1021003、HLR1021008抑制效果能达到85％。
2.修饰后siRNA在Hep 3B细胞中抑制人ASGPR-1活性
检测如表4所示的正义链和反义链进行修饰后的siRNA在Hep 3B细胞对ASGPR-1mRNA表达水平的抑制率。
按固相合成方法合成siRNA，将1×104个Hep 3B细胞接种至96孔细胞培养板中，37℃、5％CO2培养至细胞密度约90％。每孔中加入0.25μl的Lipofectamin2000，用opti-MEM稀释为25μl，siRNA储备液用opti-MEM稀释为25μL，并补加DMEM完全培养基至siRNA浓度为50nM,在37℃、5％CO2条件下继续孵育24h。提取各组总RNA并逆转录cDNA，Taqman法进行qPCR，按ΔCt法计算各组ASGPR-1mRNA相对含量水平。
实验方法与上述检测方法相同，siRNA的终浓度为50nM，检测结果如表6所示。
表6各组siRNA对Hep 3B细胞人ASGPR-1抑制结果
由表6可见，各组对Hep 3B细胞的ASGPR-1基因表达的抑制有显著效果，且HLR102203、HLR102207、HLR102208、HLR102209，HLR1022011、HLR1022015，HLR1022017，HLR1022018、HLR1022021、HLR1022022抑制效果能达到85％。
3.H01-siRNA缀合物抑制小鼠ASGPR-1表达
按上述方法合成H01-siRNA缀合物，构建人ASGPR-1的AAV8载体，取6周龄C57/BL小鼠，按照1×1011VG/只的剂量，尾静脉注射AAV8载体。14天后，皮下给予siRNA缀合物，分别在0、7天取血液，收集血浆后用ELISA法测定血浆中ASGPR-1浓度，并以0天血浆中ASGPR-1含量作为100％，计算7天血浆ASGPR-1的相对含量。
表7 siRNA缀合物在小鼠体内抑制效果
由表7可见，siRNA缀合物采用皮下给药后，可以显著降低小鼠血液中ASGPR-1蛋白水平。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。
Claims (10)
1.一种抑制ASGPR-1基因的寡核苷酸或其药学上可接受的盐，包含正义链和反义链，其特征在于，所述正义链如SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ IDNO:7或SEQ ID NO:8所示的不超过3个核苷酸差异的核苷酸序列；所述反义链如SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17或SEQ ID NO:18所示的不超过3个核苷酸差异的核苷酸序列；所述正义链与所述反义链至少80％互补。
2.根据权利要求1所述的抑制ASGPR-1基因的寡核苷酸或其药学上可接受的盐，其特征在于，所述修饰的核苷酸独立地选自2’-甲基修饰的核苷酸、2’甲氧基修饰的核苷酸、2’-氨基乙基修饰的核苷酸、2’-O-甲氧基乙基修饰的核苷酸、2’-氟修饰的核苷酸、5’-硫代磷酸酯基团的核苷酸、连接于胆固醇基衍生物或月桂酸二癸酰胺基团或VP的末端核苷酸。
3.根据权利要求2所述的寡核苷酸或其药学上可接受的盐，其特征在于，所述正义链与所述反义链包含硫代磷酸酯基修饰和乙烯基磷酸酯基修饰。
4.根据权利要求3所述的抑制ASGPR-1基因的寡核苷酸或其药学上可接受的盐，其特征在于，正义链如SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ IDNO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ IDNO:39、SEQ ID NO:40、SEQ ID NO:41或SEQ ID NO:42所示的不超过3个核苷酸差异的修饰核苷酸序列；其反义链的核苷酸序列如SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQID NO:49、SEQ ID NO:50或SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54或SEQ ID NO:55、SEQ ID NO:56、SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63或SEQ ID NO:64所示的不超过3个核苷酸差异的修饰核苷酸序列。
5.一种缀合物，其特征在于，包括配体和通过配体缀合的如权利要求1-4任一项所述寡核苷酸或其药学上可接受的盐。
6.根据权利要求5所述的缀合物，其特征在于，所述配体为去唾液酸糖蛋白受体的配体，所述配体缀合在正义链3’末端或/和5’末端；更进一步地，所述配体缀合在正义链3’末端。
7.一种组合物，其特征在于，包括如权利要求1-3任一项所述寡核苷酸或其药学上可接受的盐和药学上可接受的载体；所述寡核苷酸或其药学上可接受的盐与药学上可接受的载体重量比为1：1-500。
8.根据权利要求7所述的组合物，其特征在于，所述药学上可接受的载体为脂质体、聚合物、多肽、蛋白、单抗、外泌体或纳米材料。
9.根据权利要求7所述的组合物，其特征在于，所述药学上可接受的载体还含有辅助脂质和聚乙二醇化脂质和/或含有有机胺。
10.如权利要求1-4任一项所述抑制ASGPR-1基因的寡核苷酸或其药学上可接受的盐、如权利要求5-6任一项缀合物或如权利要求7-9任一项所述组合物在制备用于预防、治疗或缓解ASGPR-1相关疾病和症状的药物中的用途。
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