本申请是申请号为202080009787.1、申请日为2020-01-17、名称为“一种核酸、含有该核酸的组合物与缀合物及制备方法和用途”的中国发明专利申请的分案申请。This application is a divisional application of the Chinese invention patent application with application number 202080009787.1, application date 2020-01-17, and name “A nucleic acid, a composition and conjugate containing the nucleic acid, and a preparation method and use”.
技术领域Technical Field
本公开涉及一种能够抑制因子XII基因表达的核酸和含有该核酸的组合物与缀合物。本公开还涉及这些核酸、组合物与缀合物的制备方法和用途。The present disclosure relates to a nucleic acid capable of inhibiting the expression of factor XII gene and a composition and a conjugate containing the nucleic acid. The present disclosure also relates to the preparation method and use of the nucleic acid, composition and conjugate.
背景技术Background Art
遗传性血管性水肿(HAE)是一种罕见疾病，其特征在于严重肿胀的反复发作。身体最常见的肿胀区域是四肢、面部、肠道和气道。发作可能是自发性的，也可能是由于身体创伤或压力引起的。喉部(气道)水肿可能会危及生命，因为它可能导致窒息死亡。Hereditary angioedema (HAE) is a rare disease characterized by recurrent episodes of severe swelling. The most common areas of the body to swell are the limbs, face, intestines, and airways. Episodes may be spontaneous or triggered by physical trauma or stress. Laryngeal (airway) edema can be life-threatening as it can lead to death from suffocation.
因子XII是一种主要在肝脏中表达并在血液中发现的丝氨酸蛋白酶，在内源性凝血途径和激肽-激肽释放酶(kinin-kallikrein)系统中具有双重功能。kinin-kallikrein系统在炎症、血压控制、凝血和疼痛中起作用。因子XII(也称为FXII，F12或Hageman因子)的活性形式结合并切割凝血级联中的因子XI和激肽-激肽释放酶系统中的前激肽释放酶，分别产生活性形式FXI和激肽释放酶。Factor XII is a serine protease expressed primarily in the liver and found in the blood that has dual functions in the intrinsic coagulation pathway and the kinin-kallikrein system. The kinin-kallikrein system plays a role in inflammation, blood pressure control, coagulation, and pain. The active form of factor XII (also known as FXII, F12, or Hageman factor) binds to and cleaves factor XI in the coagulation cascade and prekallikrein in the kinin-kallikrein system to produce the active forms FXI and kallikrein, respectively.
因子XII是治疗HAE的关键靶点之一。通过抑制因子XII表达，能够有效抑制HAE的发生。因此，若能从基因水平沉默基因表达，阻断因子XII的生成，无疑将是最为理想的治疗手段。小干扰RNA(small interfering RNA，siRNA)可基于RNA干扰(RNA interference，RNAi)这一机制，以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。Factor XII is one of the key targets for the treatment of HAE. By inhibiting the expression of Factor XII, the occurrence of HAE can be effectively inhibited. Therefore, if gene expression can be silenced at the gene level and the production of Factor XII can be blocked, it will undoubtedly be the most ideal treatment method. Small interfering RNA (siRNA) can inhibit or block the expression of any target gene of interest in a sequence-specific manner based on the mechanism of RNA interference (RNAi), thereby achieving the purpose of treating the disease.
合适的siRNA序列和修饰及其递送系统是小RNA药物开发中的两个关键技术。Appropriate siRNA sequence and modification and its delivery system are two key technologies in the development of small RNA drugs.
发明内容Summary of the invention
在一些实施方式中，本公开提供了一种siRNA缀合物，所述缀合物具有式(308)所示的结构：In some embodiments, the present disclosure provides a siRNA conjugate having a structure shown in formula (308):
其中，in,
n1为选自1-3的整数，n3为选自0-4的整数；n1 is an integer selected from 1-3, n3 is an integer selected from 0-4;
每个m1、m2和m3各自独立地为选自2-10的整数；Each m1, m2 and m3 is independently an integer selected from 2-10;
每个R10、R11、R12、R13、R14和R15各自独立地为H，或选自于由以下基团所组成的组：C1-C10烷基、C1-C10卤代烷基以及C1-C10烷氧基；Each of R10 , R11 , R12 , R13 , R14 and R15 is independently H, or is selected from the group consisting of C1 -C10 alkyl, C1 -C10 haloalkyl and C1 -C10 alkoxy;
R3为式A59所示结构的基团：R3 is a group having a structure represented by formula A59:
其中，E1为OH、SH或BH2；Wherein, E1 is OH, SH or BH2 ;
Nu为siRNA，所述siRNA具有正义链和反义链，所述siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，所述正义链含有一段核苷酸序列I，所述反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I和所述核苷酸序列II选自如下i)-v)中的一组：Nu is siRNA, the siRNA has a sense strand and an antisense strand, each nucleotide in the siRNA is independently a modified or unmodified nucleotide, the sense strand contains a nucleotide sequence I, the antisense strand contains a nucleotide sequence II, the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complemented to form a double-stranded region, wherein the nucleotide sequence I and the nucleotide sequence II are selected from the group consisting of i)-v) below:
i)所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列I中包含位置对应于Z1的核苷酸Z3，所述核苷酸序列II中包含位置对应于Z2的核苷酸Z4，所述Z4是所述反义链5'末端的第一个核苷酸；i) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:1 and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:2 and differs by no more than 3 nucleotides, the nucleotide sequence I comprises the nucleotide Z3 corresponding to the position of Z1 , the nucleotide sequence II comprises the nucleotide Z4 corresponding to the position of Z2 , and Z4 is the first nucleotide at the 5' end of the antisense strand;
ii)所述核苷酸序列I与SEQ ID NO:61所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:62所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列I中包含位置对应于Z5的核苷酸Z7，所述核苷酸序列II中包含位置对应于Z6的核苷酸Z8，所述Z8是所述反义链5'末端的第一个核苷酸；ii) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:61 and differs in no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:62 and differs in no more than 3 nucleotides, the nucleotide sequence I comprises the nucleotide Z7 corresponding to the position Z5 , the nucleotide sequence II comprises the nucleotide Z8 corresponding to the position Z6 , and Z8 is the first nucleotide at the 5' end of the antisense strand;
iii)所述核苷酸序列I与SEQ ID NO:121所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:122所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列I中包含位置对应于Z13的核苷酸Z15，所述核苷酸序列II中包含位置对应于Z14的核苷酸Z16，所述Z16是所述反义链5'末端的第一个核苷酸；iii) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 121 and differs in no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 122 and differs in no more than 3 nucleotides, the nucleotide sequence I comprises the nucleotideZ15 corresponding to the position ofZ13 , the nucleotide sequence II comprises the nucleotideZ16 corresponding to the position ofZ14 , andZ16 is the first nucleotide at the 5' end of the antisense strand;
iv)所述核苷酸序列I与SEQ ID NO:181所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:182所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列I中包含位置对应于Z17的核苷酸Z19，所述核苷酸序列II中包含位置对应于Z18的核苷酸Z20，所述Z20是所述反义链5'末端的第一个核苷酸；iv) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 181 and differs in no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 182 and differs in no more than 3 nucleotides, the nucleotide sequence I comprises the nucleotide Z19 corresponding to the position of Z17 , and the nucleotide sequence II comprises the nucleotide Z20 corresponding to the position of Z18 , and Z20 is the first nucleotide at the 5' end of the antisense strand;
v)所述核苷酸序列I与SEQ ID NO:241所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:242所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列I中包含位置对应于Z21的核苷酸Z23，所述核苷酸序列II中包含位置对应于Z22的核苷酸Z24，所述Z24是所述反义链5'末端的第一个核苷酸；v) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 241 and differs in no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 242 and differs in no more than 3 nucleotides, the nucleotide sequence I comprises the nucleotide Z23 corresponding to the position of Z21 , the nucleotide sequence II comprises the nucleotide Z24 corresponding to the position of Z22 , and Z24 is the first nucleotide at the 5' end of the antisense strand;
R2是长度为1-20个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中R2可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2、-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)；R2 is a straight chain alkylene group having a length of 1 to 20 carbon atoms, wherein one or more carbon atoms are optionally replaced by one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2 , C2 -C10 alkenylene, C2 -C10 alkynylene, C6 -C10 arylene, C3 -C18 heterocyclylene and C5 -C10 heteroarylene; and wherein R2 may optionally have any one or more substituents selected from the group consisting of: C1 -C10 alkyl, C6 -C10 aryl, C5 -C10 heteroaryl, C1 -C10 haloalkyl, -OC1 -C10 alkyl, -OC1 -C10 alkylphenyl, -C1 -C10 alkyl-OH, -OC1 -C10 haloalkyl, -SC1 -C -C10 alkyl), -C1 -C10 alkylphenyl, -C1 -C10 alkyl-SH, -C1 -C10 haloalkyl, halogen substituent, -OH, -SH, -NH2 , -C1 -C10 alkyl-NH2 , -N(C1 -C10 alkyl)(C1 -C10 alkyl), -NH(C1 -C10 alkyl), -N(C1 -C10 alkyl)(C1 -C10 alkylphenyl), -NH(C1 -C10 alkylphenyl), cyano, nitro, -CO2 H, -C(O)O(C1 -C10 alkyl), -CON(C1 -C10 alkyl)(C1 -C10 alkyl), -CONH(C1 -C10 alkyl), -CONH2 , -NHC(O)(C1 -C 10 alkyl -C 1 -C10 alkyl), -NHC(O)(phenyl), -N(C1 -C10 alkyl)C(O)(C1 -C10 alkyl), -N(C1 -C10 alkyl)C(O)(phenyl), -C(O)C1 -C10 alkyl, -C(O)C1 -C10 alkylphenyl, -C(O)C1 -C10 haloalkyl, -OC(O)C1 -C10 alkyl, -SO2 (C1 -C10 alkyl), -SO2 (phenyl), -SO2 (C1 -C10 haloalkyl), -SO2 NH2 , -SO2 NH(C1 -C10 alkyl), -SO2 NH(phenyl), -NHSO2 (C1 -C10 alkyl), -NHSO2 (phenyl) and -NHSO2 (C1 -C10 haloalkyl);
每个L1独立地是长度为1-70个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的任何一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中，L1可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)；Each L1 is independently a straight chain alkylene group having a length of 1 to 70 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2 , C2 -C10 alkenylene, C2 -C10 alkynylene, C6 -C10 arylene, C3 -C18 heterocyclylene and C5 -C10 heteroarylene; and wherein L1 may optionally have any one or more substituents selected from the group consisting of: C1 -C10 alkyl, C6 -C10 aryl, C5 -C10 heteroaryl, C1 -C10 haloalkyl, -OC1 -C10 alkyl, -OC1 -C10 alkylphenyl, -C1 -C10 alkyl-OH, -OC1 -C10 haloalkyl, -SC1 -C10 alkyl, -SC1 -C10 alkylphenyl, -C1 -C10 alkyl-SH, -SC 1 -C 10haloalkyl, halogen substituent, -OH, -SH, -NH2 , -C1 -C10 alkyl-NH2 , -N(C1 -C10 alkyl)(C1 -C10 alkyl), -NH(C1 -C10 alkyl), -N(C1 -C10 alkyl)(C1 -C10 alkylphenyl), -NH(C1 -C10 alkylphenyl), cyano, nitro, -CO2 H, -C(O)O(C1 -C10 alkyl), -CON(C1 -C10 alkyl)(C1 -C10 alkyl), -CONH(C1 -C10 alkyl), -CONH2 , -NHC(O)(C1 -C 10 alkyl -C 1 -C10 alkyl), -NHC(O)(phenyl), -N(C1 -C10 alkyl)C(O)(C1 -C10 alkyl), -N(C1 -C10 alkyl)C(O)(phenyl), -C(O)C1 -C10 alkyl, -C(O)C1 -C10 alkylphenyl, -C(O)C1 -C10 haloalkyl, -OC(O)C1 -C10 alkyl, -SO2 (C1 -C10 alkyl), -SO2 (phenyl), -SO2 (C1 -C10 haloalkyl), -SO2 NH2 , -SO2 NH(C1 -C10 alkyl), -SO2 NH(phenyl), -NHSO2 (C1 -C10 alkyl), -NHSO2 (phenyl) and -NHSO2 (C1 -C10 haloalkyl);
表示基团共价连接的位点； represents the site of covalent attachment of a group;
M1表示靶向基团。M1 represents a targeting group.
在一些实施方式中，本公开提供了一种siRNA，所述siRNA含有正义链和反义链，所述正义链和所述反义链中的每一个核苷酸独立地为氟代修饰的核苷酸或非氟代修饰的核苷酸；所述正义链含有一段核苷酸序列I，所述反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，所述氟代修饰的核苷酸位于核苷酸序列I和核苷酸序列II中，并且，按照5'末端到3'末端的方向，在所述正义链中，所述核苷酸序列I的第7、8、9位的核苷酸为氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为非氟代修饰的核苷酸；按照5'末端到3'末端的方向，在所述反义链中，所述核苷酸序列II的第2、6、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为非氟代修饰的核苷酸，并且，In some embodiments, the present disclosure provides an siRNA, wherein the siRNA contains a sense strand and an antisense strand, wherein each nucleotide in the sense strand and the antisense strand is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide; the sense strand contains a nucleotide sequence I, and the antisense strand contains a nucleotide sequence II, wherein the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complemented to form a double-stranded region, the fluorinated modified nucleotides are located in the nucleotide sequence I and the nucleotide sequence II, and, in the direction from the 5' end to the 3' end, in the sense strand, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence I are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the sense strand are non-fluorinated modified nucleotides; in the direction from the 5' end to the 3' end, in the antisense strand, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence II are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are non-fluorinated modified nucleotides, and,
i)所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列I中包含位置对应于Z1的核苷酸Z3，所述核苷酸序列II中包含位置对应于Z2的核苷酸Z4，所述Z4是所述反义链5'末端的第一个核苷酸；或者，i) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:1 and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:2 and differs by no more than 3 nucleotides, the nucleotide sequence I contains the nucleotide Z3 corresponding to the position of Z1 , the nucleotide sequence II contains the nucleotide Z4 corresponding to the position of Z2 , and Z4 is the first nucleotide at the 5' end of the antisense strand; or,
ii)所述核苷酸序列I与SEQ ID NO:61所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:62所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列I中包含位置对应于Z5的核苷酸Z7，所述核苷酸序列II中包含位置对应于Z6的核苷酸Z8，所述Z8是所述反义链5'末端的第一个核苷酸；或者，ii) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:61 and differs in no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:62 and differs in no more than 3 nucleotides, the nucleotide sequence I comprises the nucleotide Z7 corresponding to the position of Z5 , the nucleotide sequence II comprises the nucleotide Z8 corresponding to the position of Z6 , and Z8 is the first nucleotide at the 5' end of the antisense strand; or,
iii)所述核苷酸序列I与SEQ ID NO:121所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:122所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列I中包含位置对应于Z13的核苷酸Z15，所述核苷酸序列II中包含位置对应于Z14的核苷酸Z16，所述Z16是所述反义链5'末端的第一个核苷酸；或者，iii) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 121 and differs in no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 122 and differs in no more than 3 nucleotides, the nucleotide sequence I comprises the nucleotideZ15 corresponding to the position ofZ13 , the nucleotide sequence II comprises the nucleotideZ16 corresponding to the position ofZ14 , andZ16 is the first nucleotide at the 5' end of the antisense strand; or,
iv)所述核苷酸序列I与SEQ ID NO:181所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:182所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列I中包含位置对应于Z17的核苷酸Z19，所述核苷酸序列II中包含位置对应于Z18的核苷酸Z20，所述Z20是所述反义链5'末端的第一个核苷酸；或者，iv) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO: 181 and differs in no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO: 182 and differs in no more than 3 nucleotides, the nucleotide sequence I comprises the nucleotide Z19 corresponding to the position of Z17 , and the nucleotide sequence II comprises the nucleotide Z20 corresponding to the position of Z18 , and Z20 is the first nucleotide at the 5' end of the antisense strand; or,
v)所述核苷酸序列I与SEQ ID NO:241所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:242所示的核苷酸序列长度相等，且不多于3个核苷酸差异，所述核苷酸序列I中包含位置对应于Z21的核苷酸Z23，所述核苷酸序列II中包含位置对应于Z22的核苷酸Z24，所述Z24是所述反义链5'末端的第一个核苷酸。v) the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:241 and differs in no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:242 and differs in no more than 3 nucleotides, the nucleotide sequence I comprises the nucleotideZ23 corresponding to the position ofZ21 , the nucleotide sequence II comprises the nucleotideZ24 corresponding to the position ofZ22 , andZ24 is the first nucleotide at the 5' end of the antisense strand.
在一些实施方式中，本公开提供了一种药物组合物，所述药物组合物含有本公开的siRNA和药学上可接受的载体。In some embodiments, the present disclosure provides a pharmaceutical composition comprising the siRNA of the present disclosure and a pharmaceutically acceptable carrier.
在一些实施方式中，本公开提供了一种siRNA缀合物，所述siRNA缀合物含有本公开提供的siRNA以及缀合连接至该siRNA的缀合基团。In some embodiments, the present disclosure provides a siRNA conjugate, wherein the siRNA conjugate contains the siRNA provided by the present disclosure and a conjugation group conjugated to the siRNA.
在一些实施方式中，本公开提供了本公开的siRNA和/或药物组合物和/或siRNA缀合物在制备用于治疗和/或预防遗传性血管性水肿HAE和/或血栓形成的药物中的用途。In some embodiments, the present disclosure provides use of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure in the preparation of a medicament for treating and/or preventing hereditary angioedema HAE and/or thrombosis.
在一些实施方式中，本公开提供了一种治疗和/或预防HAE和/或血栓形成的方法，所述方法包括将有效量的本公开的siRNA和/或药物组合物和/或siRNA缀合物给予患有HAE的受试者。In some embodiments, the present disclosure provides a method for treating and/or preventing HAE and/or thrombosis, the method comprising administering an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure to a subject suffering from HAE.
在一些实施方式中，本公开提供了一种抑制肝细胞中FXII基因表达的方法，该方法包括将有效量的本公开的siRNA和/或药物组合物和/或siRNA缀合物与所述肝细胞接触。In some embodiments, the present disclosure provides a method for inhibiting FXII gene expression in hepatocytes, the method comprising contacting an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure with the hepatocytes.
在一些实施方式中，本公开提供了一种试剂盒，所述试剂盒含有本公开的siRNA和/或药物组合物和/或siRNA缀合物。In some embodiments, the present disclosure provides a kit comprising the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure.
以引用的方式并入Incorporated by Reference
本说明书中提及的所有出版物、专利以及专利申请均以引用的方式并入本文，其程度与每一单独的出版物、专利或专利申请均专门并且单独地以引用的方式并入本文的程度相同。All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
有益效果Beneficial Effects
本公开提供的siRNA、含该siRNA的组合物和siRNA缀合物具有良好的稳定性，较高的基因抑制活性，和/或能显著治疗或缓解HAE症状。The siRNA, composition containing the siRNA and siRNA conjugate provided by the present disclosure have good stability, high gene inhibition activity, and/or can significantly treat or alleviate HAE symptoms.
在一些实施方式中，本公开提供的siRNA、含该siRNA的组合物或siRNA缀合物在体外细胞实验中显示出优异的靶基因抑制活性。在一些实施方式中，在50nM的剂量下，本公开提供的siRNA在人肝原代细胞中显示出高达78.70％的FXII mRNA表达量抑制率。在一些实施方式中，在50nM的剂量下，本公开提供的siRNA在C57小鼠肝原代细胞中显示出高达70.09％的FXII mRNA表达量抑制率。In some embodiments, the siRNA provided by the present disclosure, the composition containing the siRNA, or the siRNA conjugate shows excellent target gene inhibition activity in in vitro cell experiments. In some embodiments, at a dose of 50nM, the siRNA provided by the present disclosure shows an inhibition rate of up to 78.70% of FXII mRNA expression in human primary liver cells. In some embodiments, at a dose of 50nM, the siRNA provided by the present disclosure shows an inhibition rate of up to 70.09% of FXII mRNA expression in C57 mouse primary liver cells.
在一些实施方式中，在5mg/kg剂量下，本公开的siRNA缀合物在C57小鼠中显示出高达98.7％的FXIImRNA表达量抑制率。In some embodiments, at a dose of 5 mg/kg, the siRNA conjugates of the present disclosure showed an inhibition rate of up to 98.7% of FXII mRNA expression in C57 mice.
在一些实施方式中，本公开提供的siRNA、含该siRNA的组合物或siRNA缀合物未显示出明显脱靶效应。脱靶效应可以是例如抑制非靶基因的基因正常表达。据认为，如果脱靶基因表达的结合/抑制与在靶基因效果相比低于50％、40％、30％、20％或10％时，该脱靶效应就是不显著的。In some embodiments, the siRNA, the composition containing the siRNA or the siRNA conjugate provided by the present disclosure does not show obvious off-target effects. Off-target effects can be, for example, inhibition of normal gene expression of non-target genes. It is believed that if the binding/inhibition of off-target gene expression is less than 50%, 40%, 30%, 20% or 10% compared to the on-target gene effect, the off-target effect is not significant.
由此说明，本公开提供的siRNA、药物组合物以及siRNA缀合物能够抑制FXII基因的表达，有效治疗和/或预防HAE症状，具有良好的应用前景。This shows that the siRNA, pharmaceutical composition and siRNA conjugate provided by the present disclosure can inhibit the expression of FXII gene, effectively treat and/or prevent HAE symptoms, and have good application prospects.
本公开的其他特征和优点将在随后的具体实施方式部分予以详细说明。Other features and advantages of the present disclosure will be described in detail in the following detailed description.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1和图2分别是给予C57小鼠PBS和不同剂量的各缀合物后，C57小鼠肝脏组织FXIImRNA表达量(以GAPDH为内参的相对值)的散点图。Figures 1 and 2 are scatter plots of the expression levels of FXII mRNA in liver tissue of C57 mice (relative values with GAPDH as an internal reference) after administration of PBS and different doses of each conjugate to C57 mice, respectively.
具体实施方式DETAILED DESCRIPTION
以下对本公开的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本公开，并不用于限制本公开。The specific embodiments of the present disclosure are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure, and are not used to limit the present disclosure.
在本公开中，FXII mRNA是指如Genbank注册号NM_000505.3所示的序列。进一步地，若无其它说明，本公开中所使用的术语“靶基因”是指表达上述FXIImRNA的基因，术语“靶mRNA”是指上述FXIImRNA。In the present disclosure, FXII mRNA refers to the sequence shown in Genbank Accession No. NM_000505.3. Further, if not otherwise specified, the term "target gene" used in the present disclosure refers to a gene expressing the above FXII mRNA, and the term "target mRNA" refers to the above FXII mRNA.
定义definition
在上文及下文中，如无特别说明，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示与该字母s左右相邻的两个核苷酸之间为硫代磷酸酯基连接；P1表示该P1右侧相邻的一个核苷酸为5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸。在一些实施方式中，P1是表示具体修饰的VP、Ps或P，其中，字母组合VP表示该字母组合VP右侧相邻的一个核苷酸为乙烯基磷酸酯修饰的核苷酸，字母组合Ps表示该字母组合Ps右侧相邻的一个核苷酸为硫代磷酸酯修饰的核苷酸，大写字母P表示该字母P右侧相邻的一个核苷酸为5'-磷酸核苷酸。In the above and below, unless otherwise specified, capital letters C, G, U, and A represent the base composition of nucleotides; lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a fluorinated modified nucleotide; lowercase letter s represents that the two nucleotides adjacent to the left and right of the letter s are connected by a thiophosphate group; P1 represents that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide. In some embodiments, P1 is VP, Ps, or P representing a specific modification, wherein the letter combination VP represents that the nucleotide adjacent to the right of the letter combination VP is a vinyl phosphate modified nucleotide, the letter combination Ps represents that the nucleotide adjacent to the right of the letter combination Ps is a thiophosphate modified nucleotide, and the capital letter P represents that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide.
在上文及下文中，所述“氟代修饰的核苷酸”指核苷酸的核糖基2'位的羟基被氟取代形成的核苷酸，“非氟代修饰的核苷酸”指核苷酸的核糖基2'位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物。“核苷酸类似物”指能够在核酸中代替核苷酸，但结构不同于腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶脱氧核糖核苷酸的基团。如异核苷酸、桥联的核苷酸(bridged nucleic acid，简称BNA)或无环核苷酸。所述“甲氧基修饰的核苷酸”指核糖基的2'-羟基被甲氧基取代而形成的核苷酸。In the above and below, the "fluorinated modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and the "non-fluorinated modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group. "Nucleotide analog" refers to a group that can replace nucleotides in nucleic acids but has a structure different from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide or thymine deoxyribonucleotide. Such as isonucleotides, bridged nucleic acids (BNA for short) or acyclic nucleotides. The "methoxy-modified nucleotide" refers to a nucleotide in which the 2'-hydroxyl group of the ribose group is replaced by a methoxy group.
在本文的上下文中，表述“互补”或“反向互补”可互相替代使用，并具有本领域技术人员周知的含义，即，在双链核酸分子中，一条链的碱基与另一条链上的碱基以互补的方式相配对。在DNA中，嘌呤碱基腺嘌呤(A)始终与嘧啶碱基胸腺嘧啶(T)(或者在RNA中为尿嘧啶(U))相配对；嘌呤碱基鸟嘌呤(C)始终与嘧啶碱基胞嘧啶(G)相配对。每个碱基对都包括一个嘌呤和一个嘧啶。当一条链上的腺嘌呤始终与另一条链上的胸腺嘧啶(或尿嘧啶)配对，以及鸟嘌呤始终与胞嘧啶配对时，两条链被认为是彼此互补的，以及从其互补链的序列中可以推断出该链的序列。与此相应地，“错配”在本领域中意指在双链核酸中，对应位置上的碱基并未以互补的形式配对存在。In the context of this article, the expressions "complementary" or "reverse complementary" can be used interchangeably and have the meanings known to those skilled in the art, i.e., in a double-stranded nucleic acid molecule, the bases of one chain are paired with the bases on the other chain in a complementary manner. In DNA, the purine base adenine (A) is always paired with the pyrimidine base thymine (T) (or uracil (U) in RNA); the purine base guanine (C) is always paired with the pyrimidine base cytosine (G). Each base pair includes a purine and a pyrimidine. When adenine on one chain is always paired with thymine (or uracil) on the other chain, and guanine is always paired with cytosine, the two chains are considered to be complementary to each other, and the sequence of the chain can be inferred from the sequence of its complementary chain. Correspondingly, "mismatch" means in the art that in a double-stranded nucleic acid, the bases at corresponding positions are not paired in a complementary form.
在上文及下文中，如无特别说明，“基本上反向互补”是指所涉及的两段核苷酸序列之间存在不多于3个的碱基错配；“实质上反向互补”是指两段核苷酸序列之间存在不多于1个的碱基错配；“完全反向互补”是指两段核苷酸序列之间不存在碱基错配。In the above and below, unless otherwise specified, "substantially reverse complementary" means that there are no more than 3 base mismatches between the two nucleotide sequences involved; "substantially reverse complementary" means that there are no more than 1 base mismatch between the two nucleotide sequences; and "completely reverse complementary" means that there are no base mismatches between the two nucleotide sequences.
在上文及下文中，一个核苷酸序列与另外一个核苷酸序列存在“核苷酸差异”，是指前者与后者相比，相同位置的核苷酸的碱基种类发生了改变，例如，在后者中一个核苷酸碱基为A时，在前者的相同位置处的对应核苷酸碱基为U、C、G或者T的情况下，认定为两个核苷酸序列之间在该位置处存在核苷酸差异。在一些实施方式中，以无碱基核苷酸或其等同物代替原位置的核苷酸时，也可认为在该位置处产生了核苷酸差异。In the above and below, "nucleotide difference" between one nucleotide sequence and another nucleotide sequence means that the base type of the nucleotide at the same position in the former is changed compared with the latter. For example, when a nucleotide base in the latter is A, and the corresponding nucleotide base at the same position in the former is U, C, G or T, it is considered that there is a nucleotide difference at that position between the two nucleotide sequences. In some embodiments, when a nucleotide at the original position is replaced by an abasic nucleotide or its equivalent, it can also be considered that a nucleotide difference occurs at that position.
在上文及下文中，特别是在描述本公开的siRNA、含siRNA的组合物或siRNA缀合物的制备方法时，除非特别说明，所述核苷单体(nucleoside monomer)指，根据欲制备的siRNA或siRNA缀合物中核苷酸的种类和顺序，亚磷酰胺固相合成中使用的修饰或未修饰的核苷亚磷酰胺单体(unmodified or modified RNA phosphoramidites，有时RNAphosphoramidites也称为Nucleoside phosphoramidites)。亚磷酰胺固相合成为本领域技术人员所公知的RNA合成中所用的方法。本公开所用的核苷单体均可商购得到。In the above and below, especially when describing the preparation method of the siRNA, the composition containing siRNA or the siRNA conjugate of the present disclosure, unless otherwise specified, the nucleoside monomer (nucleoside monomer) refers to the modified or unmodified nucleoside phosphoramidite monomer (unmodified or modified RNA phosphoramidites, sometimes RNA phosphoramidites are also referred to as Nucleoside phosphoramidites) used in the phosphoramidite solid phase synthesis according to the type and sequence of nucleotides in the siRNA or siRNA conjugate to be prepared. Phosphoramidite solid phase synthesis is a method used in RNA synthesis known to those skilled in the art. The nucleoside monomers used in the present disclosure are all commercially available.
在本公开的上下文中，除非另有说明，“缀合”是指两个或多个各自具有特定功能的化学部分之间以共价连接的方式彼此连接；相应地，“缀合物”是指该各个化学部分之间通过共价连接而形成的化合物。进一步地，“siRNA缀合物”表示一个或多个具有特定功能的化学部分共价连接至siRNA上而形成的化合物。在下文中，有时也将本公开的siRNA缀合物简称为“缀合物”。siRNA缀合物应根据上下文，理解为siRNA缀合物的总称、式(305)和式(307)所示的siRNA缀合物总称，或式(305)、式(307)、式(308)所示的siRNA缀合物。在本公开的上下文中，“缀合分子”应当理解为可通过反应缀合至siRNA，最终形成本公开的siRNA缀合物的特定化合物。In the context of the present disclosure, unless otherwise specified, "conjugation" refers to the connection between two or more chemical moieties, each having a specific function, in a covalently linked manner; accordingly, "conjugate" refers to a compound formed by covalently linking the chemical moieties. Further, "siRNA conjugate" means a compound formed by covalently linking one or more chemical moieties having a specific function to siRNA. In the following, the siRNA conjugates of the present disclosure are sometimes referred to as "conjugates". According to the context, siRNA conjugates should be understood as the general term for siRNA conjugates, the general term for siRNA conjugates shown in formula (305) and formula (307), or the siRNA conjugates shown in formula (305), formula (307), and formula (308). In the context of the present disclosure, "conjugated molecule" should be understood as a specific compound that can be conjugated to siRNA through a reaction to ultimately form the siRNA conjugates of the present disclosure.
如本文所使用的，不介于两个字母之间或两个符号之间的短横(“-”)用于指示取代基的连接点。例如：结构式“-C1-C10烷基-NH2”中最左侧的短横是指66通过C1-C10烷基而连接。As used herein, a dash ("-") not between two letters or symbols is used to indicate the point of attachment of a substituent. For example, the leftmost dash in the formula "-C1 -C10 alkyl-NH2 " means that 66 is attached through the C1 -C10 alkyl.
如本文所使用的，“任选的”或“任选地”是指其后描述的事件或状况可以发生或不发生，并且该描述包括事件或状况发生的情况和不发生的情况。例如，“任选地取代”的“烷基”包括下文定义的“烷基”和“取代烷基”。本领域技术人员将理解的是，对于包含一个或多个取代基的任何基团，这些基团不打算引入空间上不切实际、合成上不可行和/或本身不稳定的任何取代或取代模式。As used herein, "optional" or "optionally" means that the event or condition described thereafter may or may not occur, and that the description includes instances where the event or condition occurs and instances where it does not occur. For example, "optionally substituted" "alkyl" includes "alkyl" and "substituted alkyl" as defined below. It will be understood by those skilled in the art that for any group containing one or more substituents, these groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically unfeasible, and/or inherently unstable.
如本文所使用的，“烷基”是指具有指定数量的碳原子的直链和支链，所述数量通常为1至20个碳原子，例如1至10个碳原子，如1至8个或1至6个碳原子。例如，C1-C6烷基包含1至6个碳原子的直链和支链烷基。当提及具有特定数量的碳的烷基残基时，旨在涵盖具有该数量的碳的所有支链和直链形式；因此，例如，“丁基”意味着包括正丁基、仲丁基、异丁基和叔丁基；“丙基”包括正丙基和异丙基。亚烷基是烷基的子集，指与烷基相同、但具有两个连接点的残基。As used herein, "alkyl" refers to straight and branched chains having a specified number of carbon atoms, typically 1 to 20 carbon atoms, e.g., 1 to 10 carbon atoms, such as 1 to 8 or 1 to 6 carbon atoms. For example, C1 -C6 alkyl includes straight and branched chain alkyl groups of 1 to 6 carbon atoms. When an alkyl residue having a specific number of carbons is mentioned, it is intended to encompass all branched and straight chain forms having that number of carbons; thus, for example, "butyl" is meant to include n-butyl, sec-butyl, isobutyl, and tert-butyl; "propyl" includes n-propyl and isopropyl. Alkylene is a subset of alkyl and refers to residues that are the same as alkyl, but with two points of attachment.
如本文所使用的，“烯基”是指具有至少一个碳-碳双键的不饱和支链或直链烷基，所述碳-碳双键是通过从母体烷基的相邻碳原子中除去一分子氢而获得的。该基团可以处于双键的顺式或反式构型。典型的烯基基团包括但不限于：乙烯基；丙烯基，如丙-1-烯-1-基、丙-1-烯-2-基、丙-2-烯-1-基(烯丙基)、丙-2-烯-2-基；丁烯基，例如丁-1-烯-1-基、丁-1-烯-2-基、2-甲基丙-1-烯-1-基、丁-2-烯-1-基、丁-2-烯-2-基、丁-1,3-二烯-1-基、丁-1,3-二烯-2-基等等。在某些实施方式中，烯基基团具有2到20个碳原子，而在其他实施方式中，具有2至10个、2至8个或2至6个碳原子。亚烯基是烯基的一个子集，指与烯基相同、但具有两个连接点的残基。As used herein, "alkenyl" refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon double bond, wherein the carbon-carbon double bond is obtained by removing a molecule of hydrogen from the adjacent carbon atoms of the parent alkyl group. The group can be in the cis or trans configuration of the double bond. Typical alkenyl groups include, but are not limited to, vinyl; propenyl, such as prop-1-ene-1-yl, prop-1-ene-2-yl, prop-2-ene-1-yl (allyl), prop-2-ene-2-yl; butenyl, such as but-1-ene-1-yl, but-1-ene-2-yl, 2-methylprop-1-ene-1-yl, but-2-ene-1-yl, but-2-ene-2-yl, but-1,3-diene-1-yl, but-1,3-diene-2-yl, etc. In some embodiments, the alkenyl group has 2 to 20 carbon atoms, and in other embodiments, has 2 to 10, 2 to 8 or 2 to 6 carbon atoms. Alkenylene is a subset of alkenyl and refers to residues that are identical to alkenyl but have two points of attachment.
如本文所使用的，“炔基”是指具有至少一个碳-碳三键的不饱和支链或直链烷基，所述碳-碳三键是通过从母体烷基的相邻碳原子中除去两分子氢而获得的。典型的炔基基团包括但不限于：乙炔基；丙炔基，如丙-1-炔-1-基，丙-2-炔-1-基；丁炔基，例如丁-1-炔-1-基，丁-1-炔-3-基，丁-3-炔-1-基等。在某些实施方式中，炔基具有2到20个碳原子，而在其他实施方式中，具有2至10、2至8或2至6个碳原子。亚炔基是炔基的一个子集，指的是与炔基相同、但有两个连接点的残基。As used herein, "alkynyl" refers to an unsaturated branched or straight chain alkyl group having at least one carbon-carbon triple bond obtained by removing two molecules of hydrogen from adjacent carbon atoms of the parent alkyl group. Typical alkynyl groups include, but are not limited to, ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc. In certain embodiments, the alkynyl group has 2 to 20 carbon atoms, and in other embodiments, has 2 to 10, 2 to 8, or 2 to 6 carbon atoms. Alkynylene is a subset of alkynyl and refers to residues that are identical to alkynyl but have two points of attachment.
如本文所使用的，“烷氧基”是指通过氧桥连接的指定数量碳原子的烷基，例如，甲氧基、乙氧基、丙氧基、异丙氧基、正丁氧基、仲丁氧基、叔丁氧基、戊氧基、2-戊氧基、异戊氧基、新戊氧基、己氧基、2-己氧基、3-己氧基、3-甲基戊氧基等。烷氧基通常具有1至10个、1至8个、1至6个，或1至4个通过氧桥连接的碳原子。As used herein, "alkoxy" refers to an alkyl group of the specified number of carbon atoms attached through an oxygen bridge, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, pentoxy, 2-pentoxy, isopentoxy, neopentoxy, hexoxy, 2-hexoxy, 3-hexoxy, 3-methylpentoxy, etc. Alkoxy groups typically have 1 to 10, 1 to 8, 1 to 6, or 1 to 4 carbon atoms attached through an oxygen bridge.
如本文所使用的，“芳基”是指通过从环碳原子上除去氢原子而衍生自芳香族单环或多环烃环系统形成的基团。所述芳香族单环或多环烃环系统仅含有氢和6至18个碳原子的碳，其中所述环系统中的至少一个环是完全不饱和的，即，包含根据Hückel理论的环状、离域的(4n+2)π-电子体系。芳基包括但不限于苯基、芴基和萘基等基团。亚芳基是芳基的子集，指与芳基相同、但具有两个连接点的残基。As used herein, "aryl" refers to a radical derived from an aromatic monocyclic or polycyclic hydrocarbon ring system by removing hydrogen atoms from ring carbon atoms. The aromatic monocyclic or polycyclic hydrocarbon ring system contains only hydrogen and carbons of 6 to 18 carbon atoms, wherein at least one ring in the ring system is fully unsaturated, i.e., contains a cyclic, delocalized (4n+2)π-electron system according to the Hückel theory. Aryl includes, but is not limited to, radicals such as phenyl, fluorenyl, and naphthyl. Arylene is a subset of aryl and refers to residues identical to aryl but having two points of attachment.
如本文所使用的，“环烷基”是指非芳香族碳环，通常具有3至7个环碳原子。环可以是饱和的，或具有一个或多个碳-碳双键。环烷基的实例包括环丙基、环丁基、环戊基、环戊烯基、环己基和环己烯基，以及桥联和笼状环基团，如降冰片烷(norbornane)。As used herein, "cycloalkyl" refers to a non-aromatic carbocyclic ring, typically having 3 to 7 ring carbon atoms. The ring may be saturated or have one or more carbon-carbon double bonds. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl and cyclohexenyl, as well as bridged and caged ring groups such as norbornane.
如本文所使用的，“卤素取代基”或“卤代”指氟代、氯代、溴代和碘代，术语“卤素”包括氟、氯、溴和碘。As used herein, "halogen substituent" or "halo" refers to fluoro, chloro, bromo and iodo, and the term "halogen" includes fluorine, chlorine, bromine and iodine.
如本文所使用的，“卤代烷基”是指指定数量的碳原子被一个或多个、直至最大允许数量的卤素原子取代的如上述所定义的烷基。卤代烷基的实例包括但不限于三氟甲基、二氟甲基、2-氟乙基和五氟乙基。As used herein, "haloalkyl" refers to an alkyl group as defined above in which a specified number of carbon atoms is replaced by one or more, up to the maximum permitted number of halogen atoms. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, 2-fluoroethyl and pentafluoroethyl.
“杂环基”是指稳定的3-至18-元非芳香族环基，包含2-12个碳原子和1-6个杂原子，所述杂原子选自氮、氧和硫。除非说明书中另有说明，杂环基是单环、双环、三环或四环系统，可包括稠环或桥环系统。杂环基中的杂原子可以任选地被氧化。一个或多个氮原子(如果存在的话)任选地被季铵化。杂环基是部分饱和或完全饱和的。杂环基可以通过任何环原子连接至分子的其余部分。此类杂环基的实例包括但不限于：二噁烷基、噻吩基[1,3]二硫酰基(thienyl[1,3]dithianyl)、十氢异喹啉基、咪唑啉基、咪唑烷基、异噻唑烷基、异噁唑烷基、吗啉基、八氢吲哚基、八氢异吲哚基、2-氧杂哌嗪基、2-氧杂哌啶基、2-氧杂吡咯烷基、噁唑烷基、哌啶基、哌嗪基、4-哌啶酮基、吡咯烷基、吡唑烷基、奎宁环基、噻唑烷基、四氢呋喃基、三硫酰基(trithianyl)、四氢吡喃基、硫代吗啉基(thiomorpholinyl)、硫杂吗啉基(thiamorpholinyl)、1-氧代硫吗啉基(1-oxo-thiomorpholinyl)和1,1-二氧代硫吗啉基(1,1-dioxo-thiomorpholinyl)。"Heterocyclyl" refers to a stable 3- to 18-membered non-aromatic ring radical containing 2-12 carbon atoms and 1-6 heteroatoms selected from nitrogen, oxygen and sulfur. Unless otherwise specified in the specification, a heterocyclyl is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include a fused ring or a bridged ring system. The heteroatoms in the heterocyclyl may be optionally oxidized. One or more nitrogen atoms (if present) may be optionally quaternized. The heterocyclyl is partially saturated or fully saturated. The heterocyclyl may be connected to the rest of the molecule through any ring atom. Examples of such heterocyclic groups include, but are not limited to, dioxanyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxapiperazinyl, 2-oxapiperidinyl, 2-oxapyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuranyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl.
“杂芳基”指由3-至18-元芳香环自由基衍生而成的基团，包含2个至17个碳原子和选自氮、氧和硫的1至6个杂原子。如本文所使用的，杂芳基可以是单环、双环、三环或四环系统，其中环系统中的至少一个环是完全不饱和的，即，包含根据Hückel理论的环状离域(4n+2)π-电子体系。杂芳基包括稠环或桥环系统。杂芳基中的杂原子被任选地氧化。一个或多个氮原子(如果存在的话)任选地被季铵化。杂芳基通过任何环原子附着至分子的其余部分。杂芳基的实例包括但不限于：氮杂环庚三烯基、吖啶基、苯并咪唑基、苯并吲哚基、1,3-苯并二噁唑基、苯并呋喃基、苯并噁唑基、苯并[d]噻唑基、苯并噻二唑基、苯并[b][1,4]二噁庚英基(benzo[b][1,4]dioxepinyl)、苯并[b][1,4]噁嗪基(benzo[b][1,4]oxazinyl)、1,4-苯并二噁烷基(1,4-benzodioxanyl)、苯并萘并呋喃基、苯并噁唑基、苯并间二氧杂环戊烯基(benzodioxolyl)、苯并二噁英基(benzodioxinyl)、苯并吡喃基、苯并吡喃酮基、苯并呋喃基、苯并呋喃酮基、苯并噻吩基、苯并噻吩并[3,2-d]嘧啶基、苯并三唑基、苯并[4,6]咪唑并[1,2-a]吡啶基、咔唑基、噌啉基(cinnolinyl)、环戊烷并[d]嘧啶基、6,7-二氢-5H-环戊烷并[4,5]噻吩并[2,3-d]嘧啶基、5,6-二氢苯并[h]喹唑啉基(5,6-dihydrobenzo[h]quinazolinyl)、5,6-二氢苯并[h]噌啉基(5,6dihydrobenzo[h]cinnolinyl)、6,7-二氢-5H-苯并[6,7]环庚烷并[1,2-c]哒嗪基、二苯并呋喃基、二苯并噻吩基、呋喃基、呋喃酮基、呋喃并[3,2-c]吡啶基、5,6,7,8,9,10-六氢环辛烷并[d]嘧啶基、5,6,7,8,9,10-六氢环辛烷并[d]哒嗪基、5,6,7,8,9,10-六氢环辛烷并[d]吡啶基、异噻唑基、咪唑基、吲唑基(indazolyl)、吲哚基、异吲哚基、二氢吲哚基、异二氢吲哚基、异喹啉基、吲哚嗪基(indolizinyl)、异噁唑基、5,8-甲醇-5,6,7,8-四氢喹唑啉基(5,8-methano-5,6,7,8-tetrahydroquinazolinyl)、萘啶基(naphthyridinyl)、1,6-萘啶酮基(1,6-naphthyridinonyl)、噁二唑基、2-氧杂吖庚因基(2-oxoazepinyl)、噁唑基、氧杂环丙烷基(oxiranyl)、5,6,6a,7,8,9,10,10a-八氢苯并[H]喹唑啉基、1-苯基-1H-吡咯基、吩嗪基、吩噻嗪基、吩噁嗪基、酞嗪基(phthalazinyl)、蝶啶基(pteridinyl)、嘌呤基、吡咯基、吡唑基、吡唑并[3,4-d]嘧啶基、吡啶基、吡啶并[3,2-d]嘧啶基、吡啶并[3,4-d]嘧啶基、吡嗪基、嘧啶基、哒嗪基、吡咯基、喹唑啉基、喹喔啉基(quinoxalinyl)、喹啉基、四氢喹啉基、5,6,7,8-四氢喹唑啉基、5,6,7,8-四氢苯并[4,5]噻吩并[2,3-d]嘧啶基、6,7,8,9-四氢-5H-环庚烷并[4,5]噻吩并[2,3-d]嘧啶基、5,6,7,8-四氢吡啶并[4,5-c]哒嗪基、噻唑基、噻二唑基、三唑基、四唑基、三嗪基、噻吩并[2,3-d]嘧啶基、噻吩并[3,2-d]嘧啶基、噻吩并[2,3-c]吡啶基(thieno[2,3-c]pridinyl)和噻吩基(thiophenyl/thienyl)。"Heteroaryl" refers to a group derived from a 3- to 18-membered aromatic ring radical, containing 2 to 17 carbon atoms and 1 to 6 heteroatoms selected from nitrogen, oxygen and sulfur. As used herein, heteroaryl can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, wherein at least one ring in the ring system is fully unsaturated, i.e., contains a cyclic delocalized (4n+2)π-electron system according to the Hückel theory. Heteroaryl includes fused or bridged ring systems. The heteroatoms in the heteroaryl are optionally oxidized. One or more nitrogen atoms (if present) are optionally quaternized. The heteroaryl is attached to the rest of the molecule through any ring atom. Examples of heteroaryl groups include, but are not limited to, azepine, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxazolyl, benzofuranyl, benzoxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, benzo[b][1,4]oxazinyl, 1,4-benzo dioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothiophenyl, benzothieno[3,2-d]pyrimidinyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, cyclopenta[d]pyrimidinyl, 6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidinyl, 5,6-dihydrobenzo[h]quinazolinyl, 5,6-dihydrobenzo[h]cinnolin ... 7-dihydro-5H-benzo[6,7]cyclohepta[1,2-c]pyridazinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, furano[3,2-c]pyridinyl, 5,6,7,8,9,10-hexahydrocyclooctanol[d]pyrimidinyl, 5,6,7,8,9,10-hexahydrocyclooctanol[d]pyridazinyl, 5,6,7,8,9,10-hexahydrocyclooctanol[d]pyridinyl, isothiazolyl, imidazolyl, indazolyl (ind azolyl), indolyl, isoindolyl, dihydroindolyl, isoindolyl, isoquinolinyl, indolizinyl, isoxazolyl, 5,8-methano-5,6,7,8-tetrahydroquinazolinyl, naphthyridinyl, 1,6-naphthyridinonyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 5,6,6a,7,8,9,10,10a-octahydrobenzo[H]quinazolinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyrazolo[3,4-d]pyrimidinyl, pyridinyl, pyrido[3,2-d]pyrimidinyl, pyrido[3,4-d]pyrimidinyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, quinazolinyl, quinoxalinyl, quinolinyl, tetrahydroquinolinyl, 5,6,7,8-tetrahydroquinazolinyl, 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinyl, 6,7,8,9-tetrahydro -5H-cyclohepta[4,5]thieno[2,3-d]pyrimidinyl, 5,6,7,8-tetrahydropyrido[4,5-c]pyridazinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thieno[2,3-d]pyrimidinyl, thieno[3,2-d]pyrimidinyl, thieno[2,3-c]pyridinyl (thieno[2,3-c]pridinyl) and thiophenyl (thienyl).
在本公开中可以使用各种羟基保护基团。一般来说，保护基团使化学官能团对特定的反应条件不敏感，并且可以在分子中的该官能团上添加以及去除，而不实质上损害分子的其余部分。代表性的羟基保护基团公开于Beaucage等人，Tetrahedron 1992,48,2223-2311，以及Greeneand Wuts，Protective Groups in Organic Synthesis，Chapter 2,2ded，John Wiley&Sons，New York，1991中，以引用的方式将上述文献整体并入本文。在一些实施方式中，保护基团在碱性条件下稳定，但可以在酸性条件下脱除。在一些实施方式中，本文可使用的羟基保护基的非排他性实例包括二甲氧基三苯甲基(DMT)、单甲氧基三苯甲基、9-苯基氧杂蒽-9-基(Pixyl)和9-(对甲氧基苯基)氧杂蒽-9-基(Mox)。在一些实施方式中，本文可使用的羟基保护基的非排他性实例包括Tr(三苯甲基)、MMTr(4-甲氧基三苯甲基)、DMTr(4,4'-二甲氧基三苯甲基)和TMTr(4,4',4”-三甲氧基三苯甲基)。Various hydroxyl protecting groups can be used in the present disclosure. In general, the protecting group makes the chemical functional group insensitive to specific reaction conditions, and can be added and removed on the functional group in the molecule without substantially damaging the rest of the molecule. Representative hydroxyl protecting groups are disclosed in Beaucage et al., Tetrahedron 1992, 48, 2223-2311, and Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2ded, John Wiley & Sons, New York, 1991, and the above-mentioned documents are incorporated herein by reference in their entirety. In some embodiments, the protecting group is stable under alkaline conditions, but can be removed under acidic conditions. In some embodiments, non-exclusive examples of hydroxyl protecting groups that can be used herein include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenyloxanthene-9-yl (Pixyl) and 9-(p-methoxyphenyl)oxanthene-9-yl (Mox). In some embodiments, non-exclusive examples of hydroxy protecting groups that may be used herein include Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-dimethoxytrityl), and TMTr (4,4',4"-trimethoxytrityl).
“受试者”一词，如本文所使用的，指任何动物，例如哺乳动物或有袋动物。本公开的受试者包括但不限于人类、非人灵长类(例如，恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。The term "subject", as used herein, refers to any animal, such as a mammal or marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.
如本文所使用的，“治疗”、“减轻”或“改善”可在此处互换使用。这些术语指的是获得有益的或期望的结果的方法，包括但不限于治疗益处。“治疗益处”意味着根除或改善被治疗的潜在障碍。此外，治疗益处通过根除或改善与潜在障碍相关的一个或多个生理症状，从而在受试者中观察到改善而获得，尽管受试者可能仍然受到潜在障碍的折磨。As used herein, "treat," "treat," "alleviate," or "amortize" are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results, including, but not limited to, a therapeutic benefit. "Therapeutic benefit" means eradication or amelioration of the underlying disorder being treated. Furthermore, a therapeutic benefit is obtained by eradication or amelioration of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.
如本文所使用的，“防止”和“预防”可互换使用。这些术语指获得有益或期望的结果的方法，包括但不限于预防性益处。为了获得“预防性益处”，可将缀合物或组合物给予有罹患特定疾病风险的受试者，或给予报告疾病的一种或多种生理症状的受试者，即便可能该疾病的诊断尚未作出。As used herein, "preventing" and "prevention" are used interchangeably. These terms refer to an approach to obtaining beneficial or desired results, including but not limited to a prophylactic benefit. To obtain a "prophylactic benefit," a conjugate or composition may be administered to a subject at risk for a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have been made.
本公开的siRNA含有核苷酸基团作为基本结构单元，本领域技术人员公知，所述核苷酸基团含有磷酸基团、核糖基团和碱基，在此不再赘述。The siRNA disclosed herein contains a nucleotide group as a basic structural unit. It is well known to those skilled in the art that the nucleotide group contains a phosphate group, a ribose group and a base, which will not be described in detail here.
第一种siRNAThe first siRNA
按照本公开，所述siRNA可以是第一种siRNA。According to the present disclosure, the siRNA may be a first siRNA.
所述第一种siRNA含有正义链和反义链，所述第一种siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，所述反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列长度相等，且不多于3个核苷酸差异：The first siRNA comprises a sense strand and an antisense strand, and each nucleotide in the first siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, wherein the nucleotide sequence I is equal in length to the nucleotide sequence shown in SEQ ID NO:1 and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal in length to the nucleotide sequence shown in SEQ ID NO:2 and differs by no more than 3 nucleotides:
5'-GGAACUCAAUAAAGUGCUZ1-3'(SEQ ID NO:1)；5'-GGAACUCAAUAAAGUGCUZ1 -3' (SEQ ID NO: 1);
5'-Z2AGCACUUUAUUGAGUUCC-3'(SEQ ID NO:2)，5'-Z2 AGCACUUUAUUGAGUUCC-3' (SEQ ID NO: 2),
其中，Z1为U，Z2为A，Among them,Z1 is U,Z2 is A,
并且，所述核苷酸序列I中包含位置对应于Z1的核苷酸Z3，所述核苷酸序列II中包含位置对应于Z2的核苷酸Z4，所述Z4是所述反义链5'末端的第一个核苷酸。Furthermore, the nucleotide sequence I comprises a nucleotide Z3 corresponding to position Z1 , and the nucleotide sequence II comprises a nucleotide Z4 corresponding to position Z2 , and Z4 is the first nucleotide at the 5' end of the antisense strand.
在上文与下文中，“位置对应”是指从核苷酸序列相同端起算，处于核苷酸序列中相同的位置。例如，核苷酸序列I的3'端第1个核苷酸是位置对应于SEQ ID NO:1的3'端第1个核苷酸的核苷酸。In the above and below, "position corresponding" means being at the same position in the nucleotide sequence, starting from the same end of the nucleotide sequence. For example, the first nucleotide at the 3' end of nucleotide sequence I is the nucleotide that corresponds to the first nucleotide at the 3' end of SEQ ID NO:1.
在一些实施方式中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。In some embodiments, the sense strand comprises only nucleotide sequence I, and the antisense strand comprises only nucleotide sequence II.
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:1所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列之间不多于1个核苷酸差异。In some embodiments, there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:1, and/or there is no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:2.
在一些实施方式中，所述核苷酸序列II与SEQ ID NO:2所示的核苷酸序列之间的核苷酸差异包括Z4位置处的差异，且Z4选自U、C或G。在一些实施方式中，所述核苷酸差异为Z4位置处的差异，Z4选自U、C或G。在一些实施方式中，Z3是与Z4互补的核苷酸。这些核苷酸差异并不会显著降低siRNA缀合物的靶基因抑制能力，而包含这些核苷酸差异的siRNA缀合物也在本公开的保护范围之内。In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 2 includes a difference at position Z4 , and Z4 is selected from U, C or G. In some embodiments, the nucleotide difference is a difference at position Z4 , and Z4 is selected from U, C or G. In some embodiments, Z3 is a nucleotide complementary to Z4. These nucleotide differences do not significantly reduce the target gene inhibition ability of the siRNA conjugate, and the siRNA conjugates containing these nucleotide differences are also within the scope of protection of the present disclosure.
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补。在本公开的上下文中术语“基本上反向互补”是指两个核苷酸序列之间存在不多于3个的碱基错配；“实质上反向互补”是指两个核苷酸序列之间存在不多于1个的碱基错配；“完全反向互补”是指两个核苷酸序列之间没有碱基错配。In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, substantially reverse complementary or completely reverse complementary. In the context of the present disclosure, the term "substantially reverse complementary" means that there are no more than 3 base mismatches between two nucleotide sequences; "substantially reverse complementary" means that there are no more than 1 base mismatch between two nucleotide sequences; "completely reverse complementary" means that there are no base mismatches between two nucleotide sequences.
在一些实施方式中，核苷酸序列I是SEQ ID NO:3所示的核苷酸序列，核苷酸序列II是SEQ ID NO:4所示的核苷酸序列：In some embodiments, nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:3, and nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:4:
5'-GGAACUCAAUAAAGUGCUZ3-3'(SEQ ID NO:3)；5'-GGAACUCAAUAAAGUGCUZ3-3 ' (SEQ ID NO: 3);
5'-Z4AGCACUUUAUUGAGUUCC-3'(SEQ ID NO:4)，5'-Z4 AGCACUUUAUUGAGUUCC-3' (SEQ ID NO: 4),
其中，所述Z4是反义链5'末端的第一个核苷酸，Z3选自A、U、G或C，并且Z4是与Z3互补的核苷酸；在一些实施方式中，Z3为U，Z4为A；Wherein, Z4 is the first nucleotide at the 5' end of the antisense strand, Z3 is selected from A, U, G or C, and Z4 is a nucleotide complementary to Z3 ; in some embodiments, Z3 is U and Z4 is A;
并且，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，反义链的长度为19-26个核苷酸。Furthermore, the sense strand and the antisense strand have the same or different lengths, the sense strand has a length of 19-23 nucleotides, and the antisense strand has a length of 19-26 nucleotides.
在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV长度各自为1-4个核苷酸；所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或者完全反向互补；所述核苷酸序列III连接在所述核苷酸序列I的5'末端，所述核苷酸序列IV连接在所述核苷酸序列II的3'末端。在一些实施方式中，所述核苷酸序列IV与第二段核苷酸序列实质上反向互补或者完全反向互补，该第二段核苷酸序列是指和靶mRNA中与由SEQ ID NO:1表示的核苷酸序列的5'末端相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。In some embodiments, the sense strand further comprises a nucleotide sequence III, and the antisense strand further comprises a nucleotide sequence IV, wherein the length of the nucleotide sequence III and the nucleotide sequence IV are 1-4 nucleotides respectively; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II. In some embodiments, the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, which refers to a nucleotide sequence that is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO: 1 in the target mRNA and has the same length as the nucleotide sequence IV.
在一些实施方式中，按照5'-3'的方向，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为A，核苷酸序列IV的碱基为U；此时，正义链和反义链的长度比为20/20；或者，核苷酸序列III和IV的长度均为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为CA，核苷酸序列IV的碱基组成为UG；此时，正义链和反义链的长度比为21/21；或者，核苷酸序列III和IV的长度均为3个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为GCA，核苷酸序列IV的碱基组成为UGC；此时，正义链和反义链的长度比为22/22；或者，核苷酸序列III和IV的长度均为4个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为CGCA，核苷酸序列IV的碱基组成为UGCG；此时，正义链和反义链的长度比为23/23。在一些实施方式中，所述核苷酸序列III和核苷酸序列IV的长度为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为CA，核苷酸序列IV的碱基组成为UG；此时，正义链和反义链的长度比为21/21。In some embodiments, in the 5'-3' direction, the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, and the base of the nucleotide sequence IV is U; in this case, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is CA, and the base composition of the nucleotide sequence IV is UG; in this case, the length ratio of the sense strand to the antisense strand is 21/21; or, the length of the nucleotide sequence III and IV are both 3 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is GCA, and the base composition of the nucleotide sequence IV is UGC; in this case, the length ratio of the sense strand to the antisense strand is 22/22; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is CGCA, and the base composition of the nucleotide sequence IV is UGCG; in this case, the length ratio of the sense strand to the antisense strand is 23/23. In some embodiments, the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is CA, and the base composition of the nucleotide sequence IV is UG; at this time, the length ratio of the sense strand and the antisense strand is 21/21.
在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补，因此，给出了核苷酸序列III的碱基，核苷酸序列IV的碱基也就确定了。In some embodiments, nucleotide sequence III and nucleotide sequence IV are completely reverse complementary, and therefore, given the base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.
第二种siRNAThe second siRNA
按照本公开，所述siRNA可以是第二种siRNA。According to the present disclosure, the siRNA may be a second siRNA.
所述第二种siRNA含有正义链和反义链，所述第二种siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，所述反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO:61所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:62所示的核苷酸序列长度相等，且不多于3个核苷酸差异：The second siRNA comprises a sense strand and an antisense strand, and each nucleotide in the second siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, wherein the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO:61 in length and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO:62 in length and differs by no more than 3 nucleotides:
5'-CUCAAUAAAGUGCUUUGAZ5-3'(SEQ ID NO:61)；5'-CUCAAUAAAGUGCUUUGAZ5-3 ' (SEQ ID NO: 61);
5'-Z6UCAAAGCACUUUAUUGAG-3'(SEQ ID NO:62)，5'-Z6 UCAAAGCACUUUAUUGAG-3' (SEQ ID NO: 62),
其中，Z5为A，Z6为U；Among them, Z5 is A, Z6 is U;
并且，所述核苷酸序列I中包含位置对应于Z5的核苷酸Z7，所述核苷酸序列II中包含位置对应于Z6的核苷酸Z8，所述Z8是所述反义链5'末端的第一个核苷酸。Furthermore, the nucleotide sequence I comprises a nucleotide Z7 corresponding to position Z5 , and the nucleotide sequence II comprises a nucleotide Z8 corresponding to position Z6 , and Z8 is the first nucleotide at the 5' end of the antisense strand.
在一些实施方式中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。In some embodiments, the sense strand comprises only nucleotide sequence I, and the antisense strand comprises only nucleotide sequence II.
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:61所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:62所示的核苷酸序列之间不多于1个核苷酸差异。In some embodiments, there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:61, and/or there is no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:62.
在一些实施方式中，所述核苷酸序列II与SEQ ID NO:62所示的核苷酸序列之间的核苷酸差异包括Z8位置处的差异，且Z8选自A、C或G。在一些实施方式中，所述核苷酸差异为Z8位置处的差异，Z8选自A、C或G。在一些实施方式中，Z7是与Z8互补的核苷酸。这些核苷酸差异并不会显著降低siRNA缀合物的靶基因抑制能力，而这些包含核苷酸差异的siRNA缀合物也在本公开的保护范围之内。In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 62 includes a difference at position Z8 , and Z8 is selected from A, C or G. In some embodiments, the nucleotide difference is a difference at position Z8 , and Z8 is selected from A, C or G. In some embodiments, Z7 is a nucleotide complementary to Z8. These nucleotide differences do not significantly reduce the target gene inhibition ability of the siRNA conjugate, and these siRNA conjugates containing nucleotide differences are also within the scope of protection of the present disclosure.
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补。In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary, or completely reverse complementary.
在一些实施方式中，核苷酸序列I是SEQ ID NO:63所示的核苷酸序列，核苷酸序列II是SEQ ID NO:64所示的核苷酸序列：In some embodiments, nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO:63, and nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO:64:
5'-CUCAAUAAAGUGCUUUGAZ7-3'(SEQ ID NO:63)；5'-CUCAAUAAAGUGCUUUGAZ7-3 ' (SEQ ID NO: 63);
5'-Z8UCAAAGCACUUUAUUGAG-3'(SEQ ID NO:64)，5'-Z8 UCAAAGCACUUUAUUGAG-3' (SEQ ID NO: 64),
其中，所述Z8是反义链5'末端的第一个核苷酸，Z7选自A、U、G或C，并且Z8是与Z7互补的核苷酸；在一些实施方式中，Z7为A，Z8为U；Wherein, Z8 is the first nucleotide at the 5' end of the antisense strand, Z7 is selected from A, U, G or C, and Z8 is a nucleotide complementary to Z7 ; in some embodiments, Z7 is A and Z8 is U;
并且，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，反义链的长度为19-26个核苷酸。Furthermore, the sense strand and the antisense strand have the same or different lengths, the sense strand has a length of 19-23 nucleotides, and the antisense strand has a length of 19-26 nucleotides.
在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV长度各自为1-4个核苷酸；所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或者完全反向互补；所述核苷酸序列III连接在所述核苷酸序列I的5'末端，所述核苷酸序列IV连接在所述核苷酸序列II的3'末端，所述核苷酸序列IV与第二段核苷酸序列实质上反向互补或者完全反向互补，该第二段核苷酸序列是指和靶mRNA中与由SEQ ID NO:61表示的核苷酸序列的5'末端相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。In some embodiments, the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV, and the nucleotide sequence III and the nucleotide sequence IV are each 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, which second nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO:61 in the target mRNA and has the same length as the nucleotide sequence IV.
在一些实施方式中，按照5'-3'的方向，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为A，核苷酸序列IV的碱基为U；此时，正义链和反义链的长度比为20/20；或者，核苷酸序列III和IV的长度均为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为AA，核苷酸序列IV的碱基组成为UU；此时，正义链和反义链的长度比为21/21；或者，核苷酸序列III和IV的长度均为3个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为GAA，核苷酸序列IV的碱基组成为UUC；此时，正义链和反义链的长度比为22/22；或者，核苷酸序列III和IV的长度均为4个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为GGAA，核苷酸序列IV的碱基组成为UUCC；此时，正义链和反义链的长度比为23/23。在一些实施方式中，所述核苷酸序列III和核苷酸序列IV的长度为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为AA，核苷酸序列IV的碱基组成为UU；此时，正义链和反义链的长度比为21/21。In some embodiments, in the 5'-3' direction, the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is A, and the base of the nucleotide sequence IV is U; in this case, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV are both 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is AA, and the base composition of the nucleotide sequence IV is UU; in this case, the length ratio of the sense strand to the antisense strand is 21/21; or In some embodiments, the lengths of nucleotide sequences III and IV are both 3 nucleotides, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is GAA, and the base composition of nucleotide sequence IV is UUC; in this case, the length ratio of the sense strand to the antisense strand is 22/22; or, the lengths of nucleotide sequences III and IV are both 4 nucleotides, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is GGAA, and the base composition of nucleotide sequence IV is UUCC; in this case, the length ratio of the sense strand to the antisense strand is 23/23. In some embodiments, the lengths of nucleotide sequences III and IV are 2 nucleotides, and from the 5' end to the 3' end, the base composition of nucleotide sequence III is AA, and the base composition of nucleotide sequence IV is UU; in this case, the length ratio of the sense strand to the antisense strand is 21/21.
在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补，因此，给出了核苷酸序列III的碱基，核苷酸序列IV的碱基也就确定了。In some embodiments, nucleotide sequence III and nucleotide sequence IV are completely reverse complementary, and therefore, given the base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.
第三种siRNAThe third siRNA
按照本公开，所述siRNA可以是第三种siRNA。According to the present disclosure, the siRNA may be a third siRNA.
所述第三种siRNA含有正义链和反义链，所述第三种siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，所述反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO:121所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:122所示的核苷酸序列长度相等，且不多于3个核苷酸差异：The third siRNA comprises a sense strand and an antisense strand, and each nucleotide in the third siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, wherein the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO: 121 in length and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO: 122 in length and differs by no more than 3 nucleotides:
5'-GGAGCCCAAGAAAGUGAAZ13-3'(SEQ ID NO:121)；5'-GGAGCCCAAGAAAGUGAAZ13-3 ' (SEQ ID NO: 121);
5'-Z14UUCACUUUCUUGGGCUCC-3'(SEQ ID NO:122)，5'-Z14 UUCACUUUCUUGGGCUCC-3' (SEQ ID NO: 122),
其中，Z13为A，Z14为U；Among them, Z13 is A, Z14 is U;
并且，所述核苷酸序列I中包含位置对应于Z13的核苷酸Z15，所述核苷酸序列II中包含位置对应于Z14的核苷酸Z16，所述Z16是所述反义链5'末端的第一个核苷酸。Furthermore, the nucleotide sequence I comprises a nucleotide Z15 corresponding to position Z13 , and the nucleotide sequence II comprises a nucleotide Z16 corresponding to position Z14 , and Z16 is the first nucleotide at the 5' end of the antisense strand.
在一些实施方式中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。In some embodiments, the sense strand comprises only nucleotide sequence I, and the antisense strand comprises only nucleotide sequence II.
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:121所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:122所示的核苷酸序列之间不多于1个核苷酸差异。In some embodiments, there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:121, and/or there is no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:122.
在一些实施方式中，所述核苷酸序列II与SEQ ID NO:122所示的核苷酸序列之间的核苷酸差异包括Z16位置处的差异，且Z16选自A、C或G。在一些实施方式中，所述核苷酸差异为Z16位置处的差异，Z16选自A、C或G。在一些实施方式中，Z15是与Z16互补的核苷酸。这些核苷酸差异并不会显著降低siRNA缀合物的靶基因抑制能力，而这些包含核苷酸差异的siRNA缀合物也在本公开的保护范围之内。In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 122 includes a difference at the Z16 position, and Z16 is selected from A, C or G. In some embodiments, the nucleotide difference is a difference at the Z16 position, and Z16 is selected from A, C or G. In some embodiments, Z15 is a nucleotide complementary to Z16. These nucleotide differences do not significantly reduce the target gene inhibition ability of the siRNA conjugate, and these siRNA conjugates containing nucleotide differences are also within the scope of protection of the present disclosure.
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补。In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary, or completely reverse complementary.
在一些实施方式中，核苷酸序列I是SEQ ID NO:123所示的核苷酸序列，核苷酸序列II是SEQ ID NO:124所示的核苷酸序列：In some embodiments, nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 123, and nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 124:
5'-GGAGCCCAAGAAAGUGAAZ15-3'(SEQ ID NO:123)；5'-GGAGCCCAAGAAAGUGAAZ15 -3' (SEQ ID NO: 123);
5'-Z16UUCACUUUCUUGGGCUCC-3'(SEQ ID NO:124)，5'-Z16 UUCACUUUCUUGGGCUCC-3' (SEQ ID NO: 124),
其中，所述Z16是反义链5'末端的第一个核苷酸，Z15选自A、U、G或C，并且Z16是与Z15互补的核苷酸；在一些实施方式中，Z15为A，Z16为U；Wherein, Z16 is the first nucleotide at the 5' end of the antisense strand, Z15 is selected from A, U, G or C, and Z16 is a nucleotide complementary to Z15 ; in some embodiments, Z15 is A and Z16 is U;
并且，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，反义链的长度为19-26个核苷酸。Furthermore, the sense strand and the antisense strand have the same or different lengths, the sense strand has a length of 19-23 nucleotides, and the antisense strand has a length of 19-26 nucleotides.
在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV长度各自为1-4个核苷酸；所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或者完全反向互补；所述核苷酸序列III连接在所述核苷酸序列I的5'末端，所述核苷酸序列IV连接在所述核苷酸序列II的3'末端，所述核苷酸序列IV与第二段核苷酸序列实质上反向互补或者完全反向互补，该第二段核苷酸序列是指和靶mRNA中与由SEQ ID NO:121表示的核苷酸序列的5'末端相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。In some embodiments, the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV, and the nucleotide sequence III and the nucleotide sequence IV are each 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, which second nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO: 121 in the target mRNA and has the same length as the nucleotide sequence IV.
在一些实施方式中，按照5'-3'的方向，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为U，核苷酸序列IV的碱基为A；此时，正义链和反义链的长度比为20/20；或者，核苷酸序列III和IV的长度均为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为UU，核苷酸序列IV的碱基组成为AA；此时，正义链和反义链的长度比为21/21；或者，核苷酸序列III和IV的长度均为3个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为UUU，核苷酸序列IV的碱基组成为AAA；此时，正义链和反义链的长度比为22/22；或者，核苷酸序列III和IV的长度均为4个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为GUUU，核苷酸序列IV的碱基组成为AAAC；此时，正义链和反义链的长度比为23/23。在一些实施方式中，所述核苷酸序列III和核苷酸序列IV的长度为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为AA，核苷酸序列IV的碱基组成为UU；此时，正义链和反义链的长度比为21/21。In some embodiments, in the 5'-3' direction, the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is U, and the base of the nucleotide sequence IV is A; in this case, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV are both 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is UU, and the base composition of the nucleotide sequence IV is AA; in this case, the length ratio of the sense strand to the antisense strand is 21/21; or In some embodiments, the lengths of nucleotide sequences III and IV are both 3 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of nucleotide sequence III is UUU, and the base composition of nucleotide sequence IV is AAA; in this case, the length ratio of the sense strand to the antisense strand is 22/22; or, the lengths of nucleotide sequences III and IV are both 4 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of nucleotide sequence III is GUUU, and the base composition of nucleotide sequence IV is AAAC; in this case, the length ratio of the sense strand to the antisense strand is 23/23. In some embodiments, the lengths of nucleotide sequences III and IV are 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of nucleotide sequence III is AA, and the base composition of nucleotide sequence IV is UU; in this case, the length ratio of the sense strand to the antisense strand is 21/21.
在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补，因此，给出了核苷酸序列III的碱基，核苷酸序列IV的碱基也就确定了。In some embodiments, nucleotide sequence III and nucleotide sequence IV are completely reverse complementary, and therefore, given the base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.
第四种siRNAThe fourth siRNA
按照本公开，所述siRNA可以是第四种siRNA。According to the present disclosure, the siRNA may be a fourth siRNA.
所述第五种siRNA含有正义链和反义链，所述第五种siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，所述反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO:181所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:182所示的核苷酸序列长度相等，且不多于3个核苷酸差异：The fifth siRNA comprises a sense strand and an antisense strand, and each nucleotide in the fifth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, wherein the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO: 181 in length and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO: 182 in length and differs by no more than 3 nucleotides:
5'-AGCCCAAGAAAGUGAAAGZ17-3'(SEQ ID NO:181)；5'-AGCCCAAGAAAGUGAAAGZ17 -3' (SEQ ID NO: 181);
5'-Z18CUUUCACUUUCUUGGGCU-3'(SEQ ID NO:182)，5'-Z18 CUUUCACUUUCUUGGGCU-3' (SEQ ID NO: 182),
其中，Z17为A，Z18为U，Among them, Z17 is A, Z18 is U,
并且，所述核苷酸序列I中包含位置对应于Z17的核苷酸Z19，所述核苷酸序列II中包含位置对应于Z18的核苷酸Z20，所述Z20是所述反义链5'末端的第一个核苷酸。Furthermore, the nucleotide sequence I comprises a nucleotide Z19 corresponding to position Z17 , and the nucleotide sequence II comprises a nucleotide Z20 corresponding to position Z18 , and Z20 is the first nucleotide at the 5' end of the antisense strand.
在一些实施方式中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。In some embodiments, the sense strand comprises only nucleotide sequence I, and the antisense strand comprises only nucleotide sequence II.
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:181所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:182所示的核苷酸序列之间不多于1个核苷酸差异。In some embodiments, there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:181, and/or there is no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:182.
在一些实施方式中，所述核苷酸序列II与SEQ ID NO:182所示的核苷酸序列之间的核苷酸差异包括Z20位置处的差异，且Z20选自A、C或G。在一些实施方式中，所述核苷酸差异为Z20位置处的差异，Z20选自A、C或G。在一些实施方式中，Z19是与Z20互补的核苷酸。这些核苷酸差异并不会显著降低siRNA缀合物的靶基因抑制能力，而这些包含核苷酸差异的siRNA缀合物也在本公开的保护范围之内。In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 182 includes a difference at the Z20 position, and Z20 is selected from A, C or G. In some embodiments, the nucleotide difference is a difference at the Z20 position, and Z20 is selected from A, C or G. In some embodiments, Z19 is a nucleotide complementary to Z20. These nucleotide differences do not significantly reduce the target gene inhibition ability of the siRNA conjugate, and these siRNA conjugates containing nucleotide differences are also within the scope of protection of the present disclosure.
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补。In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary, or completely reverse complementary.
在一些实施方式中，核苷酸序列I是SEQ ID NO:183所示的核苷酸序列，核苷酸序列II是SEQ ID NO:184所示的核苷酸序列：In some embodiments, nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 183, and nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 184:
5'-AGCCCAAGAAAGUGAAAGZ19-3'(SEQ ID NO:183)；5'-AGCCCAAGAAAGUGAAAGZ19 -3' (SEQ ID NO: 183);
5'-Z20CUUUCACUUUCUUGGGCU-3'(SEQ ID NO:184)，5'-Z20 CUUUCACUUUCUUGGGCU-3' (SEQ ID NO: 184),
其中，所述Z20是反义链5'末端的第一个核苷酸，Z19选自A、U、G或C，并且Z20是与Z19互补的核苷酸；在一些实施方式中，Z19为A，Z20为U；Wherein, Z20 is the first nucleotide at the 5' end of the antisense strand, Z19 is selected from A, U, G or C, and Z20 is a nucleotide complementary to Z19 ; in some embodiments, Z19 is A and Z20 is U;
并且，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，反义链的长度为19-26个核苷酸。Furthermore, the sense strand and the antisense strand have the same or different lengths, the sense strand has a length of 19-23 nucleotides, and the antisense strand has a length of 19-26 nucleotides.
在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV长度各自为1-4个核苷酸；所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或者完全反向互补；所述核苷酸序列III连接在所述核苷酸序列I的5'末端，所述核苷酸序列IV连接在所述核苷酸序列II的3'末端，所述核苷酸序列IV与第二段核苷酸序列实质上反向互补或者完全反向互补，该第二段核苷酸序列是指和靶mRNA中与由SEQ ID NO:181表示的核苷酸序列的5'末端相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。In some embodiments, the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV, and the nucleotide sequence III and the nucleotide sequence IV are each 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, which second nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO: 181 in the target mRNA and has the same length as the nucleotide sequence IV.
在一些实施方式中，按照5'-3'的方向，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为G，核苷酸序列IV的碱基为C；此时，正义链和反义链的长度比为20/20；或者，核苷酸序列III和IV的长度均为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为GG，核苷酸序列IV的碱基组成为CC；此时，正义链和反义链的长度比为21/21；或者，核苷酸序列III和IV的长度均为3个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为UGG，核苷酸序列IV的碱基组成为CCA；此时，正义链和反义链的长度比为22/22；或者，核苷酸序列III和IV的长度均为4个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为UUGG，核苷酸序列IV的碱基组成为CCAA；此时，正义链和反义链的长度比为23/23。在一些实施方式中，所述核苷酸序列III和核苷酸序列IV的长度为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为GG，核苷酸序列IV的碱基组成为CC；此时，正义链和反义链的长度比为21/21。In some embodiments, in the 5'-3' direction, the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is G, and the base of the nucleotide sequence IV is C; in this case, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequences III and IV are both 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is GG, and the base composition of the nucleotide sequence IV is CC; in this case, the length ratio of the sense strand to the antisense strand is 21/21; or In some embodiments, the lengths of nucleotide sequences III and IV are both 3 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of nucleotide sequence III is UGG, and the base composition of nucleotide sequence IV is CCA; in this case, the length ratio of the sense strand to the antisense strand is 22/22; or, the lengths of nucleotide sequences III and IV are both 4 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of nucleotide sequence III is UUGG, and the base composition of nucleotide sequence IV is CCAA; in this case, the length ratio of the sense strand to the antisense strand is 23/23. In some embodiments, the lengths of nucleotide sequences III and IV are 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of nucleotide sequence III is GG, and the base composition of nucleotide sequence IV is CC; in this case, the length ratio of the sense strand to the antisense strand is 21/21.
在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补，因此，给出了核苷酸序列III的碱基，核苷酸序列IV的碱基也就确定了。In some embodiments, nucleotide sequence III and nucleotide sequence IV are completely reverse complementary, and therefore, given the base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.
第五种siRNAThe fifth siRNA
按照本公开，所述siRNA可以是第五种siRNA。According to the present disclosure, the siRNA may be the fifth siRNA.
所述第五种siRNA含有正义链和反义链，所述第五种siRNA中的每个核苷酸各自独立地为修饰或未修饰的核苷酸，其中，所述正义链含有一段核苷酸序列I，所述反义链含有一段核苷酸序列II，所述核苷酸序列I和所述核苷酸序列II至少部分地反向互补形成双链区，其中，所述核苷酸序列I与SEQ ID NO:241所示的核苷酸序列长度相等，且不多于3个核苷酸差异，且所述核苷酸序列II与SEQ ID NO:242所示的核苷酸序列长度相等，且不多于3个核苷酸差异：The fifth siRNA comprises a sense strand and an antisense strand, and each nucleotide in the fifth siRNA is independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II, and the nucleotide sequence I and the nucleotide sequence II are at least partially reverse-complementary to form a double-stranded region, wherein the nucleotide sequence I is equal to the nucleotide sequence shown in SEQ ID NO: 241 in length and differs by no more than 3 nucleotides, and the nucleotide sequence II is equal to the nucleotide sequence shown in SEQ ID NO: 242 in length and differs by no more than 3 nucleotides:
5'-CCAAGAAAGUGAAAGACCZ21-3'(SEQ ID NO:241)；5'-CCAAGAAAGUGAAAGACCZ21 -3' (SEQ ID NO: 241);
5'-Z22GGUCUUUCACUUUCUUGG-3'(SEQ ID NO:242)，5'-Z22 GGUCUUUCACUUUCUUGG-3' (SEQ ID NO: 242),
其中，Z21为A，Z22为U；Among them, Z21 is A, Z22 is U;
所述核苷酸序列I中包含位置对应于Z21的核苷酸Z23，所述核苷酸序列II中包含位置对应于Z22的核苷酸Z24，所述Z24是所述反义链5'末端的第一个核苷酸。The nucleotide sequence I comprises a nucleotide Z23 corresponding to the position of Z21 , and the nucleotide sequence II comprises a nucleotide Z24 corresponding to the position of Z22 , wherein Z24 is the first nucleotide at the 5' end of the antisense strand.
在一些实施方式中，所述正义链仅包含核苷酸序列I，所述反义链仅包含核苷酸序列II。In some embodiments, the sense strand comprises only nucleotide sequence I, and the antisense strand comprises only nucleotide sequence II.
在一些实施方式中，所述核苷酸序列I与SEQ ID NO:241所示的核苷酸序列之间不多于1个核苷酸差异，和/或所述核苷酸序列II与SEQ ID NO:242所示的核苷酸序列之间不多于1个核苷酸差异。In some embodiments, there is no more than 1 nucleotide difference between the nucleotide sequence I and the nucleotide sequence shown in SEQ ID NO:241, and/or there is no more than 1 nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO:242.
在一些实施方式中，所述核苷酸序列II与SEQ ID NO:242所示的核苷酸序列之间的核苷酸差异包括Z24位置处的差异，且Z24选自A、C或G。在一些实施方式中，所述核苷酸差异为Z24位置处的差异，Z24选自A、C或G。在一些实施方式中，Z23是与Z24互补的核苷酸。这些核苷酸差异并不会显著降低siRNA缀合物的靶基因抑制能力，而这些包含核苷酸差异的siRNA缀合物也在本公开的保护范围之内。In some embodiments, the nucleotide difference between the nucleotide sequence II and the nucleotide sequence shown in SEQ ID NO: 242 includes a difference at the Z24 position, and Z24 is selected from A, C or G. In some embodiments, the nucleotide difference is a difference at the Z24 position, and Z24 is selected from A, C or G. In some embodiments, Z23 is a nucleotide complementary to Z24. These nucleotide differences do not significantly reduce the target gene inhibition ability of the siRNA conjugate, and these siRNA conjugates containing nucleotide differences are also within the scope of protection of the present disclosure.
在一些实施方式中，所述核苷酸序列I和所述核苷酸序列II基本上反向互补、实质上反向互补或完全反向互补。In some embodiments, the nucleotide sequence I and the nucleotide sequence II are substantially reverse complementary, essentially reverse complementary, or completely reverse complementary.
在一些实施方式中，核苷酸序列I是SEQ ID NO:303所示的核苷酸序列，核苷酸序列II是SEQ ID NO:304所示的核苷酸序列：In some embodiments, nucleotide sequence I is the nucleotide sequence shown in SEQ ID NO: 303, and nucleotide sequence II is the nucleotide sequence shown in SEQ ID NO: 304:
5'-CCAAGAAAGUGAAAGACCZ23-3'(SEQ ID NO:243)；5'-CCAAGAAAGUGAAAGACCZ23-3 ' (SEQ ID NO: 243);
5'-Z24GGUCUUUCACUUUCUUGG-3'(SEQ ID NO:244)，5'-Z24 GGUCUUUCACUUUCUUGG-3' (SEQ ID NO: 244),
其中，所述Z16是反义链5'末端的第一个核苷酸，Z23选自A、U、G或C，并且Z24是与Z23互补的核苷酸；在一些实施方式中，Z23为A，Z24为U；wherein Z16 is the first nucleotide at the 5' end of the antisense strand, Z23 is selected from A, U, G or C, and Z24 is a nucleotide complementary to Z23 ; in some embodiments, Z23 is A and Z24 is U;
并且，所述正义链和反义链长度相同或不同，所述正义链的长度为19-23个核苷酸，反义链的长度为19-26个核苷酸。Furthermore, the sense strand and the antisense strand have the same or different lengths, the sense strand has a length of 19-23 nucleotides, and the antisense strand has a length of 19-26 nucleotides.
在一些实施方式中，所述正义链还含有核苷酸序列III，所述反义链还含有核苷酸序列IV，核苷酸序列III和核苷酸序列IV长度各自为1-4个核苷酸；所述核苷酸序列III和所述核苷酸序列IV长度相等并且实质上反向互补或者完全反向互补；所述核苷酸序列III连接在所述核苷酸序列I的5'末端，所述核苷酸序列IV连接在所述核苷酸序列II的3'末端，所述核苷酸序列IV与第二段核苷酸序列实质上反向互补或者完全反向互补，该第二段核苷酸序列是指和靶mRNA中与由SEQ ID NO:241表示的核苷酸序列的5'末端相邻、且长度与所述核苷酸序列IV相同的核苷酸序列。In some embodiments, the sense strand further contains a nucleotide sequence III, and the antisense strand further contains a nucleotide sequence IV, and the nucleotide sequence III and the nucleotide sequence IV are each 1-4 nucleotides in length; the nucleotide sequence III and the nucleotide sequence IV are equal in length and are substantially reverse complementary or completely reverse complementary; the nucleotide sequence III is connected to the 5' end of the nucleotide sequence I, and the nucleotide sequence IV is connected to the 3' end of the nucleotide sequence II, and the nucleotide sequence IV is substantially reverse complementary or completely reverse complementary to a second nucleotide sequence, which second nucleotide sequence refers to a nucleotide sequence that is adjacent to the 5' end of the nucleotide sequence represented by SEQ ID NO: 241 in the target mRNA and has the same length as the nucleotide sequence IV.
在一些实施方式中，按照5'-3'的方向，所述核苷酸序列III和核苷酸序列IV的长度均为1个核苷酸，核苷酸序列III的碱基为C，核苷酸序列IV的碱基为G；此时，正义链和反义链的长度比为20/20；或者，核苷酸序列III和IV的长度均为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为GC，核苷酸序列IV的碱基组成为GC；此时，正义链和反义链的长度比为21/21；或者，核苷酸序列III和IV的长度均为3个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为AGC，核苷酸序列IV的碱基组成为GCU；此时，正义链和反义链的长度比为22/22；或者，核苷酸序列III和IV的长度均为4个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为GAGC，核苷酸序列IV的碱基组成为GCUC；此时，正义链和反义链的长度比为23/23。在一些实施方式中，所述核苷酸序列III和核苷酸序列IV的长度为2个核苷酸，按照5'末端到3'末端的方向，核苷酸序列III的碱基组成为GC，核苷酸序列IV的碱基组成为GC；此时，正义链和反义链的长度比为21/21。In some embodiments, in the 5'-3' direction, the length of the nucleotide sequence III and the nucleotide sequence IV are both 1 nucleotide, the base of the nucleotide sequence III is C, and the base of the nucleotide sequence IV is G; in this case, the length ratio of the sense strand to the antisense strand is 20/20; or, the length of the nucleotide sequence III and IV are both 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is GC, and the base composition of the nucleotide sequence IV is GC; in this case, the length ratio of the sense strand to the antisense strand is 21/21; or, the length of the nucleotide sequence III and IV are both 3 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is AGC, and the base composition of the nucleotide sequence IV is GCU; in this case, the length ratio of the sense strand to the antisense strand is 22/22; or, the length of the nucleotide sequence III and IV are both 4 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is GAGC, and the base composition of the nucleotide sequence IV is GCUC; in this case, the length ratio of the sense strand to the antisense strand is 23/23. In some embodiments, the length of the nucleotide sequence III and the nucleotide sequence IV is 2 nucleotides, and in the direction from the 5' end to the 3' end, the base composition of the nucleotide sequence III is GC, and the base composition of the nucleotide sequence IV is GC; at this time, the length ratio of the sense strand and the antisense strand is 21/21.
在一些实施方式中，核苷酸序列III和核苷酸序列IV完全反向互补，因此，给出了核苷酸序列III的碱基，核苷酸序列IV的碱基也就确定了。In some embodiments, nucleotide sequence III and nucleotide sequence IV are completely reverse complementary, and therefore, given the base of nucleotide sequence III, the base of nucleotide sequence IV is also determined.
siRNA的悬垂末端和修饰siRNA overhangs and modifications
以下，对核苷酸序列V、核酸序列、siRNA中的核苷酸修饰以及修饰序列的描述适用于上述第一种siRNA至第五种siRNA中的任意一种。即如果没有特指，下面对siRNA的描述应视为是对第一种siRNA、第二种siRNA、第三种siRNA、第四种siRNA和第五种siRNA逐一进行了描述。例如，如不特别指明具体的siRNA，“所述siRNA还含有核苷酸序列V”的意思是“第一种siRNA、第二种siRNA、第三种siRNA、第四种siRNA或第五种siRNA还含有核苷酸序列V”。Hereinafter, the description of nucleotide sequence V, nucleic acid sequence, nucleotide modification in siRNA and modified sequence is applicable to any one of the first to fifth siRNAs mentioned above. That is, if not specifically mentioned, the following description of siRNA should be regarded as describing the first siRNA, the second siRNA, the third siRNA, the fourth siRNA and the fifth siRNA one by one. For example, if the specific siRNA is not specifically mentioned, "the siRNA further contains nucleotide sequence V" means "the first siRNA, the second siRNA, the third siRNA, the fourth siRNA or the fifth siRNA further contains nucleotide sequence V".
在一些实施方式中，所述正义链和反义链长度不同，所述反义链还含有核苷酸序列V，核苷酸序列V的长度为1至3个核苷酸，连接在所述反义链的3'末端，构成反义链的3'突出端。由此，本公开提供的siRNA正义链和反义链的长度比可以是19/20、19/21、19/22、20/21、20/22、20/23、21/22、21/23、21/24、22/23、22/24、22/25、23/24、23/25或23/26。在一些实施方式中，所述核苷酸序列V的长度为2个核苷酸，由此，本公开提供的siRNA正义链和反义链的长度比可以是19/21、21/23或23/25。In some embodiments, the sense strand and the antisense strand are different in length, and the antisense strand further contains a nucleotide sequence V, the length of the nucleotide sequence V is 1 to 3 nucleotides, connected to the 3' end of the antisense strand, forming the 3' overhang of the antisense strand. Thus, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure can be 19/20, 19/21, 19/22, 20/21, 20/22, 20/23, 21/22, 21/23, 21/24, 22/23, 22/24, 22/25, 23/24, 23/25 or 23/26. In some embodiments, the length of the nucleotide sequence V is 2 nucleotides, thus, the length ratio of the sense strand and the antisense strand of the siRNA provided by the present disclosure can be 19/21, 21/23 or 23/25.
所述核苷酸序列V中的每一个核苷酸可以是任意的核苷酸，为了便于合成并节约合成成本，所述核苷酸序列V为连续的2个胸腺嘧啶脱氧核糖核苷酸(dTdT)或连续的2个尿嘧啶核糖核苷酸(UU)；或者，为了提高siRNA反义链与靶mRNA的亲和力，核苷酸序列V与靶mRNA的相应位置的核苷酸互补。因此，在一些实施方式中，本公开的siRNA的正义链和反义链的长度之比为19/21或21/23，此时，本公开的siRNA具有更好的mRNA沉默活性。Each nucleotide in the nucleotide sequence V can be any nucleotide. In order to facilitate synthesis and save synthesis costs, the nucleotide sequence V is 2 consecutive thymine deoxyribonucleotides (dTdT) or 2 consecutive uracil ribonucleotides (UU); or, in order to improve the affinity of the siRNA antisense strand to the target mRNA, the nucleotide sequence V is complementary to the nucleotides at the corresponding positions of the target mRNA. Therefore, in some embodiments, the ratio of the length of the sense strand and the antisense strand of the siRNA disclosed in the present invention is 19/21 or 21/23, at which time, the siRNA disclosed in the present invention has better mRNA silencing activity.
靶mRNA的相应位置的核苷酸是指与靶mRNA的一段核苷酸序列在5'末端相邻的核苷酸或核苷酸序列，该段靶mRNA的核苷酸序列是与核苷酸序列II实质上反向互补或完全反向互补，或者与核苷酸序列II和核苷酸序列IV构成的核苷酸序列实质上反向互补或完全反向互补的那段核苷酸序列。The nucleotides at the corresponding position of the target mRNA refer to the nucleotides or nucleotide sequences adjacent to a nucleotide sequence of the target mRNA at the 5' end, wherein the nucleotide sequence of the target mRNA is substantially reverse complementary or completely reverse complementary to the nucleotide sequence II, or is substantially reverse complementary or completely reverse complementary to the nucleotide sequence composed of the nucleotide sequence II and the nucleotide sequence IV.
在一些实施方式中，对于所述第一种siRNA，所述siRNA的正义链含有如SEQ IDNO:5所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:6所示的核苷酸序列：In some embodiments, for the first siRNA, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 5, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 6:
5'-GGAACUCAAUAAAGUGCUZ3 -3'(SEQ ID NO:5)；5'-GGAACUCAAUAAAGUGCUZ3-3 ' (SEQ ID NO: 5);
5'-Z4AGCACUUUAUUGAGUUCCUG-3'(SEQ ID NO:6)；5'-Z4 AGCACUUUAUUGAGUUCCUG-3' (SEQ ID NO: 6);
或者，所述siRNA的正义链含有如SEQ ID NO:7所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:8所示的核苷酸序列：Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:7, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:8:
5'-CAGGAACUCAAUAAAGUGCUZ3 -3'(SEQ ID NO:7)；5'-CAGGAACUCAAUAAAGUGCUZ3-3 ' (SEQ ID NO:7);
5'-Z4AGCACUUUAUUGAGUUCCUGCG-3'(SEQ ID NO:8)；5'-Z4 AGCACUUUAUUGAGUUCCUGCG-3' (SEQ ID NO:8);
其中，所述Z4是反义链5'末端的第一个核苷酸，Z3选自A、U、G或C，并且Z4是与Z3互补的核苷酸。Wherein,Z4 is the first nucleotide at the 5' end of the antisense strand,Z3 is selected from A, U, G or C, andZ4 is a nucleotide complementary toZ3 .
在一些实施方式中，对于所述第二种siRNA，所述siRNA的正义链含有如SEQ IDNO:65所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:66所示的核苷酸序列：In some embodiments, for the second siRNA, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 65, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 66:
5'-CUCAAUAAAGUGCUUUGAZ7 -3'(SEQ ID NO:65)；5'-CUCAAUAAAGUGCUUUGAZ7-3 ' (SEQ ID NO: 65);
5'-Z8UCAAAGCACUUUAUUGAGUU-3'(SEQ ID NO:66)，5'-Z8 UCAAAGCACUUUAUUGAGUU-3' (SEQ ID NO: 66),
或者，所述siRNA的正义链含有如SEQ ID NO:67所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:68所示的核苷酸序列：Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:67, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO:68:
5'-AACUCAAUAAAGUGCUUUGAZ7 -3'(SEQ ID NO:67)；5'-AACUCAAUAAAGUGCUUUGAZ7-3 ' (SEQ ID NO: 67);
5'-Z8UCAAAGCACUUUAUUGAGUUCC-3'(SEQ ID NO:68)，5'-Z8 UCAAAGCACUUUAUUGAGUUCC-3' (SEQ ID NO: 68),
其中，所述Z8是反义链5'末端的第一个核苷酸，Z7选自A、U、G或C，并且Z8是与Z7互补的核苷酸。Wherein,Z8 is the first nucleotide at the 5' end of the antisense strand,Z7 is selected from A, U, G or C, andZ8 is a nucleotide complementary toZ7 .
在一些实施方式中，对于所述第三种siRNA，所述siRNA的正义链含有如SEQ IDNO:125所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:126所示的核苷酸序列：In some embodiments, for the third siRNA, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 125, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 126:
5'-GGAGCCCAAGAAAGUGAAZ15-3'(SEQ ID NO:125)；5'-GGAGCCCAAGAAAGUGAAZ15-3 ' (SEQ ID NO: 125);
5'-Z16UUCACUUUCUUGGGCUCCAA-3'(SEQ ID NO:126)，5'-Z16 UUCACUUUCUUGGGCUCCAA-3' (SEQ ID NO: 126),
或者，所述siRNA的正义链含有如SEQ ID NO:127所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:128所示的核苷酸序列：Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 127, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 128:
5'-UUGGAGCCCAAGAAAGUGAAZ15-3'(SEQ ID NO:127)；5'-UUGGAGCCCAAGAAAGUGAAZ15-3 ' (SEQ ID NO: 127);
5'-Z16UUCACUUUCUUGGGCUCCAAAC-3'(SEQ ID NO:128)，5'-Z16 UUCACUUUCUUGGGCUCCAAAC-3' (SEQ ID NO: 128),
其中，所述Z16是反义链5'末端的第一个核苷酸，Z15选自A、U、G或C，并且Z16是与Z15互补的核苷酸。Wherein,Z16 is the first nucleotide at the 5' end of the antisense strand,Z15 is selected from A, U, G or C, andZ16 is a nucleotide complementary toZ15 .
在一些实施方式中，对于所述第四种siRNA，所述siRNA的正义链含有如SEQ IDNO:185所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:186所示的核苷酸序列：In some embodiments, for the fourth siRNA, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 185, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 186:
5'-AGCCCAAGAAAGUGAAAGZ19 -3'(SEQ ID NO:185)；5'-AGCCCAAGAAAGUGAAAGZ19 -3' (SEQ ID NO: 185);
5'-Z20CUUUCACUUUCUUGGGCUCC-3'(SEQ ID NO:186)；5'-Z20 CUUUCACUUUCUUGGGCUCC-3' (SEQ ID NO: 186);
或者，所述siRNA的正义链含有如SEQ ID NO:187所示的核苷酸序列，所述反义链含有如SEQ ID NO:188所示的核苷酸序列：Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 187, and the antisense strand contains the nucleotide sequence shown in SEQ ID NO: 188:
5'-GGAGCCCAAGAAAGUGAAAGZ19 -3'(SEQ ID NO:187)；5'-GGAGCCCAAGAAAGUGAAAGZ19 -3' (SEQ ID NO: 187);
5'-Z20CUUUCACUUUCUUGGGCUCCAA-3'(SEQ ID NO:188)；5'-Z20 CUUUCACUUUCUUGGGCUCCAA-3' (SEQ ID NO: 188);
其中，所述Z20是反义链5'末端的第一个核苷酸，Z19选自A、U、G或C，并且Z20是与Z19互补的核苷酸。Wherein,Z20 is the first nucleotide at the 5' end of the antisense strand,Z19 is selected from A, U, G or C, andZ20 is a nucleotide complementary toZ19 .
在一些实施方式中，对于所述第五种siRNA，所述siRNA的正义链含有如SEQ IDNO:245所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:246所示的核苷酸序列：In some embodiments, for the fifth siRNA, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 245, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 246:
5'-CCAAGAAAGUGAAAGACCZ23 -3'(SEQ ID NO:245)；5'-CCAAGAAAGUGAAAGACCZ23-3 ' (SEQ ID NO: 245);
5'-Z24GGUCUUUCACUUUCUUGGGC-3'(SEQ ID NO:246)；5'-Z24 GGUCUUUCACUUUCUUGGGC-3' (SEQ ID NO: 246);
或者，所述siRNA的正义链含有如SEQ ID NO:247所示的核苷酸序列，所述siRNA的反义链含有如SEQ ID NO:248所示的核苷酸序列：Alternatively, the sense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 247, and the antisense strand of the siRNA contains the nucleotide sequence shown in SEQ ID NO: 248:
5'-GCCCAAGAAAGUGAAAGACCZ23 -3'(SEQ ID NO:247)；5'-GCCCAAGAAAGUGAAAGACCZ23-3 ' (SEQ ID NO: 247);
5'-Z24GGUCUUUCACUUUCUUGGGCUC-3'(SEQ ID NO:248)；5'-Z24 GGUCUUUCACUUUCUUGGGCUC-3' (SEQ ID NO: 248);
其中，所述Z24是反义链5'末端的第一个核苷酸，Z23选自A、U、G或C，并且Z24是与Z23互补的核苷酸。Wherein, Z24 is the first nucleotide at the 5' end of the antisense strand, Z23 is selected from A, U, G or C, and Z24 is a nucleotide complementary to Z23 .
在一些实施方式中，本公开所述siRNA为siFXIIa1、siFXIIa2、siFXIIb1、siFXIIb2、siFXIId1、siFXIId2、siFXIIe1、siFXIIe2、siFXIIf1或siFXIIf2：In some embodiments, the siRNA disclosed herein is siFXIIa1, siFXIIa2, siFXIIb1, siFXIIb2, siFXIId1, siFXIId2, siFXIIe1, siFXIIe2, siFXIIf1 or siFXIIf2:
siFXIIa1siFXIIa1
正义链：5'-GGAACUCAAUAAAGUGCUU-3'(SEQ ID NO:9)Sense strand: 5'-GGAACUCAAUAAAGUGCUU-3' (SEQ ID NO: 9)
反义链：5'-AAGCACUUUAUUGAGUUCCUG-3'(SEQ ID NO:10)Antisense strand: 5'-AAGCACUUUAUUGAGUUCCUG-3' (SEQ ID NO: 10)
siFXIIa2siFXIIa2
正义链：5'-CAGGAACUCAAUAAAGUGCUU-3'(SEQ ID NO:11)Sense strand: 5'-CAGGAACUCAAUAAAGUGCUU-3' (SEQ ID NO: 11)
反义链：5'-AAGCACUUUAUUGAGUUCCUGCG-3'(SEQ ID NO:12)Antisense strand: 5'-AAGCACUUUAUUGAGUUCCUGCG-3' (SEQ ID NO: 12)
siFXIIb1siFXIIb1
正义链：5'-CUCAAUAAAGUGCUUUGAA-3'(SEQ ID NO:69)Sense strand: 5'-CUCAAUAAAGUGCUUUGAA-3' (SEQ ID NO: 69)
反义链：5'-UUCAAAGCACUUUAUUGAGUU-3'(SEQ ID NO:70)Antisense strand: 5'-UUCAAAGCACUUUAUUGAGUU-3' (SEQ ID NO: 70)
siFXIIb2siFXIIb2
正义链：5'-AACUCAAUAAAGUGCUUUGAA-3'(SEQ ID NO:71)Sense strand: 5'-AACUCAAUAAAGUGCUUUGAA-3' (SEQ ID NO: 71)
反义链：5'-UUCAAAGCACUUUAUUGAGUUCC-3'(SEQ ID NO:72)。Antisense strand: 5'-UUCAAAGCACUUUAUUGAGUUCC-3' (SEQ ID NO: 72).
siFXIId1siFXIId1
正义链：5'-GGAGCCCAAGAAAGUGAAA-3'(SEQ ID NO:129)Sense strand: 5'-GGAGCCCAAGAAAGUGAAA-3' (SEQ ID NO: 129)
反义链：5'-UUUCACUUUCUUGGGCUCCAA-3'(SEQ ID NO:130)Antisense strand: 5'-UUUCACUUUCUUGGGCUCCAA-3' (SEQ ID NO: 130)
siFXIId2siFXIId2
正义链：5'-UUGGAGCCCAAGAAAGUGAAA-3'(SEQ ID NO:131)Sense strand: 5'-UUGGAGCCCAAGAAAGUGAAA-3' (SEQ ID NO: 131)
反义链：5'-UUUCACUUUCUUGGGCUCCAAAC-3'(SEQ ID NO:132)Antisense strand: 5'-UUUCACUUUCUUGGGCUCCAAAC-3' (SEQ ID NO: 132)
siFXIIe1siFXIIe1
正义链：5'-AGCCCAAGAAAGUGAAAGA-3'(SEQ ID NO:189)Sense strand: 5'-AGCCCAAGAAAGUGAAAGA-3' (SEQ ID NO: 189)
反义链：5'-UCUUUCACUUUCUUGGGCUCC-3'(SEQ ID NO:190)Antisense strand: 5'-UCUUUCACUUUCUUGGGCUCC-3' (SEQ ID NO: 190)
siFXIIe2siFXIIe2
正义链：5'-GGAGCCCAAGAAAGUGAAAGA-3'(SEQ ID NO:191)Sense strand: 5'-GGAGCCCAAGAAAGUGAAAGA-3' (SEQ ID NO: 191)
反义链：5'-UCUUUCACUUUCUUGGGCUCCAA-3'(SEQ ID NO:192)Antisense strand: 5'-UCUUUCACUUUCUUGGGCUCCAA-3' (SEQ ID NO: 192)
siFXIIf1siFXIIf1
正义链：5'-CCAAGAAAGUGAAAGACCA-3'(SEQ ID NO:249)Sense strand: 5'-CCAAGAAAGUGAAAGACCA-3' (SEQ ID NO: 249)
反义链：5'-UGGUCUUUCACUUUCUUGGGC-3'(SEQ ID NO:250)Antisense strand: 5'-UGGUCUUUCACUUUCUUGGGC-3' (SEQ ID NO: 250)
siFXIIf2siFXIIf2
正义链：5'-GCCCAAGAAAGUGAAAGACCA-3'(SEQ ID NO:251)Sense strand: 5'-GCCCAAGAAAGUGAAAGACCA-3' (SEQ ID NO: 251)
反义链：5'-UGGUCUUUCACUUUCUUGGGCUC-3'(SEQ ID NO:252)Antisense strand: 5'-UGGUCUUUCACUUUCUUGGGCUC-3' (SEQ ID NO: 252)
在一些实施方式中，所述siRNA具有siFXIIa1、siFXIIa2、siFXIIb1、siFXIIb2、siFXIId1、siFXIId2、siFXIIe1、siFXIIe2、siFXIIf1或siFXIIf2所示的核苷酸序列(即，核酸碱基序列)。In some embodiments, the siRNA has a nucleotide sequence (i.e., a nucleic acid base sequence) shown in siFXIIa1, siFXIIa2, siFXIIb1, siFXIIb2, siFXIId1, siFXIId2, siFXIIe1, siFXIIe2, siFXIIf1, or siFXIIf2.
如前所述，本公开的siRNA中的核苷酸各自独立地为修饰或未修饰的核苷酸。在一些实施方式中，本公开的siRNA中的核苷酸为未经修饰的核苷酸；在一些实施方式中，本公开的siRNA中的部分或全部核苷酸为修饰的核苷酸，核苷酸基团上的这些修饰不会导致本公开的siRNA缀合物抑制FXII基因表达的功能明显削弱或丧失。As mentioned above, the nucleotides in the siRNA of the present disclosure are each independently modified or unmodified nucleotides. In some embodiments, the nucleotides in the siRNA of the present disclosure are unmodified nucleotides; in some embodiments, some or all of the nucleotides in the siRNA of the present disclosure are modified nucleotides, and these modifications on the nucleotide groups do not result in a significant weakening or loss of the function of the siRNA conjugate of the present disclosure in inhibiting the expression of the FXII gene.
在一些实施方式中，本公开的siRNA至少含有1个修饰的核苷酸。在本公开的上下文中，所使用的术语“修饰的核苷酸”是指核苷酸的核糖基2'位羟基被其他基团取代形成的核苷酸或核苷酸类似物，或者核苷酸上的碱基是经修饰的碱基的核苷酸。所述修饰的核苷酸不会导致siRNA抑制基因表达的功能明显削弱或丧失。例如，可以选择J.K.Watts,G.F.Deleavey,and M.J.Damha,Chemically modified siRNA:tools andapplications.Drug Discov Today,2008,13(19-20):842-55中公开的修饰的核苷酸。In some embodiments, the siRNA of the present disclosure contains at least one modified nucleotide. In the context of the present disclosure, the term "modified nucleotide" used refers to a nucleotide or nucleotide analog formed by replacing the 2' hydroxyl group of the ribose group of the nucleotide with other groups, or a nucleotide in which the base on the nucleotide is a modified base. The modified nucleotide will not cause the function of siRNA to inhibit gene expression to be significantly weakened or lost. For example, the modified nucleotide disclosed in J.K.Watts, G.F.Deleavey, and M.J.Damha, Chemically modified siRNA: tools and applications. Drug Discov Today, 2008, 13 (19-20): 842-55 can be selected.
在一些实施方式中，本公开提供的siRNA的正义链或所述反义链中的至少一个核苷酸为修饰的核苷酸，和/或至少一个磷酸酯基为具有修饰基团的磷酸酯基；换句话说，所述正义链和所述反义链中至少一条单链的磷酸-糖骨架中的磷酸酯基和/或核糖基的至少一部分为具有修饰基团的磷酸酯基和/或具有修饰基团的核糖基。In some embodiments, at least one nucleotide in the sense strand or the antisense strand of the siRNA provided by the present disclosure is a modified nucleotide, and/or at least one phosphate group is a phosphate group having a modified group; in other words, at least a portion of the phosphate group and/or ribose group in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand is a phosphate group having a modified group and/or a ribose group having a modified group.
在一些实施方式中，所述正义链和/或所述反义链中的全部核苷酸均为修饰的核苷酸。在一些实施方式中，本公开提供的siRNA的正义链和所述反义链中的每一个核苷酸独立地为氟代修饰的核苷酸或非氟代修饰的核苷酸。In some embodiments, all nucleotides in the sense strand and/or the antisense strand are modified nucleotides. In some embodiments, each nucleotide in the sense strand and the antisense strand of the siRNA provided by the present disclosure is independently a fluorinated modified nucleotide or a non-fluorinated modified nucleotide.
本公开的发明人惊奇地发现，本公开所述的siRNA在动物实验中获得了血浆中稳定性和基因沉默效率的高度平衡。The inventors of the present disclosure surprisingly found that the siRNA described in the present disclosure achieved a high balance between stability in plasma and gene silencing efficiency in animal experiments.
在一些实施方式中，所述氟代修饰的核苷酸位于核苷酸序列I和核苷酸序列II中，并且，按照5'末端到3'末端的方向，所述核苷酸序列I的第7、8、9位的核苷酸为氟代修饰的核苷酸；按照5'末端到3'末端的方向，所述核苷酸序列II的第2、6、14、16位的核苷酸为氟代修饰的核苷酸。In some embodiments, the fluorinated modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of the nucleotide sequence I are fluorinated modified nucleotides; in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14, and 16 of the nucleotide sequence II are fluorinated modified nucleotides.
在一些实施方式中，所述氟代修饰的核苷酸位于核苷酸序列I和核苷酸序列II中，所述核苷酸序列I中氟代修饰的核苷酸不多于5个，并且，按照5'末端到3'末端的方向，所述核苷酸序列I的第7、8、9位的核苷酸为氟代修饰的核苷酸；所述核苷酸序列II中氟代修饰的核苷酸不多于7个，并且，所述核苷酸序列II的第2、6、14、16位的核苷酸为氟代修饰的核苷酸。In some embodiments, the fluorinated modified nucleotides are located in nucleotide sequence I and nucleotide sequence II, the number of fluorinated modified nucleotides in the nucleotide sequence I is no more than 5, and, in the direction from the 5' end to the 3' end, the 7th, 8th, and 9th nucleotides of the nucleotide sequence I are fluorinated modified nucleotides; the number of fluorinated modified nucleotides in the nucleotide sequence II is no more than 7, and the 2nd, 6th, 14th, and 16th nucleotides of the nucleotide sequence II are fluorinated modified nucleotides.
在一些实施方式中，按照5'末端到3'末端的方向，在所述正义链中，所述核苷酸序列I的第7、8、9位或者5、7、8、9位的核苷酸为氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为非氟代修饰的核苷酸；按照5'末端到3'末端的方向，在所述反义链中，所述核苷酸序列II的第2、6、14、16位或者2、6、8、9、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为非氟代修饰的核苷酸。In some embodiments, in the direction from the 5' end to the 3' end, in the sense strand, the nucleotides at positions 7, 8, 9 or positions 5, 7, 8, 9 of the nucleotide sequence I are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the sense strand are non-fluorinated modified nucleotides; in the direction from the 5' end to the 3' end, in the antisense strand, the nucleotides at positions 2, 6, 14, 16 or positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence II are fluorinated modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are non-fluorinated modified nucleotides.
在本公开的上下文中，“氟代修饰的核苷酸”指核苷酸的核糖基2'位的羟基被氟取代形成的核苷酸，其具有以下式(7)所示的结构。“非氟代修饰的核苷酸”指核苷酸的核糖基2'位的羟基被非氟基团取代形成的核苷酸、或核苷酸类似物。在一些实施方式中，每一个非氟代修饰的核苷酸独立地选自核苷酸的核糖基2'位的羟基被非氟基团取代形成的核苷酸或核苷酸类似物中的一种。In the context of the present disclosure, "fluorinated modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by fluorine, and has a structure shown in the following formula (7). "Non-fluorinated modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group. In some embodiments, each non-fluorinated modified nucleotide is independently selected from one of the nucleotides or nucleotide analogs in which the hydroxyl group at the 2' position of the ribose group of the nucleotide is replaced by a non-fluorinated group.
这些核糖基2'位的羟基被非氟基团取代形成的核苷酸是本领域技术人员所公知的，这些核苷酸可以选自2'-烷氧基修饰的核苷酸、2'-经取代的烷氧基修饰的核苷酸、2'-烷基修饰的核苷酸、2'-经取代的烷基修饰的核苷酸、2'-氨基修饰的核苷酸、2'-经取代的氨基修饰的核苷酸、2'-脱氧核苷酸中的一种。The nucleotides formed by replacing the hydroxyl group at the 2' position of these ribose groups with non-fluorine groups are well known to those skilled in the art, and these nucleotides can be selected from 2'-alkoxy-modified nucleotides, 2'-substituted alkoxy-modified nucleotides, 2'-alkyl-modified nucleotides, 2'-substituted alkyl-modified nucleotides, 2'-amino-modified nucleotides, 2'-substituted amino-modified nucleotides, and 2'-deoxynucleotides.
在一些实施方式中，2'-烷氧基修饰的核苷酸为甲氧基修饰的核苷酸(2'-OMe)，如式(8)所示。在一些实施方式中，2'-经取代的烷氧基修饰的核苷酸，例如可以是2'-O-甲氧基乙基修饰的核苷酸(2'-MOE)，如式(9)所示。在一些实施方式中，2'-氨基修饰的核苷酸(2'-NH2)如式(10)所示。在一些实施方式中，2'-脱氧核苷酸(DNA)如式(11)所示：In some embodiments, the 2'-alkoxy modified nucleotide is a methoxy modified nucleotide (2'-OMe), as shown in formula (8). In some embodiments, the 2'-substituted alkoxy modified nucleotide, for example, can be a 2'-O-methoxyethyl modified nucleotide (2'-MOE), as shown in formula (9). In some embodiments, the 2'-amino modified nucleotide (2'-NH2 ) is as shown in formula (10). In some embodiments, the 2'-deoxynucleotide (DNA) is as shown in formula (11):
核苷酸类似物指能够在核酸中代替核苷酸，但结构不同于腺嘌呤核糖核苷酸、鸟嘌呤核糖核苷酸、胞嘧啶核糖核苷酸、尿嘧啶核糖核苷酸或胸腺嘧啶脱氧核糖核苷酸的基团。在一些实施方式中，核苷酸类似物可以是异核苷酸、桥联的核苷酸(bridged nucleicacid，简称BNA)或无环核苷酸。Nucleotide analogs refer to groups that can replace nucleotides in nucleic acids but have structures different from adenine ribonucleotides, guanine ribonucleotides, cytosine ribonucleotides, uracil ribonucleotides or thymine deoxyribonucleotides. In some embodiments, nucleotide analogs can be isonucleotides, bridged nucleic acids (BNAs) or acyclic nucleotides.
BNA是指受约束的或不能接近的核苷酸。BNA可以含有五元环、六元环、或七元环的具有“固定的”C3'-内切糖缩拢的桥联结构。通常将该桥掺入到该核糖的2'-、4'-位处以提供一个2',4'-BNA核苷酸。在一些实施方式中，BNA可以是LNA、ENA、cET BNA等，其中，LNA如式(12)所示，ENA如式(13)所示，cET BNA如式(14)所示：BNA refers to a constrained or inaccessible nucleotide. BNA may contain a five-membered ring, a six-membered ring, or a seven-membered ring with a "fixed" C3'-endosugar condensed bridge structure. The bridge is usually incorporated into the 2'-, 4'-position of the ribose to provide a 2', 4'-BNA nucleotide. In some embodiments, BNA may be LNA, ENA, cET BNA, etc., wherein LNA is shown in formula (12), ENA is shown in formula (13), and cET BNA is shown in formula (14):
无环核苷酸是核苷酸的糖环被打开形成的一类核苷酸。在一些实施方式中，无环核苷酸可以是解锁核酸(UNA)或甘油核酸(GNA)，其中，UNA如式(15)所示，GNA如式(16)所示：Acyclic nucleotides are a type of nucleotides formed by opening the sugar ring of a nucleotide. In some embodiments, the acyclic nucleotide may be an unlocked nucleic acid (UNA) or a glycerol nucleic acid (GNA), wherein UNA is as shown in formula (15) and GNA is as shown in formula (16):
上述式(15)和式(16)中，R选自H、OH或烷氧基(O-烷基)。In the above formula (15) and formula (16), R is selected from H, OH or alkoxy (O-alkyl).
异核苷酸是指核苷酸中碱基在核糖环上的位置发生改变而形成的化合物。在一些实施方式中，异核苷酸可以是碱基从核糖环的1'-位移动至2'-位或3'-位而形成的化合物，如式(17)或(18)所示：Isonucleotides refer to compounds formed by a change in the position of a base on the ribose ring in a nucleotide. In some embodiments, an isonucleotide may be a compound formed by a base moving from the 1'-position to the 2'-position or the 3'-position of the ribose ring, as shown in formula (17) or (18):
上述式(7)-式(18)化合物中，Base表示核酸碱基，例如A、U、G、C或T；R选自H、OH、F或者如上所述的非氟基团。In the compounds of formula (7) to (18) above, Base represents a nucleic acid base, such as A, U, G, C or T; and R is selected from H, OH, F or the non-fluorine group as described above.
在一些实施方式中，核苷酸类似物选自异核苷酸、LNA、ENA、cET、UNA和GNA中的一种。在一些实施方式中，每一个非氟代修饰的核苷酸均为甲氧基修饰的核苷酸，在上文和下文中，所述甲氧基修饰的核苷酸指核糖基的2'-羟基被甲氧基取代而形成的核苷酸。In some embodiments, the nucleotide analog is selected from one of isonucleotides, LNA, ENA, cET, UNA and GNA. In some embodiments, each non-fluorinated modified nucleotide is a methoxy-modified nucleotide, and in the above and below, the methoxy-modified nucleotide refers to a nucleotide formed by replacing the 2'-hydroxyl group of the ribose group with a methoxy group.
在上文及下文中，“氟代修饰的核苷酸”、“2'-氟修饰的核苷酸”、“核糖基团的2'-羟基被氟取代的核苷酸”和“具有2'-氟代核糖基的核苷酸”意义相同，均指核苷酸的2'-羟基被氟取代，而形成的具有如式(7)所示结构的化合物；“甲氧基修饰的核苷酸”、“2'-甲氧基修饰的核苷酸”、“核糖基团的2'-羟基被甲氧基取代的核苷酸”和“具有2'-甲氧基核糖基的核苷酸”意义相同，均指核苷酸核糖基团的2'-羟基被甲氧基取代而形成的具有如式(8)所示结构的化合物。In the above and below, "fluorinated modified nucleotide", "2'-fluorinated modified nucleotide", "nucleotide in which the 2'-hydroxyl group of the ribose group is substituted by fluorine" and "nucleotide having a 2'-fluorinated ribose group" have the same meaning, all referring to compounds having a structure shown in formula (7) formed by replacing the 2'-hydroxyl group of the nucleotide with fluorine; "methoxy-modified nucleotide", "2'-methoxy-modified nucleotide", "nucleotide in which the 2'-hydroxyl group of the ribose group is substituted by methoxy" and "nucleotide having a 2'-methoxyribose group" have the same meaning, all referring to compounds having a structure shown in formula (8) formed by replacing the 2'-hydroxyl group of the ribose group of the nucleotide with methoxy.
在一些实施方式中，本公开的siRNA是具有以下修饰的siRNA：按照5'末端到3'末端的方向，在所述正义链中，所述核苷酸序列I的第7、8、9位或者第5、7、8、9位的核苷酸为氟代修饰的核苷酸，所述正义链中其余位置的核苷酸为甲氧基修饰的核苷酸；在所述反义链中，所述核苷酸序列II的第2、6、14、16位或者第2、6、8、9、14、16位的核苷酸为氟代修饰的核苷酸，所述反义链中其余位置的核苷酸为甲氧基修饰的核苷酸。In some embodiments, the siRNA disclosed herein is a siRNA having the following modifications: in the direction from the 5' end to the 3' end, in the sense strand, the nucleotides at positions 7, 8, 9 or positions 5, 7, 8, 9 of the nucleotide sequence I are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the sense strand are methoxy-modified nucleotides; in the antisense strand, the nucleotides at positions 2, 6, 14, 16 or positions 2, 6, 8, 9, 14, 16 of the nucleotide sequence II are fluorine-modified nucleotides, and the nucleotides at the remaining positions in the antisense strand are methoxy-modified nucleotides.
在一些实施方式中，本公开的siRNA是具有以下修饰的siRNA：按照5'末端到3'末端的方向，所述siRNA的正义链中核苷酸序列I的第5、7、8和9位的核苷酸为氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5'末端到3'末端的方向，所述siRNA的反义链中核苷酸序列II的第2、6、8、9、14和16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸；In some embodiments, the siRNA disclosed herein is a siRNA with the following modifications: in the direction from the 5' end to the 3' end, the nucleotides at positions 5, 7, 8, and 9 of the nucleotide sequence I in the sense strand of the siRNA are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 8, 9, 14, and 16 of the nucleotide sequence II in the antisense strand of the siRNA are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides;
或者，按照5'末端到3'末端的方向，所述siRNA的正义链中核苷酸序列I的第5、7、8和9位的核苷酸为氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5'末端到3'末端的方向，所述siRNA的反义链中核苷酸序列II的第2、6、14和16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸；Alternatively, in the direction from the 5' end to the 3' end, the 5th, 7th, 8th and 9th nucleotides of the nucleotide sequence I in the sense strand of the siRNA are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the siRNA sense strand are methoxy-modified nucleotides, and, in the direction from the 5' end to the 3' end, the 2nd, 6th, 14th and 16th nucleotides of the nucleotide sequence II in the antisense strand of the siRNA are fluorinated modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides;
或者，按照5'末端到3'末端的方向，所述siRNA的正义链中核苷酸序列I的第7、8和9位的核苷酸为-氟代修饰的核苷酸，siRNA的正义链的其余位置的核苷酸为甲氧基修饰的核苷酸，并且，按照5'末端到3'末端的方向，所述siRNA的反义链中核苷酸序列II的第2、6、14和16位的核苷酸为氟代修饰的核苷酸，siRNA的反义链其余位置的核苷酸为甲氧基修饰的核苷酸。Alternatively, in the direction from the 5' end to the 3' end, the nucleotides at positions 7, 8 and 9 of the nucleotide sequence I in the sense strand of the siRNA are -fluoro-modified nucleotides, and the nucleotides at the remaining positions of the sense strand of the siRNA are methoxy-modified nucleotides, and, in the direction from the 5' end to the 3' end, the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence II in the antisense strand of the siRNA are fluorine-modified nucleotides, and the nucleotides at the remaining positions of the antisense strand of the siRNA are methoxy-modified nucleotides.
在一些实施方式中，本公开提供的siRNA为siFXIIa1-M1、siFXIIa1-M2、siFXIIa1-M3、siFXIIa2-M1、siFXIIa2-M2、siFXIIa2-M3、siFXIIb1-M1、siFXIIb1-M2、siFXIIb1-M3、siFXIIb2-M1、siFXIIb2-M2、siFXIIb2-M3、siFXIId1-M1、siFXIId1-M2、siFXIId1-M3、siFXIId2-M1、siFXIId2-M2、siFXIId2-M3、siFXIIe1-M1、siFXIIe1-M2、siFXIIe1-M3、siFXIIe2-M1、siFXIIe2-M2、siFXIIe2-M3、siFXIIf1-M1、siFXIIf1-M2、siFXIIf1-M3、siFXIIf2-M1、siFXIIf2-M2和siFXIIf2-M3中的任意一种：In some embodiments, the siRNA provided by the present disclosure is siFXIIa1-M1, siFXIIa1-M2, siFXIIa1-M3, siFXIIa2-M1, siFXIIa2-M2, siFXIIa2-M3, siFXIIb1-M1, siFXIIb1-M2, siFXIIb1-M3, siFXIIb2-M1, siFXIIb2-M2, siFXIIb2-M3, siFXIId1-M1, siFXIId1-M2, siFXIId1-M3, siFXIId2-M1, siFXIId2-M2, siFXIId2-M3, siFXIId1-M1, siFXIId2-M2, siFXIId2-M3, siFXIId1-M2, siFXIId2-M3, siFXIId1-M1, siFXIId1-M2, siFXIId2-M3, siFXIId2-M1, siFXIId2 ... IId1-M3, siFXIId2-M1, siFXIId2-M2, siFXIId2-M3, siFXIIe1-M1, siFXIIe1-M2, siFXIIe1-M3, siFXIIe2-M1, siFXIIe2-M2, siFXIIe2-M3, siFXIIf1-M1, siFXIIf1-M2, siFXIIf1-M3, siFXIIf2-M 1. Any one of siFXIIf2-M2 and siFXIIf2-M3:
siFXIIa1-M1siFXIIa1-M1
正义链：Justice Chain:
5'-GmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-GmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:13)(SEQ ID NO:13)
反义链：Antisense strand:
5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'
(SEQ ID NO:14)(SEQ ID NO:14)
siFXIIa1-M2siFXIIa1-M2
正义链：Justice Chain:
5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:15)(SEQ ID NO:15)
反义链：Antisense strand:
5'-AmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGm-3'5'-AmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGm-3'
(SEQ ID NO:16)(SEQ ID NO:16)
siFXIIa1-M3siFXIIa1-M3
正义链：Justice Chain:
5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:17)(SEQ ID NO:17)
反义链：Antisense strand:
5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'
(SEQ ID NO:18)(SEQ ID NO:18)
siFXIIa2-M1siFXIIa2-M1
正义链：Justice Chain:
5'-CmAmGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-CmAmGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:19)(SEQ ID NO:19)
反义链：Antisense strand:
5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'
(SEQ ID NO:20)(SEQ ID NO:20)
siFXIIa2-M2siFXIIa2-M2
正义链：Justice Chain:
5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:21)(SEQ ID NO:21)
反义链：Antisense strand:
5'-AmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'5'-AmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'
(SEQ ID NO:22)(SEQ ID NO:22)
siFXIIa2-M3siFXIIa2-M3
正义链：Justice Chain:
5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:23)(SEQ ID NO:23)
反义链：Antisense strand:
5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'5'-AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'
(SEQ ID NO:24)(SEQ ID NO:24)
siFXIIb1-M1siFXIIb1-M1
正义链：Justice Chain:
5'-CmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-CmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:73)(SEQ ID NO:73)
反义链：Antisense strand:
5'-UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUm-3'5'-UmUfCmAmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUm-3'
(SEQ ID NO:74)(SEQ ID NO:74)
siFXIIb1-M2siFXIIb1-M2
正义链：Justice Chain:
5'-CmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-CmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:75)(SEQ ID NO:75)
反义链：Antisense strand:
5'-UmUfCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUm-3'5'-UmUfCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUm-3'
(SEQ ID NO:76)(SEQ ID NO:76)
siFXIIb1-M3siFXIIb1-M3
正义链：Justice Chain:
5'-CmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-CmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:77)(SEQ ID NO:77)
反义链：Antisense strand:
5'-UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUm-3'5'-UmUfCmAmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUm-3'
(SEQ ID NO:78)(SEQ ID NO:78)
siFXIIb2-M1siFXIIb2-M1
正义链：Justice Chain:
5'-AmAmCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-AmAmCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:79)(SEQ ID NO:79)
反义链：Antisense strand:
5'-UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'5'-UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'
(SEQ ID NO:80)(SEQ ID NO:80)
siFXIIb2-M2siFXIIb2-M2
正义链：Justice Chain:
5'-AmAmCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-AmAmCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:81)(SEQ ID NO:81)
反义链：Antisense strand:
5'-UmUfCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'5'-UmUfCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'
(SEQ ID NO:82)(SEQ ID NO:82)
siFXIIb2-M3siFXIIb2-M3
正义链：Justice Chain:
5'-AmAmCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-AmAmCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:83)(SEQ ID NO:83)
反义链：Antisense strand:
5'-UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'5'-UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'
(SEQ ID NO:84)(SEQ ID NO:84)
siFXIId1-M1siFXIId1-M1
正义链：Justice Chain:
5'-GmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-GmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:133)(SEQ ID NO:133)
反义链：Antisense strand:
5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'
(SEQ ID NO:134)(SEQ ID NO:134)
siFXIId1-M2siFXIId1-M2
正义链：Justice Chain:
5'-GmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-GmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:135)(SEQ ID NO:135)
反义链：Antisense strand:
5'-UmUfUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAm-3'5'-UmUfUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAm-3'
(SEQ ID NO:136)(SEQ ID NO:136)
siFXIId1-M3siFXIId1-M3
正义链：Justice Chain:
5'-GmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-GmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:137)(SEQ ID NO:137)
反义链：Antisense strand:
5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'
(SEQ ID NO:138)(SEQ ID NO:138)
siFXIId2-M1siFXIId2-M1
正义链：Justice Chain:
5'-UmUmGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-UmUmGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:139)(SEQ ID NO:139)
反义链：Antisense strand:
5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'
(SEQ ID NO:140)(SEQ ID NO: 140)
siFXIId2-M2siFXIId2-M2
正义链：Justice Chain:
5'-UmUmGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-UmUmGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:141)(SEQ ID NO:141)
反义链：Antisense strand:
5'-UmUfUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'5'-UmUfUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'
(SEQ ID NO:142)(SEQ ID NO:142)
siFXIId2-M3siFXIId2-M3
正义链：Justice Chain:
5'-UmUmGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-UmUmGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:143)(SEQ ID NO:143)
反义链：Antisense strand:
5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'5'-UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'
(SEQ ID NO:144)(SEQ ID NO:144)
siFXIIe1-M1siFXIIe1-M1
正义链：Justice Chain:
5'-AmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-AmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:193)(SEQ ID NO: 193)
反义链：Antisense strand:
5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'
(SEQ ID NO:194)(SEQ ID NO: 194)
siFXIIe1-M2siFXIIe1-M2
正义链：Justice Chain:
5'-AmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-AmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:195)(SEQ ID NO: 195)
反义链：Antisense strand:
5'-UmCfUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCm-3'5'-UmCfUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCm-3'
(SEQ ID NO:196)(SEQ ID NO: 196)
siFXIIe1-M3siFXIIe1-M3
正义链：Justice Chain:
5'-AmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-AmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:197)(SEQ ID NO: 197)
反义链：Antisense strand:
5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'
(SEQ ID NO:198)(SEQ ID NO: 198)
siFXIIe2-M1siFXIIe2-M1
正义链：Justice Chain:
5'-GmGmAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-GmGmAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:199)(SEQ ID NO: 199)
反义链：Antisense strand:
5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'
(SEQ ID NO:200)(SEQ ID NO: 200)
siFXIIe2-M2siFXIIe2-M2
正义链：Justice Chain:
5'-GmGmAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-GmGmAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:201)(SEQ ID NO:201)
反义链：Antisense strand:
5'-UmCfUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'5'-UmCfUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'
(SEQ ID NO:202)(SEQ ID NO:202)
siFXIIe2-M3siFXIIe2-M3
正义链：Justice Chain:
5'-GmGmAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-GmGmAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:203)(SEQ ID NO:203)
反义链：Antisense strand:
5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'5'-UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'
(SEQ ID NO:204)(SEQ ID NO:204)
siFXIIf1-M1siFXIIf1-M1
正义链：Justice Chain:
5'-CmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-CmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:253)(SEQ ID NO:253)
反义链：Antisense strand:
5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'
(SEQ ID NO:254)(SEQ ID NO:254)
siFXIIf1-M2siFXIIf1-M2
正义链：Justice Chain:
5'-CmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-CmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:255)(SEQ ID NO:255)
反义链：Antisense strand:
5'-UmGfGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCm-3'5'-UmGfGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCm-3'
(SEQ ID NO:256)(SEQ ID NO:256)
siFXIIf1-M3siFXIIf1-M3
正义链：Justice Chain:
5'-CmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-CmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:257)(SEQ ID NO:257)
反义链：Antisense strand:
5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'
(SEQ ID NO:258)(SEQ ID NO:258)
siFXIIf2-M1siFXIIf2-M1
正义链：Justice Chain:
5'-GmCmCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-GmCmCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:259)(SEQ ID NO:259)
反义链：Antisense strand:
5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'
(SEQ ID NO:260)(SEQ ID NO:260)
siFXIIf2-M2siFXIIf2-M2
正义链：Justice Chain:
5'-GmCmCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-GmCmCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:261)(SEQ ID NO:261)
反义链：Antisense strand:
5'-UmGfGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'5'-UmGfGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'
(SEQ ID NO:262)(SEQ ID NO:262)
siFXIIf2-M3siFXIIf2-M3
正义链：Justice Chain:
5'-GmCmCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-GmCmCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:263)(SEQ ID NO:263)
反义链：Antisense strand:
5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'5'-UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'
(SEQ ID NO:264)(SEQ ID NO:264)
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸。Among them, capital letters C, G, U, and A represent the base composition of the nucleotide; the lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide.
具有上述修饰的siRNA不仅成本低，而且可使血液中的核糖核酸酶不易切割核酸，由此增加核酸的稳定性，使核酸具有更强的抵抗核酸酶水解的性能。同时，上述修饰并未显著降低siRNA的抑制性能。The siRNA with the above modification is not only low in cost, but also makes it difficult for ribonucleases in the blood to cut nucleic acids, thereby increasing the stability of nucleic acids and making them more resistant to nuclease hydrolysis. At the same time, the above modification does not significantly reduce the inhibitory performance of siRNA.
在一些实施方式中，本公开提供的siRNA的正义链和反义链中至少一条单链的磷酸-糖骨架中的磷酸酯基中的至少一部分或至少1个为具有修饰基团的磷酸酯基。在一些实施方式中，具有修饰基团的磷酸酯基为磷酸酯基中的磷酸二酯键中的至少一个氧原子被硫原子取代而形成的硫代磷酸酯基；在一些实施方式中，所述具有修饰基团的磷酸酯基为具有如式(1)所示结构的硫代磷酸酯基：In some embodiments, at least a portion or at least one of the phosphate groups in the phosphate-sugar backbone of at least one single strand in the sense strand and the antisense strand of the siRNA provided by the present disclosure is a phosphate group having a modified group. In some embodiments, the phosphate group having a modified group is a thiophosphate group formed by replacing at least one oxygen atom in the phosphodiester bond in the phosphate group with a sulfur atom; in some embodiments, the phosphate group having a modified group is a thiophosphate group having a structure as shown in formula (1):
这种修饰能稳定siRNA的双链结构，保持碱基配对的高特异性和高亲和力。This modification can stabilize the double-stranded structure of siRNA and maintain high specificity and high affinity of base pairing.
在一些实施方式中，本公开提供的siRNA中，硫代磷酸酯基连接存在于由以下位置组成的组中的至少一处：正义链或反义链任意一端的第一个和第二个核苷酸之间；正义链或反义链任意一端的第二个和第三个核苷酸之间；或上述的任意组合。在一些实施方式中，硫代磷酸酯基连接存在于除正义链5'末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯基连接存在于除正义链3'末端以外的全部上述位置处。在一些实施方式中，硫代磷酸酯基连接存在于以下位置中的至少一处：In some embodiments, in the siRNA provided by the present disclosure, the phosphorothioate linkage is present in at least one of the following positions: between the first and second nucleotides at either end of the sense strand or the antisense strand; between the second and third nucleotides at either end of the sense strand or the antisense strand; or any combination of the above. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 5' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at all of the above positions except the 3' end of the sense strand. In some embodiments, the phosphorothioate linkage is present at at least one of the following positions:
所述正义链的5'末端第1个核苷酸和第2个核苷酸之间；Between the first nucleotide and the second nucleotide at the 5' end of the sense strand;
所述正义链的5'末端第2个核苷酸和第3个核苷酸之间；Between the second nucleotide and the third nucleotide at the 5' end of the sense strand;
所述正义链的3'末端第1个核苷酸和第2个核苷酸之间；Between the first nucleotide and the second nucleotide at the 3' end of the sense strand;
所述正义链的3'末端第2个核苷酸和第3个核苷酸之间；Between the second nucleotide and the third nucleotide at the 3' end of the sense strand;
所述反义链的5'末端第1个核苷酸和第2个核苷酸之间；Between the first nucleotide and the second nucleotide at the 5' end of the antisense strand;
所述反义链的5'末端第2个核苷酸和第3个核苷酸之间；Between the second nucleotide and the third nucleotide at the 5' end of the antisense strand;
所述反义链的3'末端第1个核苷酸和第2个核苷酸之间；以及between the first nucleotide and the second nucleotide at the 3' end of the antisense strand; and
所述反义链的3'末端第2个核苷酸和第3个核苷酸之间。Between the second nucleotide and the third nucleotide at the 3' end of the antisense strand.
在一些实施方式中，本公开提供的siRNA为siFXIIa1-M1S、siFXIIa1-M2S、siFXIIa1-M3S、siFXIIa2-M1S、siFXIIa2-M2S、siFXIIa2-M3S、siFXIIb1-M1S、siFXIIb1-M2S、siFXIIb1-M3S、siFXIIb2-M1S、siFXIIb2-M2S、siFXIIb2-M3S、siFXIId1-M1S、siFXIId1-M2S、siFXIId1-M3S、siFXIId2-M1S、siFXIId2-M2S、siFXIId2-M3S、siFXIIe1-M1S、siFXIIe1-M2S、siFXIIe1-M3S、siFXIIe2-M1S、siFXIIe2-M2S、siFXIIe2-M3S、siFXIIf1-M1S、siFXIIf1-M2S、siFXIIf1-M3S、siFXIIf2-M1S、siFXIIf2-M2S和siFXIIf2-M3S中的任意一种：In some embodiments, the siRNA provided by the present disclosure is siFXIIa1-M1S, siFXIIa1-M2S, siFXIIa1-M3S, siFXIIa2-M1S, siFXIIa2-M2S, siFXIIa2-M3S, siFXIIb1-M1S, siFXIIb1-M2S, siFXIIb1-M3S, siFXIIb2-M1S, siFXIIb2-M2S, siFXIIb2-M3S, siFXIId1-M1S, siFXIId1-M2S, siFXII Any one of siFXIId1-M3S, siFXIId2-M1S, siFXIId2-M2S, siFXIId2-M3S, siFXIIe1-M1S, siFXIIe1-M2S, siFXIIe1-M3S, siFXIIe2-M1S, siFXIIe2-M2S, siFXIIe2-M3S, siFXIIf1-M1S, siFXIIf1-M2S, siFXIIf1-M3S, siFXIIf2-M1S, siFXIIf2-M2S and siFXIIf2-M3S:
siFXIIa1-M1SsiFXIIa1-M1S
正义链：Justice Chain:
5'-GmsGmsAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-GmsGmsAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:25)(SEQ ID NO:25)
反义链：Antisense strand:
5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'
(SEQ ID NO:26)(SEQ ID NO:26)
siFXIIa1-M2SsiFXIIa1-M2S
正义链：Justice Chain:
5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:27)(SEQ ID NO:27)
反义链：Antisense strand:
5'-AmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmsUmsGm-3'5'-AmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmsUmsGm-3'
(SEQ ID NO:28)(SEQ ID NO:28)
siFXIIa1-M3SsiFXIIa1-M3S
正义链：Justice Chain:
5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:29)(SEQ ID NO:29)
反义链：Antisense strand:
5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'
(SEQ ID NO:30)(SEQ ID NO:30)
siFXIIa2-M1SsiFXIIa2-M1S
正义链：Justice Chain:
5'-CmsAmsGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-CmsAmsGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:31)(SEQ ID NO:31)
反义链：Antisense strand:
5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'
(SEQ ID NO:32)(SEQ ID NO:32)
siFXIIa2-M2SsiFXIIa2-M2S
正义链：Justice Chain:
5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:33)(SEQ ID NO:33)
反义链：Antisense strand:
5'-AmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'5'-AmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'
(SEQ ID NO:34)(SEQ ID NO:34)
siFXIIa2-M3SsiFXIIa2-M3S
正义链：Justice Chain:
5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:35)(SEQ ID NO:35)
反义链：Antisense strand:
5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'5'-AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'
(SEQ ID NO:36)(SEQ ID NO:36)
siFXIIb1-M1SsiFXIIb1-M1S
正义链：Justice Chain:
5'-CmsUmsCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-CmsUmsCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:85)(SEQ ID NO:85)
反义链：Antisense strand:
5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'
(SEQ ID NO:86)(SEQ ID NO:86)
siFXIIb1-M2SsiFXIIb1-M2S
正义链：Justice Chain:
5'-CmsUmsCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-CmsUmsCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:87)(SEQ ID NO:87)
反义链：Antisense strand:
5'-UmsUfsCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmsUmsUm-3'5'-UmsUfsCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmsUmsUm-3'
(SEQ ID NO:88)(SEQ ID NO:88)
siFXIIb1-M3SsiFXIIb1-M3S
正义链：Justice Chain:
5'-CmsUmsCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-CmsUmsCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:89)(SEQ ID NO:89)
反义链：Antisense strand:
5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'
(SEQ ID NO:90)(SEQ ID NO:90)
siFXIIb2-M1SsiFXIIb2-M1S
正义链：Justice Chain:
5'-AmsAmsCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-AmsAmsCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:91)(SEQ ID NO:91)
反义链：Antisense strand:
5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'
(SEQ ID NO:92)(SEQ ID NO:92)
siFXIIb2-M2SsiFXIIb2-M2S
正义链：Justice Chain:
5'-AmsAmsCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-AmsAmsCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:93)(SEQ ID NO:93)
反义链：Antisense strand:
5'-UmsUfsCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'5'-UmsUfsCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'
(SEQ ID NO:94)(SEQ ID NO:94)
siFXIIb2-M3SsiFXIIb2-M3S
正义链：Justice Chain:
5'-AmsAmsCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-AmsAmsCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:95)(SEQ ID NO:95)
反义链：Antisense strand:
5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'5'-UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'
(SEQ ID NO:96)(SEQ ID NO:96)
siFXIId1-M1SsiFXIId1-M1S
正义链：Justice Chain:
5'-GmsGmsAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-GmsGmsAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:145)(SEQ ID NO:145)
反义链：Antisense strand:
5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'
(SEQ ID NO:146)(SEQ ID NO:146)
siFXIId1-M2SsiFXIId1-M2S
正义链：Justice Chain:
5'-GmsGmsAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-GmsGmsAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:147)(SEQ ID NO:147)
反义链：Antisense strand:
5'-UmsUfsUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmsAmsAm-3'5'-UmsUfsUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmsAmsAm-3'
(SEQ ID NO:148)(SEQ ID NO: 148)
siFXIId1-M3SsiFXIId1-M3S
正义链：Justice Chain:
5'-GmsGmsAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-GmsGmsAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:149)(SEQ ID NO: 149)
反义链：Antisense strand:
5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'
(SEQ ID NO:150)(SEQ ID NO: 150)
siFXIId2-M1SsiFXIId2-M1S
正义链：Justice Chain:
5'-UmsUmsGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-UmsUmsGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:151)(SEQ ID NO:151)
反义链：Antisense strand:
5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'
(SEQ ID NO:152)(SEQ ID NO:152)
siFXIId2-M2SsiFXIId2-M2S
正义链：Justice Chain:
5'-UmsUmsGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-UmsUmsGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:153)(SEQ ID NO:153)
反义链：Antisense strand:
5'-UmsUfsUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'5'-UmsUfsUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'
(SEQ ID NO:154)(SEQ ID NO: 154)
siFXIId2-M3SsiFXIId2-M3S
正义链：Justice Chain:
5'-UmsUmsGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-UmsUmsGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:155)(SEQ ID NO:155)
反义链：Antisense strand:
5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'5'-UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'
(SEQ ID NO:156)(SEQ ID NO:156)
siFXIIe1-M1SsiFXIIe1-M1S
正义链：Justice Chain:
5'-AmsGmsCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-AmsGmsCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:205)(SEQ ID NO:205)
反义链：Antisense strand:
5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'
(SEQ ID NO:206)(SEQ ID NO:206)
siFXIIe1-M2SsiFXIIe1-M2S
正义链：Justice Chain:
5'-AmsGmsCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-AmsGmsCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:207)(SEQ ID NO:207)
反义链：Antisense strand:
5'-UmsCfsUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmsCmsCm-3'5'-UmsCfsUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmsCmsCm-3'
(SEQ ID NO:208)(SEQ ID NO:208)
siFXIIe1-M3SsiFXIIe1-M3S
正义链：Justice Chain:
5'-AmsGmsCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-AmsGmsCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:209)(SEQ ID NO:209)
反义链：Antisense strand:
5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'
(SEQ ID NO:210)(SEQ ID NO:210)
siFXIIe2-M1SsiFXIIe2-M1S
正义链：Justice Chain:
5'-GmsGmsAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-GmsGmsAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:211)(SEQ ID NO:211)
反义链：Antisense strand:
5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'
(SEQ ID NO:212)(SEQ ID NO:212)
siFXIIe2-M2SsiFXIIe2-M2S
正义链：Justice Chain:
5'-GmsGmsAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-GmsGmsAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:213)(SEQ ID NO:213)
反义链：Antisense strand:
5'-UmsCfsUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'5'-UmsCfsUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'
(SEQ ID NO:214)(SEQ ID NO:214)
siFXIIe2-M3SsiFXIIe2-M3S
正义链：Justice Chain:
5'-GmsGmsAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-GmsGmsAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:215)(SEQ ID NO:215)
反义链：Antisense strand:
5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'5'-UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'
(SEQ ID NO:216)(SEQ ID NO:216)
siFXIIf1-M1SsiFXIIf1-M1S
正义链：Justice Chain:
5'-CmsCmsAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-CmsCmsAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:265)(SEQ ID NO:265)
反义链：Antisense strand:
5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'
(SEQ ID NO:266)(SEQ ID NO:266)
siFXIIf1-M2SsiFXIIf1-M2S
正义链：Justice Chain:
5'-CmsCmsAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-CmsCmsAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:267)(SEQ ID NO:267)
反义链：Antisense strand:
5'-UmsGfsGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmsGmsCm-3'5'-UmsGfsGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmsGmsCm-3'
(SEQ ID NO:268)(SEQ ID NO:268)
siFXIIf1-M3SsiFXIIf1-M3S
正义链：Justice Chain:
5'-CmsCmsAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-CmsCmsAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:269)(SEQ ID NO:269)
反义链：Antisense strand:
5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'
(SEQ ID NO:270)(SEQ ID NO:270)
siFXIIf2-M1SsiFXIIf2-M1S
正义链：Justice Chain:
5'-GmsCmsCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-GmsCmsCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:271)(SEQ ID NO:271)
反义链：Antisense strand:
5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'
(SEQ ID NO:272)(SEQ ID NO:272)
siFXIIf2-M2SsiFXIIf2-M2S
正义链：Justice Chain:
5'-GmsCmsCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-GmsCmsCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:273)(SEQ ID NO:273)
反义链：Antisense strand:
5'-UmsGfsGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'5'-UmsGfsGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'
(SEQ ID NO:274)(SEQ ID NO:274)
siFXIIf2-M3SsiFXIIf2-M3S
正义链：Justice Chain:
5'-GmsCmsCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-GmsCmsCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:275)(SEQ ID NO:275)
反义链：Antisense strand:
5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'5'-UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'
(SEQ ID NO:276)(SEQ ID NO:276)
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母左右两个核苷酸之间为硫代磷酸酯基连接。Among them, capital letters C, G, U, and A represent the base composition of the nucleotide; the lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; the lowercase letter s indicates that the two nucleotides on the left and right of the letter are connected by a thiophosphate group.
在一些实施方式中，所述siRNA反义链的5'末端核苷酸为5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸。In some embodiments, the 5' terminal nucleotide of the antisense strand of the siRNA is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide.
常用的所述5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸是本领域技术人员所公知的，如5'-磷酸核苷酸可具有如下式(2)所示的结构：The commonly used 5'-phosphate nucleotides or 5'-phosphate analogue modified nucleotides are well known to those skilled in the art. For example, the 5'-phosphate nucleotides may have a structure as shown in the following formula (2):
再如，Anastasia Khvorova and Jonathan K.Watts,The chemical evolutionof oligonucleotide therapies of clinical utility.Nature Biotechnology,2017,35(3):238-48中公开了如下4种5'-磷酸类似物修饰的核苷酸：For example, Anastasia Khvorova and Jonathan K. Watts, The chemical evolution of oligonucleotide therapies of clinical utility. Nature Biotechnology, 2017, 35(3): 238-48 disclose the following four 5'-phosphate analog-modified nucleotides:
其中，R选自H、OH、甲氧基、氟；Base表示核酸碱基，选自A、U、C、G或T。Wherein, R is selected from H, OH, methoxy, and fluorine; Base represents a nucleic acid base, selected from A, U, C, G, or T.
在一些实施方式中，5'-磷酸核苷酸为式(2)所示的含有5'-磷酸修饰的核苷酸，5'-磷酸类似物修饰的核苷酸为含有乙烯基磷酸酯(5'-(E)-vinylphosphonate，E-VP)修饰的核苷酸，如式(3)所示，或者为硫代磷酸酯修饰的核苷酸，如式(5)所示。In some embodiments, the 5'-phosphate nucleotide is a nucleotide containing a 5'-phosphate modification as shown in formula (2), and the 5'-phosphate analog modified nucleotide is a nucleotide containing a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modification as shown in formula (3), or a nucleotide modified with a thiophosphate as shown in formula (5).
在一些实施方式中，本公开提供的siRNA为siFXIIa1-M1P1、siFXIIa1-M2P1、siFXIIa1-M3P1、siFXIIa2-M1P1、siFXIIa2-M2P1、siFXIIa2-M3P1、siFXIIa1-M1SP1、siFXIIa1-M2SP1、siFXIIa1-M3SP1、siFXIIa2-M1SP1、siFXIIa2-M2SP1、siFXIIa2-M3SP1、siFXIIa1U-M1P1、siFXIIa1U-M2P1、siFXIIa1U-M3P1、siFXIIa2U-M1P1、siFXIIa2U-M2P1、siFXIIa2U-M3P1、siFXIIa1U-M1SP1、siFXIIa1U-M2SP1、siFXIIa1U-M3SP1、siFXIIa2U-M1SP1、siFXIIa2U-M2SP1、siFXIIa2U-M3SP1、siFXIIb1-M1P1、siFXIIb1-M2P1、siFXIIb1-M3P1、siFXIIb2-M1P1、siFXIIb2-M2P1、siFXIIb2-M3P1、siFXIIb1-M1SP1、siFXIIb1-M2SP1、siFXIIb1-M3SP1、siFXIIb2-M1SP1、siFXIIb2-M2SP1、siFXIIb2-M3SP1、siFXIId1-M1P1、siFXIId1-M2P1、siFXIId1-M3P1、siFXIId2-M1P1、siFXIId2-M2P1、siFXIId2-M3P1、siFXIId1-M1SP1、siFXIId1-M2SP1、siFXIId1-M3SP1、siFXIId2-M1SP1、siFXIId2-M2SP1、siFXIId2-M3SP1、siFXIIe1-M1P1、siFXIIe1-M2P1、siFXIIe1-M3P1、siFXIIe2-M1P1、siFXIIe2-M2P1、siFXIIe2-M3P1、siFXIIe1-M1SP1、siFXIIe1-M2SP1、siFXIIe1-M3SP1、siFXIIe2-M1SP1、siFXIIe2-M2SP1、siFXIIe2-M3SP1、siFXIIf1-M1P1、siFXIIf1-M2P1、siFXIIf1-M3P1、siFXIIf2-M1P1、siFXIIf2-M2P1、siFXIIf2-M3P1、siFXIIf1-M1SP1、siFXIIf1-M2SP1、siFXIIf1-M3SP1、siFXIIf2-M1SP1、siFXIIf2-M2SP1和siFXIIf2-M3SP1中的任意一种：In some embodiments, the siRNA provided by the present disclosure is siFXIIa1-M1P1, siFXIIa1-M2P1, siFXIIa1-M3P1, siFXIIa2-M1P1, siFXIIa2-M2P1, siFXIIa2-M3P1, siFXIIa1-M1SP1, siFXIIa1-M2SP1, siFXIIa1-M3SP1, siFXIIa2-M1SP1, siFXIIa2-M2SP1, siFXIIa2-M3SP1, siFXIIa1U-M1P1, siFXIIa1U-M2P1, siFXIIa1U-M3P1, siFXIIa2U-M1P1, siFXIIa2U-M2P1, siFXIIa2U-M3P1, siFXIIa1U-M1SP1, siFXIIa1U-M2SP1, siFXIIa1U-M3SP1, siFXIIa2U-M1SP1, siFXIIa2U-M2SP1, siFXIIa2U-M3SP1, siFXIIb1-M1P1, siFXIIb1-M2P1, siFXIIb1-M3P1, siFXIIb 2-M1P1, siFXIIb2-M2P1, siFXIIb2-M3P1, siFXIIb1-M1SP1, siFXIIb1-M2SP1, siFXIIb1-M3SP1, siFXIIb2-M1SP1, siFXIIb2-M2SP1, s iFXIIb2-M3SP1, siFXIId1-M1P1, siFXIId1-M2P1, siFXIId1-M3P1, siFXIId2-M1P1, siFXIId2-M2P1, siFXIId2-M3P1, siFXIId1-M1SP1, siFXIId1-M2SP1, siFXIId1-M3SP1, siFXIId2-M1SP1, si FXIId2-M2SP1, siFXIId2-M3SP1, siFXIIe1-M1P1, siFXIIe1-M2P1, siFXIIe1-M3P1, siFXIIe2-M1P1, siFXIIe2-M2P1, siFXIIe2-M3P1 、siFXIIe1-M1SP1、siFXIIe1-M2SP1、siFXIIe1-M3SP1、siFXIIe2-M1SP1、siFXIIe2-M2SP1、siFXIIe2-M3SP1、siFXIIf1-M1P1、siFXIIf1-M2P1、siFXIIf1-M3P1、siFXIIf2-M1P1、siFXIIf2-M2P1、siFXIIf2-M3P1、siFXIIf1-M1SP1、siFXIIf1-M2SP1、siFXIIf1-M3SP1、siFXIIf2-M1SP1、siFXIIf2-M2SP1和siFXIIf2-M3SP1:
siFXIIa1-M1P1siFXIIa1-M1P1
正义链：Justice Chain:
5'-GmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-GmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:37)(SEQ ID NO:37)
反义链：Antisense strand:
5'-P1AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'5'-P1AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'
(SEQ ID NO:38)(SEQ ID NO:38)
siFXIIa1-M2P1siFXIIa1-M2P1
正义链：Justice Chain:
5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:39)(SEQ ID NO:39)
反义链：Antisense strand:
5'-P1AmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGm-3'5'-P1AmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGm-3'
(SEQ ID NO:40)(SEQ ID NO:40)
siFXIIa1-M3P1siFXIIa1-M3P1
正义链：Justice Chain:
5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:41)(SEQ ID NO:41)
反义链：Antisense strand:
5'-P1AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'5'-P1AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'
(SEQ ID NO:42)(SEQ ID NO:42)
siFXIIa2-M1P1siFXIIa2-M1P1
正义链：Justice Chain:
5'-CmAmGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-CmAmGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:43)(SEQ ID NO:43)
反义链：Antisense strand:
5'-P1AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'5'-P1AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'
(SEQ ID NO:44)(SEQ ID NO:44)
siFXIIa2-M2P1siFXIIa2-M2P1
正义链：Justice Chain:
5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:45)(SEQ ID NO:45)
反义链：Antisense strand:
5'-P1AmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'5'-P1AmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'
(SEQ ID NO:46)(SEQ ID NO:46)
siFXIIa2-M3P1siFXIIa2-M3P1
正义链：Justice Chain:
5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:47)(SEQ ID NO:47)
反义链：Antisense strand:
5'-P1AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'5'-P1AmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'
(SEQ ID NO:48)(SEQ ID NO:48)
siFXIIa1-M1SP1siFXIIa1-M1SP1
正义链：Justice Chain:
5'-GmsGmsAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-GmsGmsAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:49)(SEQ ID NO:49)
反义链：Antisense strand:
5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'
(SEQ ID NO:50)(SEQ ID NO:50)
siFXIIa1-M2SP1siFXIIa1-M2SP1
正义链：Justice Chain:
5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:51)(SEQ ID NO:51)
反义链：Antisense strand:
5'-P1AmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmsUmsGm-3'5'-P1AmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmsUmsGm-3'
(SEQ ID NO:52)(SEQ ID NO:52)
siFXIIa1-M3SP1siFXIIa1-M3SP1
正义链：Justice Chain:
5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:53)(SEQ ID NO:53)
反义链：Antisense strand:
5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'
(SEQ ID NO:54)(SEQ ID NO:54)
siFXIIa2-M1SP1siFXIIa2-M1SP1
正义链：Justice Chain:
5'-CmsAmsGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-CmsAmsGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:55)(SEQ ID NO:55)
反义链：Antisense strand:
5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'
(SEQ ID NO:56)(SEQ ID NO:56)
siFXIIa2-M2SP1siFXIIa2-M2SP1
正义链：Justice Chain:
5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:57)(SEQ ID NO:57)
反义链：Antisense strand:
5'-P1AmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'5'-P1AmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'
(SEQ ID NO:58)(SEQ ID NO:58)
siFXIIa2-M3SP1siFXIIa2-M3SP1
正义链：Justice Chain:
5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmUm-3'
(SEQ ID NO:59)(SEQ ID NO:59)
反义链：Antisense strand:
5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'5'-P1AmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'
(SEQ ID NO:60)(SEQ ID NO:60)
siFXIIa1U-M1P1siFXIIa1U-M1P1
正义链：Justice Chain:
5'-GmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'5'-GmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'
(SEQ ID NO:335)(SEQ ID NO:335)
反义链：Antisense strand:
5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'
(SEQ ID NO:336)(SEQ ID NO:336)
siFXIIa1U-M2P1siFXIIa1U-M2P1
正义链：Justice Chain:
5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'
(SEQ ID NO:337)(SEQ ID NO:337)
反义链：Antisense strand:
5'-P1UmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGm-3'5'-P1UmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGm-3'
(SEQ ID NO:338)(SEQ ID NO:338)
siFXIIa1U-M3P1siFXIIa1U-M3P1
正义链：Justice Chain:
5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'5'-GmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'
(SEQ ID NO:339)(SEQ ID NO:339)
反义链：Antisense strand:
5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGm-3'
(SEQ ID NO:340)(SEQ ID NO:340)
siFXIIa2U-M1P1siFXIIa2U-M1P1
正义链：Justice Chain:
5'-CmAmGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'5'-CmAmGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'
(SEQ ID NO:341)(SEQ ID NO:341)
反义链：Antisense strand:
5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'
(SEQ ID NO:342)(SEQ ID NO:342)
siFXIIa2U-M2P1siFXIIa2U-M2P1
正义链：Justice Chain:
5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'
(SEQ ID NO:343)(SEQ ID NO:343)
反义链：Antisense strand:
5'-P1UmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'5'-P1UmAfGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'
(SEQ ID NO:344)(SEQ ID NO:344)
siFXIIa2U-M3P1siFXIIa2U-M3P1
正义链：Justice Chain:
5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'5'-CmAmGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'
(SEQ ID NO:345)(SEQ ID NO:345)
反义链：Antisense strand:
5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'5'-P1UmAfGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmCmGm-3'
(SEQ ID NO:346)(SEQ ID NO:346)
siFXIIa1U-M1SP1siFXIIa1U-M1SP1
正义链：Justice Chain:
5'-GmsGmsAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'5'-GmsGmsAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'
(SEQ ID NO:347)(SEQ ID NO:347)
反义链：Antisense strand:
5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'
(SEQ ID NO:348)(SEQ ID NO:348)
siFXIIa1U-M2SP1siFXIIa1U-M2SP1
正义链：Justice Chain:
5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'
(SEQ ID NO:349)(SEQ ID NO:349)
反义链：Antisense strand:
5'-P1UmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmsUmsGm-3'5'-P1UmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmsUmsGm-3'
(SEQ ID NO:350)(SEQ ID NO:350)
siFXIIa1U-M3SP1siFXIIa1U-M3SP1
正义链：Justice Chain:
5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'5'-GmsGmsAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'
(SEQ ID NO:351)(SEQ ID NO:351)
反义链：Antisense strand:
5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmsUmsGm-3'
(SEQ ID NO:352)(SEQ ID NO:352)
siFXIIa2U-M1SP1siFXIIa2U-M1SP1
正义链：Justice Chain:
5'-CmsAmsGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'5'-CmsAmsGmGmAmAmCmUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'
(SEQ ID NO:353)(SEQ ID NO:353)
反义链：Antisense strand:
5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'
(SEQ ID NO:354)(SEQ ID NO:354)
siFXIIa2U-M2SP1siFXIIa2U-M2SP1
正义链：Justice Chain:
5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'
(SEQ ID NO:355)(SEQ ID NO:355)
反义链：Antisense strand:
5'-P1UmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'5'-P1UmsAfsGmCmAmCfUmUfUfAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'
(SEQ ID NO:356)(SEQ ID NO:356)
siFXIIa2U-M3SP1siFXIIa2U-M3SP1
正义链：Justice Chain:
5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'5'-CmsAmsGmGmAmAmCfUmCfAfAfUmAmAmAmGmUmGmCmUmAm-3'
(SEQ ID NO:357)(SEQ ID NO:357)
反义链：Antisense strand:
5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'5'-P1UmsAfsGmCmAmCfUmUmUmAmUmUmGmAfGmUfUmCmCmUmGmsCmsGm-3'
(SEQ ID NO:358)(SEQ ID NO:358)
siFXIIb1-M1P1siFXIIb1-M1P1
正义链：Justice Chain:
5'-CmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-CmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:97)(SEQ ID NO:97)
反义链：Antisense strand:
5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUm-3'5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUm-3'
(SEQ ID NO:98)(SEQ ID NO:98)
siFXIIb1-M2P1siFXIIb1-M2P1
正义链：Justice Chain:
5'-CmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-CmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:99)(SEQ ID NO:99)
反义链：Antisense strand:
5'-P1UmUfCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUm-3'5'-P1UmUfCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUm-3'
(SEQ ID NO:100)(SEQ ID NO: 100)
siFXIIb1-M3P1siFXIIb1-M3P1
正义链：Justice Chain:
5'-CmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-CmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:101)(SEQ ID NO: 101)
反义链：Antisense strand:
5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUm-3'5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUm-3'
(SEQ ID NO:102)(SEQ ID NO: 102)
siFXIIb2-M1P1siFXIIb2-M1P1
正义链：Justice Chain:
5'-AmAmCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-AmAmCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:103)(SEQ ID NO: 103)
反义链：Antisense strand:
5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'
(SEQ ID NO:104)(SEQ ID NO: 104)
siFXIIb2-M2P1siFXIIb2-M2P1
正义链：Justice Chain:
5'-AmAmCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-AmAmCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:105)(SEQ ID NO: 105)
反义链：Antisense strand:
5'-P1UmUfCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'5'-P1UmUfCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'
(SEQ ID NO:106)(SEQ ID NO: 106)
siFXIIb2-M3P1siFXIIb2-M3P1
正义链：Justice Chain:
5'-AmAmCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-AmAmCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:107)(SEQ ID NO: 107)
反义链：Antisense strand:
5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'5'-P1UmUfCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmCmCm-3'
(SEQ ID NO:108)(SEQ ID NO: 108)
siFXIIb1-M1SP1siFXIIb1-M1SP1
正义链：Justice Chain:
5'-CmsUmsCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-CmsUmsCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:109)(SEQ ID NO: 109)
反义链：Antisense strand:
5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'
(SEQ ID NO:110)(SEQ ID NO:110)
siFXIIb1-M2SP1siFXIIb1-M2SP1
正义链：Justice Chain:
5'-CmsUmsCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-CmsUmsCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:111)(SEQ ID NO:111)
反义链：Antisense strand:
5'-P1UmsUfsCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmsUmsUm-3'5'-P1UmsUfsCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmsUmsUm-3'
(SEQ ID NO:112)(SEQ ID NO:112)
siFXIIb1-M3SP1siFXIIb1-M3SP1
正义链：Justice Chain:
5'-CmsUmsCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-CmsUmsCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:113)(SEQ ID NO:113)
反义链：Antisense strand:
5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmsUmsUm-3'
(SEQ ID NO:114)(SEQ ID NO:114)
siFXIIb2-M1SP1siFXIIb2-M1SP1
正义链：Justice Chain:
5'-AmsAmsCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-AmsAmsCmUmCmAmAmUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:115)(SEQ ID NO:115)
反义链：Antisense strand:
5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'
(SEQ ID NO:116)(SEQ ID NO:116)
SiFXIIb2-M2SP1SiFXIIb2-M2SP1
正义链：Justice Chain:
5'-AmsAmsCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-AmsAmsCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:117)(SEQ ID NO:117)
反义链：Antisense strand:
5'-P1UmsUfsCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'5'-P1UmsUfsCmAmAmAfGmCfAfCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'
(SEQ ID NO:118)(SEQ ID NO:118)
SiFXIIb2-M3SP1SiFXIIb2-M3SP1
正义链：Justice Chain:
5'-AmsAmsCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'5'-AmsAmsCmUmCmAmAfUmAfAfAfGmUmGmCmUmUmUmGmAmAm-3'
(SEQ ID NO:119)(SEQ ID NO:119)
反义链：Antisense strand:
5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'5'-P1UmsUfsCmAmAmAfGmCmAmCmUmUmUmAfUmUfGmAmGmUmUmsCmsCm-3'
(SEQ ID NO:120)(SEQ ID NO: 120)
siFXIId1-M1P1siFXIId1-M1P1
正义链：Justice Chain:
5'-GmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-GmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:157)(SEQ ID NO:157)
反义链：Antisense strand:
5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'
(SEQ ID NO:158)(SEQ ID NO:158)
siFXIId1-M2P1siFXIId1-M2P1
正义链：Justice Chain:
5'-GmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-GmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:159)(SEQ ID NO:159)
反义链：Antisense strand:
5'-P1UmUfUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAm-3'5'-P1UmUfUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAm-3'
(SEQ ID NO:160)(SEQ ID NO: 160)
siFXIId1-M3P1siFXIId1-M3P1
正义链：Justice Chain:
5'-GmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-GmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:161)(SEQ ID NO: 161)
反义链：Antisense strand:
5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAm-3'
(SEQ ID NO:162)(SEQ ID NO: 162)
siFXIId2-M1P1siFXIId2-M1P1
正义链：Justice Chain:
5'-UmUmGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-UmUmGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:163)(SEQ ID NO:163)
反义链：Antisense strand:
5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'
siFXIId2-M2P1siFXIId2-M2P1
正义链：Justice Chain:
5'-UmUmGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-UmUmGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:165)(SEQ ID NO:165)
反义链：Antisense strand:
5'-P1UmUfUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'5'-P1UmUfUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'
(SEQ ID NO:166)(SEQ ID NO: 166)
siFXIId2-M3P1siFXIId2-M3P1
正义链：Justice Chain:
5'-UmUmGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-UmUmGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:167)(SEQ ID NO: 167)
反义链：Antisense strand:
5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'5'-P1UmUfUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmAmCm-3'
(SEQ ID NO:168)(SEQ ID NO: 168)
siFXIId1-M1SP1siFXIId1-M1SP1
正义链：Justice Chain:
5'-GmsGmsAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-GmsGmsAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:169)(SEQ ID NO: 169)
反义链：Antisense strand:
5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'
(SEQ ID NO:170)(SEQ ID NO: 170)
siFXIId1-M2SP1siFXIId1-M2SP1
正义链：Justice Chain:
5'-GmsGmsAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-GmsGmsAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:171)(SEQ ID NO: 171)
反义链：Antisense strand:
5'-P1UmsUfsUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmsAmsAm-3'5'-P1UmsUfsUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmsAmsAm-3'
(SEQ ID NO:172)(SEQ ID NO: 172)
siFXIId1-M3SP1siFXIId1-M3SP1
正义链：Justice Chain:
5'-GmsGmsAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-GmsGmsAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:173)(SEQ ID NO: 173)
反义链：Antisense strand:
5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmsAmsAm-3'
(SEQ ID NO:174)(SEQ ID NO: 174)
siFXIId2-M1SP1siFXIId2-M1SP1
正义链：Justice Chain:
5'-UmsUmsGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-UmsUmsGmGmAmGmCmCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:175)(SEQ ID NO: 175)
反义链：Antisense strand:
5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'
(SEQ ID NO:176)(SEQ ID NO: 176)
siFXIId2-M2SP1siFXIId2-M2SP1
正义链：Justice Chain:
5'-UmsUmsGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-UmsUmsGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:177)(SEQ ID NO: 177)
反义链：Antisense strand:
5'-P1UmsUfsUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'5'-P1UmsUfsUmCmAmCfUmUfUfCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'
(SEQ ID NO:178)(SEQ ID NO: 178)
siFXIId2-M3SP1siFXIId2-M3SP1
正义链：Justice Chain:
5'-UmsUmsGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'5'-UmsUmsGmGmAmGmCfCmCfAfAfGmAmAmAmGmUmGmAmAmAm-3'
(SEQ ID NO:179)(SEQ ID NO: 179)
反义链：Antisense strand:
5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'5'-P1UmsUfsUmCmAmCfUmUmUmCmUmUmGmGfGmCfUmCmCmAmAmsAmsCm-3'
(SEQ ID NO:180)(SEQ ID NO: 180)
siFXIIe1-M1P1siFXIIe1-M1P1
正义链：Justice Chain:
5'-AmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-AmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:217)(SEQ ID NO:217)
反义链：Antisense strand:
5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'
(SEQ ID NO:218)(SEQ ID NO:218)
siFXIIe1-M2P1siFXIIe1-M2P1
正义链：Justice Chain:
5'-AmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-AmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:219)(SEQ ID NO:219)
反义链：Antisense strand:
5'-P1UmCfUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCm-3'5'-P1UmCfUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCm-3'
(SEQ ID NO:220)(SEQ ID NO:220)
siFXIIe1-M3P1siFXIIe1-M3P1
正义链：Justice Chain:
5'-AmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-AmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:221)(SEQ ID NO:221)
反义链：Antisense strand:
5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCm-3'
(SEQ ID NO:222)(SEQ ID NO:222)
siFXIIe2-M1P1siFXIIe2-M1P1
正义链：Justice Chain:
5'-GmGmAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-GmGmAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:223)(SEQ ID NO:223)
反义链：Antisense strand:
5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'
(SEQ ID NO:224)(SEQ ID NO:224)
siFXIIe2-M2P1siFXIIe2-M2P1
正义链：Justice Chain:
5'-GmGmAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-GmGmAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:225)(SEQ ID NO:225)
反义链：Antisense strand:
5'-P1UmCfUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'5'-P1UmCfUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'
(SEQ ID NO:226)(SEQ ID NO:226)
siFXIIe2-M3P1siFXIIe2-M3P1
正义链：Justice Chain:
5'-GmGmAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-GmGmAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:227)(SEQ ID NO:227)
反义链：Antisense strand:
5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'5'-P1UmCfUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmAmAm-3'
(SEQ ID NO:228)(SEQ ID NO:228)
siFXIIe1-M1SP1siFXIIe1-M1SP1
正义链：Justice Chain:
5'-AmsGmsCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-AmsGmsCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:229)(SEQ ID NO:229)
反义链：Antisense strand:
5'-P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'5'-P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'
(SEQ ID NO:230)(SEQ ID NO:230)
siFXIIe1-M2SP1siFXIIe1-M2SP1
正义链：Justice Chain:
5'-AmsGmsCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-AmsGmsCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:231)(SEQ ID NO:231)
反义链：Antisense strand:
5'-P1UmsCfsUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmsCmsCm-3'5'-P1UmsCfsUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmsCmsCm-3'
(SEQ ID NO:232)(SEQ ID NO:232)
siFXIIe1-M3SP1siFXIIe1-M3SP1
正义链：Justice Chain:
5'-AmsGmsCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-AmsGmsCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:233)(SEQ ID NO:233)
反义链：Antisense strand:
5'-P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'5'-P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmsCmsCm-3'
(SEQ ID NO:234)(SEQ ID NO:234)
siFXIIe2-M1SP1siFXIIe2-M1SP1
正义链：Justice Chain:
5'-GmsGmsAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-GmsGmsAmGmCmCmCmAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:235)(SEQ ID NO:235)
反义链：Antisense strand:
5'-P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'5'-P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'
(SEQ ID NO:236)(SEQ ID NO:236)
siFXIIe2-M2SP1siFXIIe2-M2SP1
正义链：Justice Chain:
5'-GmsGmsAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-GmsGmsAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:237)(SEQ ID NO:237)
反义链：Antisense strand:
5'-P1UmsCfsUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'5'-P1UmsCfsUmUmUmCfAmCfUfUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'
(SEQ ID NO:238)(SEQ ID NO:238)
siFXIIe2-M3SP1siFXIIe2-M3SP1
正义链：Justice Chain:
5'-GmsGmsAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'5'-GmsGmsAmGmCmCmCfAmAfGfAfAmAmGmUmGmAmAmAmGmAm-3'
(SEQ ID NO:239)(SEQ ID NO:239)
反义链：Antisense strand:
5'-P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'5'-P1UmsCfsUmUmUmCfAmCmUmUmUmCmUmUfGmGfGmCmUmCmCmsAmsAm-3'
(SEQ ID NO:240)(SEQ ID NO:240)
siFXIIf1-M1P1siFXIIf1-M1P1
正义链：Justice Chain:
5'-CmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-CmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:277)(SEQ ID NO:277)
反义链：Antisense strand:
5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'
(SEQ ID NO:278)(SEQ ID NO:278)
siFXIIf1-M2P1siFXIIf1-M2P1
正义链：Justice Chain:
5'-CmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-CmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:279)(SEQ ID NO:279)
反义链：Antisense strand:
5'-P1UmGfGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCm-3'5'-P1UmGfGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCm-3'
(SEQ ID NO:280)(SEQ ID NO:280)
siFXIIf1-M3P1siFXIIf1-M3P1
正义链：Justice Chain:
5'-CmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-CmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:281)(SEQ ID NO:281)
反义链：Antisense strand:
5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCm-3'
(SEQ ID NO:282)(SEQ ID NO:282)
siFXIIf2-M1P1siFXIIf2-M1P1
正义链：Justice Chain:
5'-GmCmCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-GmCmCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:283)(SEQ ID NO:283)
反义链：Antisense strand:
5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'
(SEQ ID NO:284)(SEQ ID NO:284)
siFXIIf2-M2P1siFXIIf2-M2P1
正义链：Justice Chain:
5'-GmCmCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-GmCmCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:285)(SEQ ID NO:285)
反义链：Antisense strand:
5'-P1UmGfGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'5'-P1UmGfGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'
(SEQ ID NO:286)(SEQ ID NO:286)
siFXIIf2-M3P1siFXIIf2-M3P1
正义链：Justice Chain:
5'-GmCmCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-GmCmCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:287)(SEQ ID NO:287)
反义链：Antisense strand:
5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'5'-P1UmGfGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmUmCm-3'
(SEQ ID NO:288)(SEQ ID NO:288)
siFXIIf1-M1SP1siFXIIf1-M1SP1
正义链：Justice Chain:
5'-CmsCmsAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-CmsCmsAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:289)(SEQ ID NO:289)
反义链：Antisense strand:
5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'
(SEQ ID NO:290)(SEQ ID NO:290)
siFXIIf1-M2SP1siFXIIf1-M2SP1
正义链：Justice Chain:
5'-CmsCmsAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-CmsCmsAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:291)(SEQ ID NO:291)
反义链：Antisense strand:
5'-P1UmsGfsGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmsGmsCm-3'5'-P1UmsGfsGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmsGmsCm-3'
(SEQ ID NO:292)(SEQ ID NO:292)
siFXIIf1-M3SP1siFXIIf1-M3SP1
正义链：Justice Chain:
5'-CmsCmsAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-CmsCmsAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:293)(SEQ ID NO:293)
反义链：Antisense strand:
5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmsGmsCm-3'
(SEQ ID NO:294)(SEQ ID NO:294)
siFXIIf2-M1SP1siFXIIf2-M1SP1
正义链：Justice Chain:
5'-GmsCmsCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-GmsCmsCmCmAmAmGmAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:295)(SEQ ID NO:295)
反义链：Antisense strand:
5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'
(SEQ ID NO:296)(SEQ ID NO:296)
siFXIIf2-M2SP1siFXIIf2-M2SP1
正义链：Justice Chain:
5'-GmsCmsCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-GmsCmsCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:297)(SEQ ID NO:297)
反义链：Antisense strand:
5'-P1UmsGfsGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'5'-P1UmsGfsGmUmCmUfUmUfCfAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'
(SEQ ID NO:298)(SEQ ID NO:298)
siFXIIf2-M3SP1siFXIIf2-M3SP1
正义链：Justice Chain:
5'-GmsCmsCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'5'-GmsCmsCmCmAmAmGfAmAfAfGfUmGmAmAmAmGmAmCmCmAm-3'
(SEQ ID NO:299)(SEQ ID NO:299)
反义链：Antisense strand:
5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'5'-P1UmsGfsGmUmCmUfUmUmCmAmCmUmUmUfCmUfUmGmGmGmCmsUmsCm-3'
(SEQ ID NO:300)(SEQ ID NO:300)
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母左右两个核苷酸之间为硫代磷酸酯基连接；P1表示该P1右侧相邻的一个核苷酸为5'-磷酸核苷酸或5'-磷酸类似物修饰的核苷酸。在一些实施方式中，P1是表示具体修饰的VP、Ps或P，其中，字母组合VP表示该字母组合VP右侧相邻的一个核苷酸为乙烯基磷酸酯(5'-(E)-vinylphosphonate，E-VP)修饰的核苷酸，字母组合Ps表示该字母组合Ps右侧相邻的一个核苷酸为硫代磷酸酯修饰的核苷酸，大写字母P表示该字母P右侧相邻的一个核苷酸为5'-磷酸核苷酸。Among them, the capital letters C, G, U, and A represent the base composition of the nucleotide; the lowercase letter m represents that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; the lowercase letter f represents that the nucleotide adjacent to the left of the letter f is a fluorinated modified nucleotide; the lowercase letter s represents that the two nucleotides on the left and right of the letter are connected by a thiophosphate group; P1 represents that the nucleotide adjacent to the right of P1 is a 5'-phosphate nucleotide or a 5'-phosphate analog modified nucleotide. In some embodiments, P1 is VP, Ps, or P representing a specific modification, wherein the letter combination VP represents that the nucleotide adjacent to the right of the letter combination VP is a vinyl phosphate (5'-(E)-vinylphosphonate, E-VP) modified nucleotide, the letter combination Ps represents that the nucleotide adjacent to the right of the letter combination Ps is a thiophosphate modified nucleotide, and the capital letter P represents that the nucleotide adjacent to the right of the letter P is a 5'-phosphate nucleotide.
本公开的发明人意外发现，本公开提供的siRNA不仅具有显著增强的血浆和溶酶体稳定性，还保留很高的基因抑制活性。The inventors of the present disclosure unexpectedly discovered that the siRNA provided by the present disclosure not only has significantly enhanced plasma and lysosomal stability, but also retains high gene inhibition activity.
本公开提供的siRNA可以通过本领域常规的siRNA制备方法(例如固相合成和液相合成的方法)得到。其中，固相合成已经有商业化订制服务。可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入本公开所述的siRNA中，制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本领域技术人员所熟知的。The siRNA provided by the present disclosure can be obtained by conventional siRNA preparation methods in the art (e.g., solid phase synthesis and liquid phase synthesis methods). Among them, solid phase synthesis already has commercial customization services. The modified nucleotide groups can be introduced into the siRNA described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art.
药物组合物Pharmaceutical composition
本公开提供了一种药物组合物，所述药物组合物含有如上所述的siRNA作为活性成分和药学上可接受的载体。The present disclosure provides a pharmaceutical composition, which contains the siRNA as described above as an active ingredient and a pharmaceutically acceptable carrier.
所述药学上可接受的载体可以是siRNA给药领域常规使用的载体，例如但不限于磁性纳米粒(magnetic nanoparticles，如基于Fe3O4或Fe2O3的纳米粒)、碳纳米管(carbonnanotubes)、介孔硅(mesoporous silicon)、磷酸钙纳米粒(calcium phosphatenanoparticles)、聚乙烯亚胺(polyethylenimine，PEI)、聚酰胺型树形高分子(polyamidoamine(PAMAM)dendrimer)、聚赖氨酸(poly(L-lysine)，PLL)、壳聚糖(chitosan)、1,2-二油酰基-3-三甲铵丙烷(1,2-dioleoyl-3-trimethylammonium-propane，DOTAP)、聚D型或L型乳酸/羟基乙酸共聚物(poly(D&L-lactic/glycolic acid)copolymer，PLGA)、聚(氨乙基乙撑磷酸酯)(poly(2-aminoethyl ethylene phosphate)，PPEEA)和聚(甲基丙烯酸-N,N-二甲氨基乙酯)(poly(2-dimethylaminoethylmethacrylate)，PDMAEMA)以及它们的衍生物中的一种或多种。The pharmaceutically acceptable carrier can be a carrier conventionally used in the field of siRNA administration, such as, but not limited to, magnetic nanoparticles (such as nanoparticles based onFe3O4 orFe2O3 ), carbon nanotubes, mesoporous silicon, calcium phosphate nanoparticles, polyethyleneimine (PEI), polyamidoamine (PAMAM) dendrimer, poly(L-lysine), chitosan, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), poly (D&L-lactic/glycolic acid) copolymer (PLGA), poly (2-aminoethyl ethylene phosphate ... Phosphate), PPEEA) and poly(methacrylate-N,N-dimethylaminoethyl ester) (poly(2-dimethylaminoethylmethacrylate), PDMAEMA) and one or more of their derivatives.
在一些实施方式中，所述药物组合物中，对siRNA和药学上可接受的载体的含量没有特别要求，在一些实施方式中，siRNA与药学上可接受的载体的重量比可以为1:(1-500)，在一些的实施方式中，上述重量比为1:(1-50)。In some embodiments, there are no special requirements for the content of siRNA and pharmaceutically acceptable carrier in the pharmaceutical composition. In some embodiments, the weight ratio of siRNA to pharmaceutically acceptable carrier can be 1:(1-500). In some embodiments, the above weight ratio is 1:(1-50).
在一些实施方式中，所述药物组合物中，还可以包含药学上可接受的其它辅料，该辅料可以为本领域常规采用的各种制剂或化合物的一种或多种。例如，所述药学上可接受的其它辅料可以包括pH缓冲液、保护剂和渗透压调节剂中的至少一种。In some embodiments, the pharmaceutical composition may further include other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds conventionally used in the art. For example, the other pharmaceutically acceptable excipients may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.
所述pH缓冲液可以为pH值7.5-8.5的三羟甲基胺基甲烷盐酸盐缓冲液和/或pH值5.5-8.5的磷酸盐缓冲液，例如可以为pH值5.5-8.5的磷酸盐缓冲液。The pH buffer may be a tris(hydroxymethyl)aminomethane hydrochloride buffer with a pH value of 7.5-8.5 and/or a phosphate buffer with a pH value of 5.5-8.5, for example, a phosphate buffer with a pH value of 5.5-8.5.
所述保护剂可以为肌醇、山梨醇、蔗糖、海藻糖、甘露糖、麦芽糖、乳糖和葡萄糖中的至少一种。以所述药物组合物的总重量为基准，所述保护剂的含量可以为0.01-30重量％。The protective agent may be at least one of inositol, sorbitol, sucrose, trehalose, mannose, maltose, lactose and glucose. Based on the total weight of the pharmaceutical composition, the content of the protective agent may be 0.01-30% by weight.
所述渗透压调节剂可以为氯化钠和/或氯化钾。所述渗透压调节剂的含量使所述药物组合物的渗透压为200-700毫渗摩尔/千克(mOsm/kg)。根据所需渗透压，本领域技术人员可以容易地确定所述渗透压调节剂的含量。The osmotic pressure regulator can be sodium chloride and/or potassium chloride. The content of the osmotic pressure regulator makes the osmotic pressure of the pharmaceutical composition 200-700 milliosmole/kilogram (mOsm/kg). According to the desired osmotic pressure, those skilled in the art can easily determine the content of the osmotic pressure regulator.
在一些实施方式中，所述药物组合物可以为液体制剂，例如注射液；也可以为冻干粉针剂，实施给药时与液体辅料混合，配制成液体制剂。所述液体制剂可以但不限于用于皮下、肌肉或静脉注射给药，也可以但不限于通过喷雾给药到肺脏、或通过喷雾经肺脏给药到其它脏器组织(如肝脏)。在一些实施方式中，所述药物组合物用于静脉注射给药。In some embodiments, the pharmaceutical composition can be a liquid preparation, such as an injection; or a lyophilized powder injection, which is mixed with a liquid excipient during administration to prepare a liquid preparation. The liquid preparation can be, but is not limited to, administered subcutaneously, intramuscularly or intravenously, and can also be, but is not limited to, administered to the lungs by spraying, or administered to other organs (such as the liver) through the lungs by spraying. In some embodiments, the pharmaceutical composition is used for intravenous administration.
在一些实施方式中，所述药物组合物可以为脂质体制剂的形式。在一些实施方式中，所述脂质体制剂中使用的药学上可接受的载体包含含胺的转染化合物(下文也可将其称为有机胺)、辅助脂质和/或聚乙二醇化脂质。其中，所述有机胺、辅助脂质和聚乙二醇化脂质可分别选自于CN103380113A(通过引用的方式将其整体并入本文)中所描述的含胺的转染化合物或其药学上可接受的盐或衍生物、辅助脂质和聚乙二醇化脂质中的一种或多种。In some embodiments, the pharmaceutical composition can be in the form of a liposome preparation. In some embodiments, the pharmaceutically acceptable carrier used in the liposome preparation comprises an amine-containing transfection compound (hereinafter also referred to as an organic amine), an auxiliary lipid and/or a pegylated lipid. Wherein, the organic amine, the auxiliary lipid and the pegylated lipid can be selected from one or more of the amine-containing transfection compound or its pharmaceutically acceptable salt or derivative, the auxiliary lipid and the pegylated lipid described in CN103380113A (which is incorporated herein by reference in its entirety).
在一些实施方式中，所述有机胺可为CN103380113A中描述的如式(201)所示的化合物或其药学上可接受的盐：In some embodiments, the organic amine may be a compound described in CN103380113A as shown in formula (201) or a pharmaceutically acceptable salt thereof:
其中：in:
每个X101和X102各自独立地是O、S、N-A或C-A，其中A是氢或C1-C20烃链；Each X101 and X102 is independently O, S, NA or CA, wherein A is hydrogen or a C1-C20 hydrocarbon chain;
每个Y101和Z101各自独立地是C＝O、C＝S、S＝O、CH-OH或SO2；Each of Y101 and Z101 is independently C═O, C═S, S═O, CH—OH or SO2 ;
每个R101、R102、R103、R104、R105、R106和R107各自独立地是氢，环状或无环的、被取代的或未被取代的、支链或直链脂族基团，环状或无环的、被取代的或未被取代的、支链或直链杂脂族基团，被取代的或未被取代的、支链或直链酰基，被取代的或未被取代的、支链或直链芳基，被取代的或未被取代的、支链或直链杂芳基；each of R101 , R102 , R103 , R104 , R105 , R106 and R107 is independently hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or straight-chain heteroaliphatic group, a substituted or unsubstituted, branched or straight-chain acyl group, a substituted or unsubstituted, branched or straight-chain aryl group, a substituted or unsubstituted, branched or straight-chain heteroaryl group;
x是1-10的整数；x is an integer from 1 to 10;
n是1-3的整数，m是0-20的整数，p是0或1；其中，如果m＝p＝0，则R102是氢；n is an integer of 1-3, m is an integer of 0-20, and p is 0 or 1; wherein, if m=p=0, R102 is hydrogen;
并且，如果n或m中的至少一个是2，那么R103和在式(201)中的氮形成如式(202)或式(203)所示的结构：And, if at least one of n or m is 2, then R103 and the nitrogen in formula (201) form a structure as shown in formula (202) or formula (203):
其中，g、e和f各自独立地是1-6的整数，“HCC”代表烃链，且每个*N代表式(201)中的氮原子。wherein g, e and f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each *N represents a nitrogen atom in formula (201).
在一些实施方式中，R103是多胺。在其它实施方式中，R103是缩酮。在一些实施方式中，在式(201)中的R101和R102中的每一个独立地是任意的被取代的或未被取代的、支链或直链烷基或烯基，所述烷基或烯基具有3至约20个碳原子，诸如8至约18个碳原子，和0至4个双键，诸如0至2个双键。In some embodiments, R103 is a polyamine. In other embodiments, R103 is a ketal. In some embodiments, each of R101 and R102 in formula (201) is independently any substituted or unsubstituted, branched or linear alkyl or alkenyl group having 3 to about 20 carbon atoms, such as 8 to about 18 carbon atoms, and 0 to 4 double bonds, such as 0 to 2 double bonds.
在一些实施方式中，如果n和m中的每一个独立地具有1或3的值，那么R103可以是下述式(204)-式(213)中的任一个：In some embodiments, if each of n and m independently has a value of 1 or 3, then R103 can be any one of the following formulas (204) to (213):
其中，式(204)-式(213)中，g、e和f各自独立地是1-6的整数，每个“HCC”代表烃链，且每个*显示R103与在式(201)中的氮原子的可能连接点，其中在任意*位置上的每个H可以被替换以实现与在式(201)中的氮原子的连接。Wherein, in formula (204)-formula (213), g, e and f are each independently an integer of 1-6, each "HCC" represents a hydrocarbon chain, and each * shows a possible connection point between R103 and the nitrogen atom in formula (201), wherein each H at any * position can be replaced to achieve connection with the nitrogen atom in formula (201).
其中，式(201)所示化合物可以根据CN103380113A中的描述制备。Among them, the compound represented by formula (201) can be prepared according to the description in CN103380113A.
在一些实施方式中，所述有机胺为如式(214)所示的有机胺和/或如式(215)所示的有机胺：In some embodiments, the organic amine is an organic amine as shown in formula (214) and/or an organic amine as shown in formula (215):
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；The auxiliary lipid is cholesterol, a cholesterol analog and/or a cholesterol derivative;
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。The PEGylated lipid is 1,2-dipalmitamide-sn-glycerol-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)]-2000.
在一些实施方式中，所述药物组合物中，所述有机胺、所述辅助脂质和所述聚乙二醇化脂质三者之间的摩尔比为(19.7-80)：(19.7-80)：(0.3-50)，例如可以为(50-70)：(20-40)：(3-20)。In some embodiments, in the pharmaceutical composition, the molar ratio of the organic amine, the auxiliary lipid and the pegylated lipid is (19.7-80):(19.7-80):(0.3-50), for example, it can be (50-70):(20-40):(3-20).
在一些实施方式中，由本公开的siRNA与上述含胺的转染试剂形成的药物组合物颗粒具有约30nm至约200nm的平均直径，通常为约40nm至约135nm，更通常地，该脂质体颗粒的平均直径是约50nm至约120nm、约50nm至约100nm、约60nm至约90nm或约70nm至约90nm，例如，该脂质体颗粒的平均直径是约30、40、50、60、70、75、80、85、90、100、110、120、130、140、150或160nm。In some embodiments, the pharmaceutical composition particles formed by the siRNA of the present disclosure and the above-mentioned amine-containing transfection reagent have an average diameter of about 30 nm to about 200 nm, typically about 40 nm to about 135 nm, more typically, the average diameter of the liposome particles is about 50 nm to about 120 nm, about 50 nm to about 100 nm, about 60 nm to about 90 nm, or about 70 nm to about 90 nm, for example, the average diameter of the liposome particles is about 30, 40, 50, 60, 70, 75, 80, 85, 90, 100, 110, 120, 130, 140, 150 or 160 nm.
在一些实施方式中，由本公开的siRNA与上述含胺的转染试剂形成的药物组合物中，siRNA与全部脂质(例如有机胺、辅助脂质和/或聚乙二醇化脂质)的重量比(重量/重量比)在从约1:1至约1:50、从约1:1至约1:30、从约1:3至约1:20、从约1:4至约1:18、从约1:5至约1:17、从约1:5至约1:15、从约1:5至约1:12、从约1:6至约1:12或从约1:6至约1:10的范围内，例如，本公开的siRNA与全部脂质的重量比为约1:5、1:6、1:7、1:8、1:9、1:10、1:11、1:12、1:13、1:14、1:15、1:16、1:17或1:18。In some embodiments, in the pharmaceutical composition formed by the siRNA of the present disclosure and the above-mentioned amine-containing transfection reagent, the weight ratio (weight/weight ratio) of siRNA to total lipids (e.g., organic amines, helper lipids and/or pegylated lipids) is from about 1:1 to about 1:50, from about 1:1 to about 1:30, from about 1:3 to about 1:20, from about 1:4 to about 1:18, from about 1:5 to about 1:17, from about 1:5 to about 1:15, from about 1:5 to about 1:12, from about 1:6 to about 1:12, or from about 1:6 to about 1:10, for example, the weight ratio of the siRNA of the present disclosure to total lipids is about 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17 or 1:18.
在一些实施方式中，所述药物组合物在销售时各组分可以独立存在，在使用时可以液体制剂的形式存在。在一些实施方式中，本公开提供的siRNA与上述药学上可接受的载体形成的药物组合物可以按照已知的各种方法制备，只是用本公开提供的siRNA替代现有siRNA即可；在一些实施方式中，可以按照如下方法制备：In some embodiments, the components of the pharmaceutical composition can exist independently when sold, and can exist in the form of a liquid preparation when used. In some embodiments, the pharmaceutical composition formed by the siRNA provided by the present disclosure and the above-mentioned pharmaceutically acceptable carrier can be prepared according to various known methods, except that the siRNA provided by the present disclosure is substituted for the existing siRNA; in some embodiments, it can be prepared according to the following method:
将有机胺、辅助脂质和聚乙二醇化脂质按照上述摩尔比悬浮于醇中并混匀得到脂质溶液；醇的用量使得到的脂质溶液的总质量浓度为2-25mg/mL，例如可以为8-18mg/mL。所述醇选自药学上可接受的醇，诸如在室温附近为液体的醇，例如，乙醇、丙二醇、苯甲醇、甘油、聚乙二醇200，聚乙二醇300，聚乙二醇400中的一种或多种，例如可以为乙醇。The organic amine, the auxiliary lipid and the PEGylated lipid are suspended in alcohol according to the above molar ratio and mixed to obtain a lipid solution; the amount of alcohol used is such that the total mass concentration of the obtained lipid solution is 2-25 mg/mL, for example, 8-18 mg/mL. The alcohol is selected from pharmaceutically acceptable alcohols, such as alcohols that are liquid at room temperature, for example, ethanol, propylene glycol, benzyl alcohol, glycerol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, one or more, for example, ethanol.
将本公开提供的siRNA溶解于缓冲盐溶液中，得到siRNA水溶液。缓冲盐溶液的浓度为0.05-0.5M，例如可以为0.1-0.2M，调节缓冲盐溶液的pH至4.0-5.5，例如可以为5.0-5.2，缓冲盐溶液的用量使siRNA的浓度不超过0.6mg/mL，例如可以为0.2-0.4mg/mL。所述缓冲盐选自可溶性醋酸盐、可溶性柠檬酸盐中的一种或多种，例如可以为醋酸钠和/或醋酸钾。The siRNA provided by the present disclosure is dissolved in a buffered saline solution to obtain an siRNA aqueous solution. The concentration of the buffered saline solution is 0.05-0.5M, for example, 0.1-0.2M, and the pH of the buffered saline solution is adjusted to 4.0-5.5, for example, 5.0-5.2, and the amount of the buffered saline solution is such that the concentration of the siRNA does not exceed 0.6mg/mL, for example, 0.2-0.4mg/mL. The buffer salt is selected from one or more of soluble acetates and soluble citrates, for example, sodium acetate and/or potassium acetate.
将脂质溶液和siRNA水溶液混合，将混合后得到的产物在40-60℃孵育至少2分钟，例如可以为5-30分钟，得到孵育后的脂质体制剂。脂质溶液和siRNA水溶液的体积比为1：(2-5)。The lipid solution and the siRNA aqueous solution are mixed, and the mixed product is incubated at 40-60° C. for at least 2 minutes, for example, 5-30 minutes, to obtain an incubated liposome preparation. The volume ratio of the lipid solution to the siRNA aqueous solution is 1:(2-5).
将孵育后的脂质体制剂浓缩或稀释，去除杂质，除菌，得到本公开提供的药物组合物，其理化参数为pH值为6.5-8，包封率不低于80％，粒径为40-200nm，多分散指数不高于0.30，渗透压为250-400mOsm/kg；例如理化参数可以为pH值为7.2-7.6，包封率不低于90％，粒径为60-100nm，多分散指数不高于0.20，渗透压为300-400mOsm/kg。The incubated liposome preparation is concentrated or diluted, impurities are removed, and sterilized to obtain the pharmaceutical composition provided by the present disclosure, whose physicochemical parameters are pH value of 6.5-8, encapsulation efficiency of not less than 80%, particle size of 40-200nm, polydispersity index of not more than 0.30, and osmotic pressure of 250-400mOsm/kg; for example, the physicochemical parameters can be pH value of 7.2-7.6, encapsulation efficiency of not less than 90%, particle size of 60-100nm, polydispersity index of not more than 0.20, and osmotic pressure of 300-400mOsm/kg.
其中，浓缩或稀释可以在去除杂质之前、之后或同时进行。去除杂质的方法可以采用现有各种方法，例如可以使用切相流系统、中空纤维柱，在100K Da条件下超滤，超滤交换溶液为pH7.4的磷酸盐缓冲液(PBS)。除菌的方法可以采用现有各种方法，例如可以在0.22μm滤器上过滤除菌。The concentration or dilution can be performed before, after or simultaneously with the removal of impurities. The impurity removal method can adopt various existing methods, such as using a tangential flow system, a hollow fiber column, ultrafiltration under 100K Da conditions, and the ultrafiltration exchange solution is a phosphate buffered saline (PBS) with a pH of 7.4. The sterilization method can adopt various existing methods, such as filtration sterilization on a 0.22μm filter.
siRNA缀合物siRNA conjugates
本公开提供了一种siRNA缀合物，所述siRNA缀合物含有上述siRNA以及缀合连接至该siRNA的缀合基团。The present disclosure provides a siRNA conjugate, wherein the siRNA conjugate contains the above-mentioned siRNA and a conjugation group conjugated to the siRNA.
一般来说，所述缀合基团包含药学上可接受的至少一个靶向基团和任选的接头(linker)，并且，所述siRNA、所述接头和所述靶向基团依次连接。在一些实施方式中，所述靶向基团为1-6个。在一些实施方式中，所述靶向基团为2-4个。所述siRNA分子可以非共价或共价缀合至所述缀合基团，例如可以共价缀合至所述缀合基团。siRNA与缀合基团的缀合位点可以在siRNA正义链的3'端或5'端，也可在反义链的5'端，还可以在siRNA的内部序列中。在一些实施方式中，所述siRNA与缀合基团的缀合位点在siRNA正义链的3'末端。In general, the conjugated group comprises at least one pharmaceutically acceptable targeting group and an optional linker, and the siRNA, the linker and the targeting group are connected in sequence. In some embodiments, the targeting group is 1-6. In some embodiments, the targeting group is 2-4. The siRNA molecule can be non-covalently or covalently conjugated to the conjugated group, for example, it can be covalently conjugated to the conjugated group. The conjugation site of siRNA and conjugated group can be at the 3' end or 5' end of the siRNA sense strand, also at the 5' end of the antisense strand, and also in the internal sequence of siRNA. In some embodiments, the conjugation site of siRNA and conjugated group is at the 3' end of the siRNA sense strand.
在一些实施方式中，所述缀合基团可以连接在核苷酸的磷酸基团、2'-位羟基或者碱基上。在一些实施方式中，所述缀合基团还可以连接在3'-位羟基上，此时核苷酸之间采用2'-5'磷酸二酯键连接。当缀合基团连接在siRNA链的末端时，所述缀合基团通常连接在核苷酸的磷酸基团上；当缀合基团连接在siRNA的内部序列时，所述缀合基团通常连接在核糖糖环或者碱基上。各种连接方式可以参考文献：Muthiah Manoharan et.al.siRNAconjugates carrying sequentially assembled trivalent N-acetylgalactosaminelinked through nucleosides elicit robust gene silencing in vivo inhepatocytes.ACS Chemical biology,2015,10(5):1181-7.In some embodiments, the conjugate group can be connected to the phosphate group, 2'-hydroxyl group or base of the nucleotide. In some embodiments, the conjugate group can also be connected to the 3'-hydroxyl group, in which case the nucleotides are connected by a 2'-5' phosphodiester bond. When the conjugate group is connected to the end of the siRNA chain, the conjugate group is usually connected to the phosphate group of the nucleotide; when the conjugate group is connected to the internal sequence of the siRNA, the conjugate group is usually connected to the ribose sugar ring or the base. Various connection methods can be referred to in the literature: Muthiah Manoharan et.al.siRNA conjugates carrying sequentially assembled trivalent N-acetylgalactosamine linked through nucleosides elicit robust gene silencing in vivo inhepatocytes.ACS Chemical biology, 2015, 10(5): 1181-7.
在一些实施方式中，所述siRNA与缀合基团间可以通过酸不稳定的、或可还原的化学键相连，在细胞内涵体的酸性环境下，这些化学键可降解，从而使siRNA成为自由状态。对于不可降解的缀合方式，缀合基团可连接在siRNA的正义链，从而尽量降低缀合对siRNA活性的影响。In some embodiments, the siRNA and the conjugated group can be connected by acid-labile or reducible chemical bonds, which can be degraded in the acidic environment of the cell endosome, thereby making the siRNA free. For non-degradable conjugation methods, the conjugated group can be connected to the sense strand of the siRNA, thereby minimizing the effect of conjugation on the activity of the siRNA.
在一些实施方式中，所述药学上可接受的靶向基团可以是siRNA给药领域常规使用的配体，例如WO2009082607A2中描述的各种配体，以引用的方式将其全部公开内容并入本文。In some embodiments, the pharmaceutically acceptable targeting group may be a ligand conventionally used in the field of siRNA administration, such as various ligands described in WO2009082607A2, the entire disclosure of which is incorporated herein by reference.
在一些实施方式中，所述药学上可接受的靶向基团可以选自以下靶向分子或其衍生物形成的配体中的一种或多种：亲脂分子，例如胆固醇、胆汁酸、维生素(例如维生素E)、不同链长的脂质分子；聚合物，例如聚乙二醇；多肽，例如透膜肽；适配体；抗体；量子点；糖类，例如乳糖、聚乳糖、甘露糖、半乳糖、N-乙酰半乳糖胺(GalNAc)；叶酸(folate)；肝实质细胞表达的受体配体，例如去唾液酸糖蛋白、去唾液酸糖残基、脂蛋白(如高密度脂蛋白、低密度脂蛋白等)、胰高血糖素、神经递质(如肾上腺素)、生长因子、转铁蛋白等。In some embodiments, the pharmaceutically acceptable targeting group can be selected from one or more of the ligands formed by the following targeting molecules or their derivatives: lipophilic molecules, such as cholesterol, bile acid, vitamins (such as vitamin E), lipid molecules of different chain lengths; polymers, such as polyethylene glycol; polypeptides, such as membrane-permeable peptides; aptamers; antibodies; quantum dots; carbohydrates, such as lactose, polylactose, mannose, galactose, N-acetylgalactosamine (GalNAc); folic acid (folate); receptor ligands expressed by hepatic parenchymal cells, such as asialoglycoproteins, asialosugar residues, lipoproteins (such as high-density lipoproteins, low-density lipoproteins, etc.), glucagon, neurotransmitters (such as adrenaline), growth factors, transferrin, etc.
在一些实施方式中，所述的每个配体独立地选自一个能够与细胞表面受体结合的配体。在一些实施方式中，至少一个配体是能够与肝细胞表面受体结合的配体。在一些实施方式中，至少一个配体是能够与哺乳动物细胞表面受体结合的配体。在一些实施方式中，至少一个配体是能够与人肝细胞表面受体结合的配体。在一些实施方式中，至少一个配体是能够与肝表面去唾液酸糖蛋白受体(ASGPR)结合的配体。这些配体的种类为本领域技术人员所公知，其作用一般是与靶细胞表面的特异性受体相结合，介导与配体连接的siRNA递送至靶细胞。In some embodiments, each ligand is independently selected from a ligand that can bind to a cell surface receptor. In some embodiments, at least one ligand is a ligand that can bind to a hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand that can bind to a mammalian cell surface receptor. In some embodiments, at least one ligand is a ligand that can bind to a human hepatocyte surface receptor. In some embodiments, at least one ligand is a ligand that can bind to a liver surface asialoglycoprotein receptor (ASGPR). The types of these ligands are well known to those skilled in the art, and their function is generally to bind to a specific receptor on the surface of the target cell, mediating the delivery of the siRNA connected to the ligand to the target cell.
在一些实施方式中，所述药学上可接受的靶向基团可以是与哺乳动物肝细胞表面上的去唾液酸糖蛋白受体(ASGPR)结合的任意一种配体。在一些实施方式中，每个配体独立地为去唾液酸糖蛋白，例如去唾液酸血清类粘蛋白(asialoorosomucoid，ASOR)或去唾液酸胎球蛋白(asialofetuin，ASF)。在一些实施方式中，所述配体为糖或糖的衍生物。In some embodiments, the pharmaceutically acceptable targeting group can be any ligand that binds to an asialoglycoprotein receptor (ASGPR) on the surface of a mammalian hepatocyte. In some embodiments, each ligand is independently an asialoglycoprotein, such as asialo serum mucin (ASOR) or asialo fetuin (ASF). In some embodiments, the ligand is a sugar or a derivative of a sugar.
在一些实施方式中，至少一个配体是糖。在一些实施方式中，每个配体均是糖。在一些实施方式中，至少一个配体是单糖、多糖、修饰的单糖、修饰的多糖或糖衍生物。在一些实施方式中，至少一个所述配体可以是单糖，双糖或三糖。在一些实施方式中，至少有一个配体是修饰的糖。在一些实施方式中，每一个配体均为修饰的糖。在一些实施方式中，每个配体均独立地选自多糖、修饰的多糖、单糖、修饰的单糖、多糖衍生物或单糖衍生物。在一些实施方式中，每一个或至少一个配体选自于由以下糖所组成的组：葡萄糖及其衍生物、甘露聚糖及其衍生物、半乳糖及其衍生物、木糖及其衍生物、核糖及其衍生物、岩藻糖及其衍生物、乳糖及其衍生物、麦芽糖及其衍生物，阿拉伯糖及其衍生物、果糖及其衍生物和唾液酸。In some embodiments, at least one ligand is a sugar. In some embodiments, each ligand is a sugar. In some embodiments, at least one ligand is a monosaccharide, a polysaccharide, a modified monosaccharide, a modified polysaccharide or a sugar derivative. In some embodiments, at least one of the ligands may be a monosaccharide, a disaccharide or a trisaccharide. In some embodiments, at least one ligand is a modified sugar. In some embodiments, each ligand is independently selected from a polysaccharide, a modified polysaccharide, a monosaccharide, a modified monosaccharide, a polysaccharide derivative or a monosaccharide derivative. In some embodiments, each or at least one ligand is selected from the group consisting of the following sugars: glucose and its derivatives, mannan and its derivatives, galactose and its derivatives, xylose and its derivatives, ribose and its derivatives, fucose and its derivatives, lactose and its derivatives, maltose and its derivatives, arabinose and its derivatives, fructose and its derivatives and sialic acid.
在一些实施方式中，每个所述配体可独立地选自D-吡喃甘露糖、L-吡喃甘露糖、D-阿拉伯糖、D-呋喃木糖、L-呋喃木糖、D-葡萄糖、L-葡萄糖、D-半乳糖、L-半乳糖、α-D-呋喃甘露糖、β-D-呋喃甘露糖、α-D-吡喃甘露糖、β-D-吡喃甘露糖、α-D-吡喃葡萄糖、β-D-吡喃葡萄糖、α-D-呋喃葡萄糖、β-D-呋喃葡萄糖、α-D-呋喃果糖、α-D-吡喃果糖、α-D-吡喃半乳糖、β-D-吡喃半乳糖、α-D-呋喃半乳糖、β-D-呋喃半乳糖、葡糖胺、唾液酸、半乳糖胺、N-乙酰半乳糖胺、N-三氟乙酰半乳糖胺、N-丙酰半乳糖胺、N-正丁酰半乳糖胺、N-异丁酰半乳糖胺、2-氨基-3-O-[(R)-1-羧乙基]-2-脱氧-β-D-吡喃葡萄糖、2-脱氧-2-甲基氨基-L-吡喃葡萄糖、4,6-二脱氧-4-甲酰胺基-2,3-二-O-甲基-D-吡喃甘露糖、2-脱氧-2-磺氨基-D-吡喃葡萄糖、N-乙醇酰基-α-神经氨酸、5-硫代-β-D-吡喃葡萄糖、2,3,4-三-O-乙酰基-1-硫代-6-O-三苯甲基-α-D-吡喃葡萄糖苷甲酯、4-硫代-β-D-吡喃半乳糖、3,4,6,7-四-O-乙酰基-2-脱氧-1,5-二硫代-α-D-吡喃葡庚糖苷乙酯、2,5-脱水-D-阿洛糖腈、核糖、D-核糖、D-4-硫代核糖、L-核糖或L-4-硫代核糖。所述配体的其它选择可参见例如CN105378082A的记载，以引用的方式将其全部公开内容并入本文。In some embodiments, each of the ligands can be independently selected from D-mannopyranose, L-mannopyranose, D-arabinose, D-xylofuranose, L-xylofuranose, D-glucose, L-glucose, D-galactose, L-galactose, α-D-mannofuranose, β-D-mannofuranose, α-D-mannopyranose, β-D-mannopyranose, α-D-glucose, β-D-glucose, Sugar, α-D-glucofuranose, β-D-glucofuranose, α-D-fructofuranose, α-D-fructopyranose, α-D-galactopyranose, β-D-galactopyranose, α-D-galactofuranose, β-D-galactofuranose, glucosamine, sialic acid, galactosamine, N-acetylgalactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-butyrylgalactosamine, N-isobutyrylgalactosamine , 2-amino-3-O-[(R)-1-carboxyethyl]-2-deoxy-β-D-glucopyranose, 2-deoxy-2-methylamino-L-glucopyranose, 4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose, 2-deoxy-2-sulfoamino-D-glucopyranose, N-glycolyl-α-neuraminic acid, 5-thio-β-D-glucopyranose, 2, 3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside methyl ester, 4-thio-β-D-galactopyranose, 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucopyranoside heptanoside ethyl ester, 2,5-anhydro-D-allose nitrile, ribose, D-ribose, D-4-thioribose, L-ribose or L-4-thioribose. Other selections of the ligand can refer to, for example, the records of CN105378082A, and all of its disclosures are incorporated herein by reference.
在一些实施方式中，所述siRNA缀合物中药学上可接受的靶向基团可以是半乳糖或N-乙酰半乳糖胺，其中，半乳糖或N-乙酰半乳糖胺分子可以是一价、二价、三价、四价。应当理解的是，这里所述的一价、二价、三价、四价分别指siRNA分子与含有作为靶向基团的半乳糖或N-乙酰半乳糖胺基团的缀合分子形成siRNA缀合物后，该siRNA缀合物中siRNA基团与半乳糖或N-乙酰半乳糖胺基团的摩尔比为1:1、1:2、1:3或1:4。在一些实施方式中，所述药学上可接受的靶向基团是N-乙酰半乳糖胺。在一些实施方式中，当本公开所述的siRNA与含有N-乙酰半乳糖胺的缀合基团缀合时，N-乙酰半乳糖胺分子是三价或四价。在一些实施方式中，当本公开所述的siRNA与含有N-乙酰半乳糖胺的缀合基团缀合时，N-乙酰半乳糖胺分子是三价。In some embodiments, the pharmaceutically acceptable targeting group in the siRNA conjugate can be galactose or N-acetylgalactosamine, wherein the galactose or N-acetylgalactosamine molecule can be monovalent, divalent, trivalent, or tetravalent. It should be understood that the monovalent, divalent, trivalent, and tetravalent herein refer to the siRNA molecule and the conjugated molecule containing the galactose or N-acetylgalactosamine group as the targeting group to form the siRNA conjugate, and the molar ratio of the siRNA group to the galactose or N-acetylgalactosamine group in the siRNA conjugate is 1:1, 1:2, 1:3, or 1:4. In some embodiments, the pharmaceutically acceptable targeting group is N-acetylgalactosamine. In some embodiments, when the siRNA described in the present disclosure is conjugated to a conjugated group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent or tetravalent. In some embodiments, when the siRNA described in the present disclosure is conjugated to a conjugation group containing N-acetylgalactosamine, the N-acetylgalactosamine molecule is trivalent.
靶向基团可经由合适的接头与siRNA分子相连，本领域技术人员可以根据靶向基团的具体类型选择合适的接头。这些接头、靶向基团的种类以及与siRNA的连接方式，可参见WO2015006740A2的公开内容，通过引用的方式将其整体内容并入本文。The targeting group can be connected to the siRNA molecule via a suitable linker, and those skilled in the art can select a suitable linker according to the specific type of the targeting group. These linkers, the types of targeting groups and the connection mode with siRNA can be found in the disclosure of WO2015006740A2, the entire contents of which are incorporated herein by reference.
在一些实施方式中，当所述靶向基团为N-乙酰半乳糖胺时，合适的接头可以为如式(301)所示的结构：In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be a structure as shown in formula (301):
其中，in,
k为1-3的整数；k is an integer from 1 to 3;
LA为具有如式(302)所示结构的包含酰胺键的链状部分，每个所述LA在其两端分别与一个所述靶向基团和所述LC部分通过醚键相连接：LA is a chain portion containing an amide bond having a structure as shown in formula (302), and each of theLA is connected to one of the targeting groups and theLC portion via an ether bond at both ends thereof:
LB为具有如式(303)所示结构的包含N-酰基吡咯烷的链状部分，所述链状部分在其一端具有羰基并与所述LC部分通过酰胺键相连接，在另一端具有氧基并与所述siRNA通过磷酸酯键相连接：LB is a chain portion containing N-acylpyrrolidine having a structure as shown in formula (303), wherein the chain portion has a carbonyl group at one end and is connected to theLC portion via an amide bond, and has an oxygen group at the other end and is connected to the siRNA via a phosphate bond:
LC为基于羟甲基氨基甲烷、二羟甲基氨基甲烷或三羟甲基氨基甲烷的2-4价连接基团，所述LC经由氧原子与各个所述LA部分通过醚键相连接，并且经由氮原子与所述LB部分通过酰胺键相连接。LC is a 2-4 valent linking group based on hydroxymethylaminomethane, dihydroxymethylaminomethane or trihydroxymethylaminomethane, and is connected to eachLA part through an ether bond via an oxygen atom, and is connected to theLB part through anamide bond via a nitrogen atom.
在一些实施方式中，当n＝3，LC为基于三羟甲基氨基甲烷的4价连接基团时，由作为接头的-(LA)3三羟甲基氨基甲烷-LB-连接N-乙酰半乳糖胺分子和siRNA分子所形成的siRNA缀合物，其结构如下式(304)所示：In some embodiments, when n=3,LC is a tetravalent linking group based on tris(hydroxymethylaminomethane), the siRNA conjugate formed by connecting the N-acetylgalactosamine molecule and the siRNA molecule using -(LA )3tris (hydroxymethylaminomethane)-LB- as a linker has a structure as shown in the following formula (304):
式中，双螺旋结构表示siRNA。In the formula, the double helix structure represents siRNA.
同样，siRNA与缀合基团的缀合位点可以在siRNA正义链的3'端或5'端，也可在反义链的5'端，还可以在siRNA的内部序列中。Likewise, the conjugation site between siRNA and the conjugation group can be at the 3' end or 5' end of the sense strand of siRNA, at the 5' end of the antisense strand, or in the internal sequence of siRNA.
在一些实施方式中，本公开所述siRNA的正义链3'末端通过接头-(LA)3三羟甲基氨基甲烷-LB-与三个N-乙酰半乳糖胺(GalNAc)分子共价缀合，得到siRNA分子与GalNAc分子的摩尔比为1:3的siRNA缀合物，下文也可将其称为(GalNAc)3-siRNA，其结构如下式(305)所示：In some embodiments, the 3' end of the sense strand of the siRNA disclosed herein is covalently conjugated to three N-acetylgalactosamine (GalNAc) molecules via a linker -(LA )3 trishydroxymethylaminomethane-LB- to obtain a siRNA conjugate having a molar ratio of siRNA molecules to GalNAc molecules of 1:3, which may also be referred to as (GalNAc)3 -siRNA hereinafter, and has a structure as shown in the following formula (305):
其中，双螺旋结构表示所述siRNA，并且所述接头连接至所述siRNA的正义链3'末端。The double helix structure represents the siRNA, and the linker is connected to the 3' end of the positive strand of the siRNA.
在一些实施方式中，当所述靶向基团为N-乙酰半乳糖胺时，合适的接头可以为如式(306)所示的结构：In some embodiments, when the targeting group is N-acetylgalactosamine, a suitable linker may be a structure as shown in formula (306):
其中，in,
l为0-3的整数；l is an integer from 0 to 3;
*表示接头上通过醚键与靶向基团连接的位点；* indicates the site on the linker that is connected to the targeting group via an ether bond;
#表示接头上通过磷酸酯键与siRNA连接的位点。# indicates the site on the linker that is connected to the siRNA via a phosphate bond.
在一些实施方式中，当l＝2时，所述siRNA缀合物具有如式(307)所示的结构：In some embodiments, when l=2, the siRNA conjugate has a structure as shown in formula (307):
其中，双螺旋结构表示所述siRNA，并且所述接头连接至所述siRNA的正义链3'末端。The double helix structure represents the siRNA, and the linker is connected to the 3' end of the positive strand of the siRNA.
上述缀合物可以通过现有技术中已经详细描述的方法进行合成。例如，WO2015006740A2中详细描述了多种缀合物的制备方法。通过本领域技术人员熟知的方式，获得本公开的siRNA缀合物。如WO2014025805A1中记载了式(305)所示结构的制备方法，Rajeev等人在ChemBioChem 2015,16,903-908中描述了式(307)所示结构的制备方法。The above conjugates can be synthesized by methods that have been described in detail in the prior art. For example, WO2015006740A2 describes in detail the preparation methods of various conjugates. The siRNA conjugates disclosed herein are obtained by methods well known to those skilled in the art. For example, WO2014025805A1 describes the preparation method of the structure shown in formula (305), and Rajeev et al. describe the preparation method of the structure shown in formula (307) in ChemBioChem 2015, 16, 903-908.
在一些实施方式中，所述siRNA缀合物具有如式(308)所示的结构：In some embodiments, the siRNA conjugate has a structure as shown in formula (308):
其中：in:
n1为选自1-3的整数，n3为选自0-4的整数；n1 is an integer selected from 1-3, n3 is an integer selected from 0-4;
每个m1、m2和m3各自独立地为选自2-10的整数；Each m1, m2 and m3 is independently an integer selected from 2-10;
每个R10、R11、R12、R13、R14和R15各自独立地为H，或选自于由以下基团所组成的组：C1-C10烷基、C1-C10卤代烷基以及C1-C10烷氧基；Each of R10 , R11 , R12 , R13 , R14 and R15 is independently H, or is selected from the group consisting of C1 -C10 alkyl, C1 -C10 haloalkyl and C1 -C10 alkoxy;
R3为式A59所示结构的基团：R3 is a group having a structure represented by formula A59:
其中，E1为OH、SH或BH2，Nu为前述本公开的siRNA；wherein E1 is OH, SH or BH2 , and Nu is the aforementioned siRNA disclosed herein;
R2是长度为1-20个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的任何一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中，R2可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)；R2 is a straight chain alkylene group having a length of 1 to 20 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2 , C2 -C10 alkenylene, C2 -C10 alkynylene, C6 -C10 arylene, C3 -C18 heterocyclylene and C5 -C10 heteroarylene; and wherein R2 may optionally have any one or more substituents selected from the group consisting of: C1 -C10 alkyl, C6 -C10 aryl, C5 -C10 heteroaryl, C1 -C10 haloalkyl, -OC1 -C10 alkyl, -OC1 -C10 alkylphenyl, -C1 -C10 alkyl-OH, -OC1 -C10 haloalkyl, -SC1 -C -C10 alkyl), -SC1 -C10 alkylphenyl, -C1 -C10 alkyl-SH, -SC 1 -C 10haloalkyl, halogen substituent, -OH, -SH, -NH2 , -C1 -C10 alkyl-NH2 , -N(C1 -C10 alkyl)(C1 -C10 alkyl), -NH(C1 -C10 alkyl), -N(C1 -C10 alkyl)(C1 -C10 alkylphenyl), -NH(C1 -C10 alkylphenyl), cyano, nitro, -CO2 H, -C(O)O(C1 -C10 alkyl), -CON(C1 -C10 alkyl)(C1 -C10 alkyl), -CONH(C1 -C10 alkyl), -CONH2 , -NHC(O)(C1 -C 10 alkyl -C 1 -C10 alkyl), -NHC(O)(phenyl), -N(C1 -C10 alkyl)C(O)(C1 -C10 alkyl), -N(C1 -C10 alkyl)C(O)(phenyl), -C(O)C1 -C10 alkyl, -C(O)C1 -C10 alkylphenyl, -C(O)C1 -C10 haloalkyl, -OC(O)C1 -C10 alkyl, -SO2 (C1 -C10 alkyl), -SO2 (phenyl), -SO2 (C1 -C10 haloalkyl), -SO2 NH2 , -SO2 NH(C1 -C10 alkyl), -SO2 NH(phenyl), -NHSO2 (C1 -C10 alkyl), -NHSO2 (phenyl) and -NHSO2 (C1 -C10 haloalkyl);
每个L1是长度为1-70个碳原子的直链亚烷基，其中一个或多个碳原子任选地被选自于以下基团所组成的组中的任何一个或多个所替换：C(O)、NH、O、S、CH＝N、S(O)2、C2-C10亚烯基、C2-C10亚炔基、C6-C10亚芳基、C3-C18亚杂环基和C5-C10亚杂芳基；并且其中，L1可任选地具有由以下基团所组成的组中的任何一个或多个的取代基：C1-C10烷基、C6-C10芳基、C5-C10杂芳基、C1-C10卤代烷基、-OC1-C10烷基、-OC1-C10烷基苯基、-C1-C10烷基-OH、-OC1-C10卤代烷基、-SC1-C10烷基、-SC1-C10烷基苯基、-C1-C10烷基-SH、-SC1-C10卤代烷基、卤素取代基、-OH、-SH、-NH2、-C1-C10烷基-NH2、-N(C1-C10烷基)(C1-C10烷基)、-NH(C1-C10烷基)、-N(C1-C10烷基)(C1-C10烷基苯基)、-NH(C1-C10烷基苯基)、氰基、硝基、-CO2H、-C(O)O(C1-C10烷基)、-CON(C1-C10烷基)(C1-C10烷基)、-CONH(C1-C10烷基)、-CONH2，-NHC(O)(C1-C10烷基)、-NHC(O)(苯基)、-N(C1-C10烷基)C(O)(C1-C10烷基)、-N(C1-C10烷基)C(O)(苯基)、-C(O)C1-C10烷基、-C(O)C1-C10烷基苯基、-C(O)C1-C10卤烷基、-OC(O)C1-C10烷基、-SO2(C1-C10烷基)、-SO2(苯基)、-SO2(C1-C10卤代烷基)、-SO2NH2、-SO2NH(C1-C10烷基)、-SO2NH(苯基)、-NHSO2(C1-C10烷基)、-NHSO2(苯基)和-NHSO2(C1-C10卤代烷基)。Each L1 is a straight chain alkylene group having a length of 1 to 70 carbon atoms, wherein one or more carbon atoms are optionally replaced by any one or more selected from the group consisting of: C(O), NH, O, S, CH=N, S(O)2 , C2 -C10 alkenylene, C2 -C10 alkynylene, C6 -C10 arylene, C3 -C18 heterocyclylene and C5 -C10 heteroarylene; and wherein L1 may optionally have any one or more substituents selected from the group consisting of: C1 -C10 alkyl, C6 -C10 aryl, C5 -C10 heteroaryl, C1 -C10 haloalkyl, -OC1 -C10 alkyl, -OC1 -C10 alkylphenyl, -C1 -C10 alkyl-OH, -OC1 -C10 haloalkyl, -SC1 -C 10 -C10 alkyl), -SC1 -C10 alkylphenyl, -C1 -C10 alkyl-SH, -SC 1 -C 10haloalkyl, halogen substituent, -OH, -SH, -NH2 , -C1 -C10 alkyl-NH2 , -N(C1 -C10 alkyl)(C1 -C10 alkyl), -NH(C1 -C10 alkyl), -N(C1 -C10 alkyl)(C1 -C10 alkylphenyl), -NH(C1 -C10 alkylphenyl), cyano, nitro, -CO2 H, -C(O)O(C1 -C10 alkyl), -CON(C1 -C10 alkyl)(C1 -C10 alkyl), -CONH(C1 -C10 alkyl), -CONH2 , -NHC(O)(C1 -C 10 alkyl-C10 alkyl), -SO 2 (phenyl), -SO 2 (C1 -C10 haloalkyl), -SO2 NH2 , -SO 2 NH (C1-C10 alkyl),-SO2NH(phenyl), -NHSO 2(C1-C10alkyl),-NHSO2(phenyl)and-NHSO 2( C1-C10 haloalkyl).
在一些实施方式中，L1可选自于由A1-A26基团或其任意组合所组成的组，其中A1-A26的结构和定义如下所示：In some embodiments,L1 can be selected from the group consisting of A1-A26 groups or any combination thereof, wherein the structures and definitions of A1-A26 are as follows:
其中，j1为1-20的整数；j2为1-20的整数；Wherein, j1 is an integer from 1 to 20; j2 is an integer from 1 to 20;
R'为C1-C10烷基；R' is a C1 -C10 alkyl group;
Ra选自式A27-A45基团或其任意组合所组成的组：Ra is selected from the group consisting of formula A27-A45 groups or any combination thereof:
Rb为C1-C10烷基；表示基团共价连接的位点。Rb is a C1 -C10 alkyl group; Indicates the site to which a group is covalently attached.
技术人员会理解的是，尽管为了方便起见，L1被定义为线性亚烷基，但是它可能不是线性基团或者名称不同，例如由于上述替换和/或置换而产生的胺或烯基。为了本公开内容的目的，L1的长度是连接两个连接点的链中的原子数。为此目的，将替换所述直链亚烷基的碳原子而得到的环(如亚杂环基或亚杂芳基)计为一个原子。The skilled person will appreciate that althoughL is defined as a linear alkylene for convenience, it may not be a linear group or be named differently, such as an amine or alkenyl resulting from the above substitutions and/or replacements. For the purposes of this disclosure, the length ofL is the number of atoms in the chain connecting the two points of attachment. For this purpose, a ring (such as a heterocyclylene or heteroarylene) resulting from the replacement of a carbon atom of the linear alkylene is counted as one atom.
M1表示靶向基团，其定义和可选择的范围与上述靶向基团相同。在一些实施方式中，每个M1独立地选自对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体中的一种。M1 represents a targeting group, and its definition and selectable range are the same as those of the above-mentioned targeting groups. In some embodiments, eachM1 is independently selected from one of the ligands having affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells.
当M1为对哺乳动物肝脏细胞表面上的去唾液酸糖蛋白受体具有亲合力的配体时，在一些实施方式中，n1可以是1-3的整数，n3可以是0-4的整数，保证所述缀合物中M1靶向基团的个数至少为2；在一些实施方式中，n1+n3≥2，这样可以使得M1靶向基团的个数至少为3，从而使得M1靶向基团与肝表面去唾液酸糖蛋白受体更容易结合，进而促进所述缀合物通过内吞作用进入细胞。实验表明，当M1靶向基团的个数大于3个时，M1靶向基团与肝表面去唾液酸糖蛋白受体结合的容易程度增加并不明显，因此，从合成容易程度、结构/工艺成本和递送效率等多方面综合考虑，在一些实施方式中，n1为1-2的整数，n3为0-1的整数，且n1+n3＝2-3。WhenM1 is a ligand with affinity for the asialoglycoprotein receptor on the surface of mammalian liver cells, in some embodiments, n1 can be an integer of 1-3, and n3 can be an integer of 0-4, ensuring that the number ofM1 targeting groups in the conjugate is at least 2; in some embodiments, n1+n3≥2, so that the number ofM1 targeting groups can be at least 3, so that theM1 targeting group is more likely to bind to the asialoglycoprotein receptor on the surface of the liver, thereby promoting the conjugate to enter the cell through endocytosis. Experiments show that when the number ofM1 targeting groups is greater than 3, the ease of binding of theM1 targeting group to the asialoglycoprotein receptor on the surface of the liver is not significantly increased. Therefore, considering the ease of synthesis, structure/process cost and delivery efficiency, in some embodiments, n1 is an integer of 1-2, n3 is an integer of 0-1, and n1+n3=2-3.
在一些实施方式中，每个m1、m2和m3独立地选自2-10的整数时，可以使多个M1靶向基团之间的空间位置适合M1靶向基团与肝表面去唾液酸糖蛋白受体的结合，为了使本公开提供的缀合物更为简单，更容易合成和/或降低成本，在一些实施方式中，每个m1、m2和m3各自独立地为2-5的整数，在一些实施方式中，m1＝m2＝m3。In some embodiments, when each m1, m2 and m3 is independently selected from an integer of 2-10, the spatial position between the multipleM1 targeting groups can be suitable for the binding of theM1 targeting group to the liver surface asialoglycoprotein receptor. In order to make the conjugate provided by the present disclosure simpler, easier to synthesize and/or reduce the cost, in some embodiments, each m1, m2 and m3 is independently an integer of 2-5. In some embodiments, m1=m2=m3.
本领域技术人员可以理解，当每个R10、R11、R12、R13、R14和R15各自独立地选自H、C1-C10烷基、C1-C10卤代烷基、以及C1-C10烷氧基中的一种时，不会改变本公开的缀合物的性质，均可以实现本公开的目的。在一些实施方式中，每个R10、R11、R12、R13、R14和R15各自独立地选自H、甲基和乙基。在一些实施方式中，每个R10、R11、R12、R13、R14和R15均为H。Those skilled in the art will appreciate that when each R10 , R11 , R12 , R13 , R14 and R15 is independently selected from one of H, C1 -C10 alkyl, C1 -C10 haloalkyl, and C1 -C10 alkoxy, the properties of the conjugate of the present disclosure will not be changed, and the purpose of the present disclosure can be achieved. In some embodiments, each R10 , R11 , R12 , R13 , R14 and R15 is independently selected from H, methyl and ethyl. In some embodiments, each R10 , R11 , R12 , R13 , R14 and R15 is H.
R3为式A59所示结构的基团，其中，E1为OH、SH或BH2，基于制备原料易获取性的考虑，在一些实施方式中，E1为OH或SH。R3 is a group of the structure represented by formula A59, wherein E1 is OH, SH or BH2 . Based on the consideration of the availability of raw materials for preparation, in some embodiments, E1 is OH or SH.
R2的选择是为了实现与含氮骨架上的N原子与A59的连接。在本公开的上下文中，“含氮骨架”是指连接有R10、R11、R12、R13、R14和R15的碳原子与N原子互相连接的链状结构。因此，R2可以是任何能够以适当方式将A59基团连接至含氮骨架上的N原子的连接基团。在一些实施方式中，在通过固相合成的工艺制备式(308)所示的siRNA缀合物的情况下，R2基团中需要同时含有与含氮骨架上的N原子连接的连接位点和与R3中的P原子相连接的连接位点。在一些实施方式中，R2中所述与含氮骨架上的N原子连接的位点与N形成酰胺键，所述与R3上的P原子连接的位点与P原子形成磷酸酯键；在一些实施方式中，R2可以是B5、B6、B5'或B6'：The selection of R2 is to achieve the connection with the N atom on the nitrogen-containing skeleton and A59. In the context of the present disclosure, "nitrogen-containing skeleton" refers to a chain structure in which the carbon atoms connected to R10 , R11 , R12 , R13 , R14 and R15 are connected to the N atom. Therefore, R2 can be any connecting group that can connect the A59 group to the N atom on the nitrogen-containing skeleton in an appropriate manner. In some embodiments, when the siRNA conjugate shown in formula (308) is prepared by a solid phase synthesis process, the R2 group needs to contain both a connecting site connected to the N atom on the nitrogen-containing skeleton and a connecting site connected to the P atom in R3. In some embodiments, the site in R2 that connects to the N atom on the nitrogen-containing skeleton forms an amide bond with N, and the site that connects to the P atom on R3 forms a phosphate bond with the P atom; in some embodiments, R2 can be B5, B6, B5' or B6':
其中，表示基团共价键连接的位点。in, Indicates the site of covalent attachment of a group.
q2的取值范围可以是1-10的整数，在一些实施方式中，q2为1-5的整数。The value range of q2 can be an integer from 1 to 10. In some embodiments, q2 is an integer from 1 to 5.
L1的作用是将M1靶向基团与含氮骨架上的N原子连接，为式(308)所示的siRNA缀合物提供肝靶向功能。在一些实施方式中，L1选自式A1-A26基团中的一种或多种的连接组合。在一些实施方式中，L1选自A1、A4、A5、A6、A8、A10、A11和A13中的一种或多种的连接组合。在一些实施方式中，L1选自A1、A4、A8、A10和A11中至少2个的连接组合。在一些实施方式中，L1选自A1、A8、A10中至少2个的连接组合。The role ofL1 is to connect theM1 targeting group to the N atom on the nitrogen-containing skeleton, providing liver targeting function for the siRNA conjugate shown in formula (308). In some embodiments,L1 is selected from a combination of one or more of the formula A1-A26 groups. In some embodiments,L1 is selected from a combination of one or more of A1, A4, A5, A6, A8, A10, A11 and A13. In some embodiments,L1 is selected from a combination of at least two of A1, A4, A8, A10 and A11. In some embodiments,L1 is selected from a combination of at least two of A1, A8, A10.
在一些实施方式中，L1的长度可以为3-25个原子，3-20个原子、4-15个原子或5-12个原子。在一些实施方式中，L1的长度为3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、30个、35个、40个、45个、50个、55个、60个原子。In some embodiments,L1 can be 3-25 atoms, 3-20 atoms, 4-15 atoms, or 5-12 atoms in length. In some embodiments,L1 is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 atoms in length.
在一些实施方式中，j1为2-10的整数，在一些实施方式中，j1为3-5的整数。在一些实施方式中，j2为2-10的整数，在一些实施方式中，j2为3-5的整数。R'为C1-C4烷基，在一些实施方式中，R'为甲基、乙基和异丙基中的一种。Ra为A27、A28、A29、A30和A31中的一种，在一些实施方式中，Ra为A27或A28。Rb为C1-C5的烷基，在一些实施方式中，Rb为甲基、乙基、异丙基和丁基中的一种。在一些实施方式中，在式A1-A26中各自对j1、j2、R'、Ra、Rb进行选择，以实现M1靶向基团与含氮骨架上的N连接，并使M1靶向基团之间的空间位置更适合M1靶向基团与肝表面去唾液酸糖蛋白受体结合。In some embodiments, j1 is an integer of 2-10, and in some embodiments, j1 is an integer of 3-5. In some embodiments, j2 is an integer of 2-10, and in some embodiments, j2 is an integer of 3-5. R' is a C1 -C4 alkyl group, and in some embodiments, R' is one of a methyl group, an ethyl group, and an isopropyl group. Ra is one of A27, A28, A29, A30, and A31, and in some embodiments, Ra is A27 or A28. Rb is a C1 -C5 alkyl group, and in some embodiments, Rb is one of a methyl group, an ethyl group, an isopropyl group, and a butyl group. In some embodiments, j1, j2, R', Ra, and Rb are selected in formulas A1-A26 to achieve the connection between the M1 targeting group and the N on the nitrogen-containing skeleton, and to make the spatial position between the M1 targeting groups more suitable for the M1 targeting group to bind to the liver surface asialoglycoprotein receptor.
在一些实施方式中，该缀合物具有式(403)、(404)、(405)、(406)、(407)、(408)、(409)、(410)、(411)、(412)、(413)、(414)、(415)、(416)、(417)、(418)、(419)、(420)、(421)或(422)所示的结构：In some embodiments, the conjugate has a structure shown in formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), or (422):
在一些实施方式中，式A59中的P原子可以连接到siRNA序列中任何可能的位置，例如，式A59中的P原子可以连接到siRNA正义链或反义链的任何一个核苷酸上；在一些实施方式中，式A59中的P原子连接到siRNA正义链的任何一个核苷酸上。在一些实施方式中，式A59中的P原子连接到siRNA正义链或反义链的端部；在一些实施方式中，式A59中的P原子连接到siRNA正义链的端部。所述端部指所述正义链或所述反义链中从其一端起算的前4个核苷酸。在一些实施方式中，式A59中的P原子连接到siRNA正义链或反义链的末端；在一些实施方式中，式A59中的P原子连接到siRNA正义链的3'末端。在连接至siRNA的正义链的上述位置的情况下，式(308)所示的siRNA缀合物进入细胞后，在解旋时，可以释放出单独的siRNA反义链，以阻断FXII mRNA翻译蛋白质的过程，抑制因子XII(FXII)基因表达。In some embodiments, the P atom in formula A59 can be connected to any possible position in the siRNA sequence, for example, the P atom in formula A59 can be connected to any nucleotide of the sense strand or antisense strand of the siRNA; in some embodiments, the P atom in formula A59 is connected to any nucleotide of the sense strand of the siRNA. In some embodiments, the P atom in formula A59 is connected to the end of the sense strand or antisense strand of the siRNA; in some embodiments, the P atom in formula A59 is connected to the end of the sense strand of the siRNA. The end refers to the first 4 nucleotides of the sense strand or antisense strand from one end thereof. In some embodiments, the P atom in formula A59 is connected to the end of the sense strand or antisense strand of the siRNA; in some embodiments, the P atom in formula A59 is connected to the 3' end of the sense strand of the siRNA. In the case of connecting to the above-mentioned position of the sense strand of the siRNA, after the siRNA conjugate shown in formula (308) enters the cell, when unwinding, a separate siRNA antisense strand can be released to block the process of FXII mRNA translation into protein and inhibit the expression of factor XII (FXII) gene.
在一些实施方式中，式A59中的P原子可以连接到siRNA中的核苷酸上任何可能的位置，例如，核苷酸的5'位、核苷酸的2'位、核苷酸的3'位或核苷酸的碱基上。在一些实施方式中，式A59中的P原子可通过形成磷酸二酯键连接至所述siRNA中的核苷酸的2'位、3'位或5'位。在一些实施方式中，式A59中的P原子连接在siRNA正义链3'末端核苷酸的3'羟基脱氢后形成的氧原子上(此时，A59中的P原子也可以看作是siRNA中含有的磷酸基团中的P原子)，或者式A59中的P原子通过取代siRNA正义链中的一个核苷酸的2'-羟基中的氢与核苷酸连接，或者式A59中的P原子通过取代siRNA正义链5'末端核苷酸的5'羟基中的氢与核苷酸连接。In some embodiments, the P atom in formula A59 can be connected to any possible position on the nucleotide in the siRNA, for example, the 5' position of the nucleotide, the 2' position of the nucleotide, the 3' position of the nucleotide or the base of the nucleotide. In some embodiments, the P atom in formula A59 can be connected to the 2' position, 3' position or 5' position of the nucleotide in the siRNA by forming a phosphodiester bond. In some embodiments, the P atom in formula A59 is connected to the oxygen atom formed after the 3' hydroxyl dehydrogenation of the 3' terminal nucleotide of the siRNA sense strand (at this time, the P atom in A59 can also be regarded as the P atom in the phosphate group contained in the siRNA), or the P atom in formula A59 is connected to the nucleotide by replacing the hydrogen in the 2'-hydroxyl of a nucleotide in the siRNA sense strand, or the P atom in formula A59 is connected to the nucleotide by replacing the hydrogen in the 5' hydroxyl of the 5' terminal nucleotide of the siRNA sense strand.
本公开的发明人意外发现，本公开的siRNA缀合物在具有显著提高的血浆中稳定性、低脱靶效应的同时，还表现出并未明显降低的FXII mRNA沉默活性。因此，在一些实施方式中，本公开的siRNA缀合物中的siRNA可以为表1a、1b、1d、1e和1f中示出的siRNA中的任意一种。The inventors of the present disclosure unexpectedly discovered that the siRNA conjugates of the present disclosure not only have significantly improved stability in plasma and low off-target effects, but also show no significantly reduced FXII mRNA silencing activity. Therefore, in some embodiments, the siRNA in the siRNA conjugates of the present disclosure can be any one of the siRNAs shown in Tables 1a, 1b, 1d, 1e and 1f.
表1a本公开缀合物中的第一种siRNA序列Table 1a The first siRNA sequence in the conjugates of the present disclosure
表1b本公开缀合物中的第二种siRNA序列Table 1b Second siRNA sequences in the conjugates of the present disclosure
表1d本公开缀合物中的第三种siRNA序列Table 1d The third siRNA sequence in the conjugate of the present disclosure
表1e本公开缀合物中的第四种siRNA序列Table 1e The fourth siRNA sequence in the conjugate of the present disclosure
表1f本公开缀合物中的第五种siRNA序列Table 1f The fifth siRNA sequence in the conjugate of the present disclosure
本公开所述siRNA或siRNA缀合物中，每个相邻核苷酸之间由磷酸二酯键或硫代磷酸二酯键连接，磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子带有负电荷，它可以以羟基或巯基的形式存在，羟基或巯基中的氢离子也可以部分或全部被阳离子取代。所述阳离子可以是任意的阳离子，如金属阳离子，铵离子NH4+，有机铵阳离子中的一种。出于提高溶解性考虑，在一种实施方式中，所述阳离子选自碱金属离子、三级胺形成的铵阳离子和季铵阳离子中的一种或多种。碱金属离子可以是K+和/或Na+，三级胺形成的阳离子可以是三乙胺形成的铵离子和/或N,N-二异丙基乙胺形成的铵离子。因此，本公开所述siRNA或siRNA缀合物可以至少部分以盐的形式存在。在一种方式中，磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子至少部分与钠离子结合，本公开所述siRNA或siRNA缀合物以钠盐或部分钠盐的形式存在。In the siRNA or siRNA conjugate disclosed in the present invention, each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate diester bond, and the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or the phosphorothioate diester bond carries a negative charge, which can exist in the form of a hydroxyl group or a thiol group, and the hydrogen ion in the hydroxyl group or the thiol group can also be partially or completely replaced by a cation. The cation can be any cation, such as a metal cation, an ammonium ionNH4+ , or one of an organic ammonium cation. In order to improve solubility, in one embodiment, the cation is selected from one or more of an alkali metal ion, an ammonium cation formed by a tertiary amine, and a quaternary ammonium cation. The alkali metal ion can be K+ and/or Na+ , and the cation formed by the tertiary amine can be an ammonium ion formed by triethylamine and/or an ammonium ion formed by N,N-diisopropylethylamine. Therefore, the siRNA or siRNA conjugate disclosed in the present invention can exist at least partially in the form of a salt. In one embodiment, the non-bridging oxygen atoms or sulfur atoms in the phosphodiester bond or the phosphorothioate diester bond are at least partially bound to sodium ions, and the siRNA or siRNA conjugate described in the present disclosure exists in the form of a sodium salt or a partial sodium salt.
本领域技术人员清楚知晓的是，可以通过使用具有相应修饰的核苷单体来将修饰的核苷酸基团引入所述的siRNA中。制备具有相应修饰的核苷单体的方法及将修饰的核苷酸基团引入siRNA的方法也是本领域技术人员所熟知的。所有修饰的核苷单体均可以商购得到或者采用已知方法制备得到。It is well known to those skilled in the art that the modified nucleotide groups can be introduced into the siRNA by using nucleoside monomers with corresponding modifications. The method of preparing nucleoside monomers with corresponding modifications and the method of introducing the modified nucleotide groups into siRNA are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or prepared by known methods.
式(308)所示的siRNA缀合物的制备Preparation of siRNA conjugate represented by formula (308)
可以采用任意合理的合成路线制备式(308)所示的siRNA缀合物。The siRNA conjugate of formula (308) can be prepared using any reasonable synthetic route.
在一些实施方式中，式(308)所示的siRNA缀合物可以采用如下方法制备，该方法包括在亚磷酰胺固相合成的条件下，分别按照siRNA正义链和反义链的核苷酸种类和顺序，按照3'到5'的方向将核苷单体依次连接，每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应；分离出siRNA的正义链和反义链，退火，其中，所述siRNA为上述本公开的siRNA；In some embodiments, the siRNA conjugate represented by formula (308) can be prepared by the following method, which comprises, under the conditions of phosphoramidite solid phase synthesis, sequentially connecting nucleoside monomers in the 3' to 5' direction according to the nucleotide types and sequences of the sense strand and antisense strand of the siRNA, wherein the connection of each nucleoside monomer comprises four steps of deprotection, coupling, capping, oxidation or sulfurization; separating the sense strand and antisense strand of the siRNA, and annealing, wherein the siRNA is the siRNA disclosed above;
并且，该方法还包括在偶联反应条件和偶联试剂存在下，将式(321)所示的化合物与核苷单体或连接在固相载体上的核苷酸序列接触，使式(321)所示的化合物经偶联反应连接至核苷酸序列。下文中，式(321)所示的化合物也称作缀合分子。Furthermore, the method further comprises contacting the compound represented by formula (321) with a nucleoside monomer or a nucleotide sequence connected to a solid support under coupling reaction conditions and in the presence of a coupling reagent, so that the compound represented by formula (321) is connected to the nucleotide sequence through a coupling reaction. Hereinafter, the compound represented by formula (321) is also referred to as a conjugated molecule.
其中：in:
R4为能够结合至式(308)所示化合物中Nu代表的siRNA的基团。在一些实施方式中，R4为能够通过共价键结合至Nu代表的siRNA的基团。在一些实施方式中，R4为能够经反应而通过磷酸二酯键缀合至Nu代表的siRNA的任意官能团的基团；R4 is a group that can bind to the siRNA represented by Nu in the compound represented by formula (308). In some embodiments, R4 is a group that can bind to the siRNA represented by Nu through a covalent bond. In some embodiments, R4 is a group that can react and conjugate to any functional group of the siRNA represented by Nu through a phosphodiester bond;
每个S1独立地是M1中全部活性羟基被YCOO-基团取代而形成的基团，其中，每个Y独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种；在一些实施方式中，Y为甲基。EachS1 is independently a group formed by replacing all active hydroxyl groups inM1 with YCOO- groups, wherein each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halogenated phenyl and alkylphenyl; in some embodiments, Y is methyl.
n1、n3、m1、m2、m3、R10、R11、R12、R13、R14、R15、L1、M1各自的定义和可选择的范围如前所述。The definitions and selectable ranges of n1, n3, m1, m2, m3, R10 , R11 , R12 , R13 , R14 , R15 , L1 , and M1 are as described above.
R4的选择是为了实现与含氮骨架上的N原子的连接，并且为合成式(308)所示的siRNA缀合物提供合适的反应位点。在一些实施方式中，R4中包括R2连接基团或经保护的R2连接基团，以及可通过反应与siRNA形成A59所示结构的官能团。R4 is selected to achieve connection with the N atom on the nitrogen-containing backbone and to provide a suitable reaction site for synthesizing the siRNA conjugate shown in formula (308). In some embodiments,R4 includes anR2 linking group or a protectedR2 linking group and a functional group that can react with siRNA to form the structure shown in A59.
在一些实施方式中，R4包含可与Nu代表的siRNA或核苷单体上的基团形成亚磷酸酯的第1官能团以及可与羟基或氨基反应形成共价键的第2官能团或者含有由所述共价键连接的固相载体。在一些实施方式中，所述第1官能团为亚磷酰胺、羟基或被保护的羟基。在一些实施方式中，所述第2官能团为亚磷酰胺、羧基或羧酸盐。在一些实施方式中，所述第2官能团为经由共价键连接至分子其他部分的固相载体，所述共价键由羟基或氨基形成。在一些实施方式中，所述固相载体经由磷酸酯键、羧酸酯键或酰胺键连接。在一些实施方式中，所述固相载体为树脂。In some embodiments, R4 comprises a first functional group that can form a phosphite with a group on the siRNA or nucleoside monomer represented by Nu and a second functional group that can react with a hydroxyl or amino group to form a covalent bond or contains a solid support connected by the covalent bond. In some embodiments, the first functional group is a phosphoramidite, a hydroxyl or a protected hydroxyl. In some embodiments, the second functional group is a phosphoramidite, a carboxyl or a carboxylate. In some embodiments, the second functional group is a solid support connected to other parts of the molecule via a covalent bond, and the covalent bond is formed by a hydroxyl or an amino group. In some embodiments, the solid support is connected via a phosphate bond, a carboxylate bond or an amide bond. In some embodiments, the solid support is a resin.
在一些实施方式中，所述第1官能团含有羟基、-ORk或式(C3)所示的基团；所述第2官能团含有式(C1)、(C2)、(C3)、(C1')或(C3')所示的结构：In some embodiments, the first functional group contains a hydroxyl group, -ORk , or a group represented by formula (C3); the second functional group contains a structure represented by formula (C1), (C2), (C3), (C1') or (C3'):
式中，q1为1-4的整数，X为O或NH，M+为阳离子，Rk为羟基保护基团，SPS表示固相载体，表示基团共价连接的位点。In the formula,q1 is an integer of 1-4, X is O or NH, M+ is a cation,Rk is a hydroxyl protecting group, SPS represents a solid phase support, Indicates the site to which a group is covalently attached.
在一些实施方式中，所述第1官能团含有亚磷酰胺基团，如式(C3)所示，该亚磷酰胺基团可以与核苷酸上的任意位置的羟基，如2'位羟基或3'位羟基发生偶联反应形成亚磷酸酯，并经氧化或硫化形成式A59所示的磷酸二酯键或硫代磷酸酯键，将缀合分子缀合至siRNA。此时，即使所述第2官能团并不存在，式(321)化合物也能够缀合至核苷酸，不影响式(308)所示的siRNA缀合物的获得。在此情况下，在经由亚磷酰胺固相合成等方法获得siRNA的正义链或反义链后，使式(321)化合物与核苷酸序列中末端核苷酸上的羟基反应，并在后续的氧化或硫化过程中形成磷酸二酯键连接或硫代磷酸酯连接，将式(321)化合物缀合至siRNA。In some embodiments, the first functional group contains a phosphoramidite group, as shown in formula (C3), which can react with a hydroxyl group at any position on the nucleotide, such as the 2' hydroxyl group or the 3' hydroxyl group, to form a phosphite, and then oxidize or sulfurize to form a phosphodiester bond or a phosphorothioate bond as shown in formula A59, and conjugate the conjugate molecule to the siRNA. In this case, even if the second functional group does not exist, the compound of formula (321) can be conjugated to the nucleotide, and does not affect the acquisition of the siRNA conjugate shown in formula (308). In this case, after obtaining the sense strand or antisense strand of the siRNA by a method such as phosphoramidite solid phase synthesis, the compound of formula (321) is reacted with the hydroxyl group on the terminal nucleotide in the nucleotide sequence, and a phosphodiester bond or a phosphorothioate bond is formed in the subsequent oxidation or sulfurization process, and the compound of formula (321) is conjugated to the siRNA.
在一些实施方式中，所述第1官能团含有被保护的羟基。在一些实施方式中，所述第2官能团包含可与固相载体反应的基团，所述反应提供包含固相载体的缀合分子。在一些实施方式中，所述第2官能团含有羧基、羧酸盐或亚磷酰胺，如式(C1)、(C2)或(C3)所示，当所述第2官能团包含羧基或羧酸盐时，式(321)化合物与固相载体，例如树脂上的羟基或氨基进行酯化反应或酰胺化反应，形成经羧酸酯键连接的包含固相载体的缀合分子。当所述第2官能团包含亚磷酰胺官能团时，式(321)化合物与通用固相载体，例如树脂上的羟基发生偶联反应，并经氧化形成经磷酸二酯键连接的包含固相载体的缀合分子。随后，以上述连接固相载体后的产物作为起始，按照亚磷酰胺固相合成方法依次连接核苷单体，获得连接有缀合基团的siRNA的正义链或反义链。在亚磷酰胺固相合成过程中，所述第1官能团发生脱保护，随后在偶联反应条件下与核苷单体上的亚磷酰胺基团发生偶联。In some embodiments, the first functional group contains a protected hydroxyl group. In some embodiments, the second functional group contains a group that can react with a solid phase carrier, and the reaction provides a conjugated molecule containing a solid phase carrier. In some embodiments, the second functional group contains a carboxyl group, a carboxylate or a phosphoramidite, as shown in formula (C1), (C2) or (C3). When the second functional group contains a carboxyl group or a carboxylate, the compound of formula (321) undergoes an esterification reaction or an amidation reaction with a solid phase carrier, such as a hydroxyl group or an amino group on a resin, to form a conjugated molecule containing a solid phase carrier connected via a carboxylate ester bond. When the second functional group contains a phosphoramidite functional group, the compound of formula (321) undergoes a coupling reaction with a universal solid phase carrier, such as a hydroxyl group on a resin, and is oxidized to form a conjugated molecule containing a solid phase carrier connected via a phosphodiester bond. Subsequently, starting with the product after the above connection to the solid phase carrier, nucleoside monomers are sequentially connected according to the phosphoramidite solid phase synthesis method to obtain a sense chain or antisense chain of siRNA connected to a conjugated group. During the phosphoramidite solid phase synthesis process, the first functional group is deprotected and then coupled with the phosphoramidite group on the nucleoside monomer under coupling reaction conditions.
在一些实施方式中，所述第1官能团含有羟基或被保护的羟基；所述第2官能团含有经羧酸酯键连接的固相载体或经酰胺键连接的固相载体、或者经磷酸酯键连接的固相载体，如式(C1')或(C3')所示。此时，由式(321)化合物代替固相载体作为起始，按照亚磷酰胺固相合成方法依次连接核苷单体，获得连接有缀合基团的siRNA的正义链或反义链。In some embodiments, the first functional group contains a hydroxyl group or a protected hydroxyl group; the second functional group contains a solid phase carrier connected via a carboxylate bond, a solid phase carrier connected via an amide bond, or a solid phase carrier connected via a phosphate bond, as shown in formula (C1') or (C3'). In this case, the compound of formula (321) is used as a starting point instead of the solid phase carrier, and nucleoside monomers are sequentially connected according to the phosphoramidite solid phase synthesis method to obtain the sense strand or antisense strand of the siRNA connected with a conjugated group.
在一些实施方式中，羧酸盐可以表示为-COO-M+，其中，M+是阳离子，例如选自金属阳离子，铵阳离子NH4+，有机铵阳离子中的一种。在一种实施方式中，所述金属离子选自碱金属离子中的一种，如K+或Na+。出于提高溶解性、使反应顺利进行的考虑，在一些实施方式中，有机铵离子为三级胺形成的铵阳离子或季铵阳离子，如，三乙胺形成的铵离子或N,N-二异丙基乙胺形成的铵离子。在一些实施方式中，羧酸盐是三乙胺羧酸盐或N,N-二异丙基乙胺羧酸盐。In some embodiments, the carboxylate can be represented as -COO- M+ , wherein M+ is a cation, for example, selected from a metal cation, an ammonium cation NH4+ , and an organic ammonium cation. In one embodiment, the metal ion is selected from an alkali metal ion, such as K+ or Na+ . In order to improve solubility and allow the reaction to proceed smoothly, in some embodiments, the organic ammonium ion is an ammonium cation or a quaternary ammonium cation formed by a tertiary amine, such as an ammonium ion formed by triethylamine or an ammonium ion formed by N, N-diisopropylethylamine. In some embodiments, the carboxylate is triethylamine carboxylate or N, N-diisopropylethylamine carboxylate.
在一些实施方式中，R4含有式(B9)、(B10)、(B9')、(B10')、(B11)、(B12)、(B11')或(B12')所示的结构：In some embodiments,R4 comprises a structure represented by formula (B9), (B10), (B9'), (B10'), (B11), (B12), (B11') or (B12'):
其中，q1为1-4的整数，q2为1-10的整数，X为O或NH，M+为阳离子，Rk为羟基保护基团，SPS表示固相载体，表示基团共价连接的位点。在一些实施方式中，q1为1或2。在一些实施方式中，q2为1-5的整数。在一些实施方式中，R4含有式(B9)或(B10)所示的结构。在一些实施方式中，R4含有式(B11)或(B12)所示的结构。whereinq1 is an integer of 1-4,q2 is an integer of 1-10, X is O or NH, M+ is a cation,Rk is a hydroxyl protecting group, SPS represents a solid phase support, In some embodiments, q1 is 1 or 2. In some embodiments, q2 is an integer from 1 to 5. In some embodiments, R4 contains the structure shown in formula (B9) or (B10). In some embodiments, R4 contains the structure shown in formula (B11) or (B12).
在一些实施方式中，Rk是Tr(三苯甲基)、MMTr(4-甲氧基三苯甲基)、DMTr(4,4'-双甲氧基三苯甲基)、TMTr(4,4',4'-三甲氧基三苯甲基)中的一种或多种。在一些实施方式中，Rk可以是DMTr，即4,4'-双甲氧基三苯甲基(4,4'-dimethoxytrityl)。In some embodiments, Rk is one or more of Tr (trityl), MMTr (4-methoxytrityl), DMTr (4,4'-bismethoxytrityl), TMTr (4,4',4'-trimethoxytrityl). In some embodiments, Rk may be DMTr, i.e., 4,4'-bismethoxytrityl.
L1的定义如前所述。The definition ofL1 is as described above.
在一些实施方式中，L1被用于将M1靶向基团连接至含氮骨架上的N原子，从而为式(308)所示的siRNA缀合物提供肝靶向功能。在一些实施方式中，L1包含A1-A26中的任一个或其组合。In some embodiments,L1 is used to connect theM1 targeting group to the N atom on the nitrogen-containing skeleton, thereby providing liver targeting function for the siRNA conjugate represented by formula (308). In some embodiments,L1 comprises any one of A1-A26 or a combination thereof.
根据上述描述，本领域技术人员容易理解的是，相较于本领域公知的亚磷酰胺固相合成方法而言，可通过上述第1官能团以及任选的第2官能团，获得将缀合分子连接至核苷酸序列的任意可能的位置的式(308)所示的siRNA缀合物，例如，缀合分子连接至核苷酸序列的端部，缀合分子连接至核苷酸序列的末端。相应地，除非另有说明，以下涉及缀合物和/或缀合分子的制备的描述中，当提及“脱保护”、“偶联”、“盖帽”、“氧化”、“硫化”等反应时，应当理解为本领域公知的亚磷酰胺核酸固相合成方法中所涉及的反应条件和试剂也同样适用于这些反应。示例性的反应条件和试剂将在后文详细描述。According to the above description, it is easy for a person skilled in the art to understand that, compared with the phosphoramidite solid phase synthesis method known in the art, the siRNA conjugate shown in formula (308) can be obtained by connecting the conjugate molecule to any possible position of the nucleotide sequence through the above-mentioned first functional group and the optional second functional group, for example, the conjugate molecule is connected to the end of the nucleotide sequence, and the conjugate molecule is connected to the end of the nucleotide sequence. Accordingly, unless otherwise specified, in the following description of the preparation of conjugates and/or conjugate molecules, when referring to reactions such as "deprotection", "coupling", "capping", "oxidation", and "sulfurization", it should be understood that the reaction conditions and reagents involved in the phosphoramidite nucleic acid solid phase synthesis method known in the art are also applicable to these reactions. Exemplary reaction conditions and reagents will be described in detail later.
在一些实施方式中，每个S1独立地是M1。在一些实施方式中，每个S1独立地是M1中至少一个活性羟基被羟基保护基团保护而形成的基团。在一些实施方式中，每个S1独立地是M1中任何存在的活性羟基全部被羟基保护基团保护而形成的基团。在一些实施方式中，任何本领域技术人员已知的羟基保护基团均可被用于保护M1中的活性羟基。在一些实施方式中，被保护的羟基可以式YCOO-表示，其中，每个Y独立地选自于由C1-C10烷基和C6-C10芳基所组成的组，所述C1-C10烷基和C6-C10芳基任选地被一个或多个取代基取代，所述取代基选自于由卤素和C1-C6烷基所组成的组。在一些实施方式中，每个Y独立地选自于由以下基团所组成的组：甲基、三氟甲基、二氟甲基、单氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤苯基，以及C1-C6烷基苯基。In some embodiments, each S1 is independently M1. In some embodiments, each S1 is independently a group formed by at least one active hydroxyl group in M1 being protected by a hydroxyl protecting group. In some embodiments, each S1 is independently a group formed by all active hydroxyl groups existing in M1 being protected by hydroxyl protecting groups. In some embodiments, any hydroxyl protecting group known to those skilled in the art can be used to protect the active hydroxyl group in M1. In some embodiments, the protected hydroxyl group can be represented by the formula YCOO-, wherein each Y is independently selected from the group consisting of C1 -C10 alkyl and C6 -C10 aryl, and the C1 -C10 alkyl and C6 -C10 aryl are optionally substituted by one or more substituents selected from the group consisting of halogen and C1 -C6 alkyl. In some embodiments, each Y is independently selected from the group consisting of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl, and C1 -C6 alkylphenyl.
在一些实施方式中，每个S1各自独立地选自于由式A46-A54所组成的组：In some embodiments, eachS1 is independently selected from the group consisting of formula A46-A54:
在一些实施方式中，S1为式A49或A50。In some embodiments,S1 is formula A49 or A50.
在一些实施方式中，每个Y独立地选自甲基、三氟甲基、二氟甲基、一氟甲基、三氯甲基、二氯甲基、一氯甲基、乙基、正丙基、异丙基、苯基、卤代苯基以及烷基苯基中的一种；在一些实施方式中，Y为甲基。In some embodiments, each Y is independently selected from one of methyl, trifluoromethyl, difluoromethyl, monofluoromethyl, trichloromethyl, dichloromethyl, monochloromethyl, ethyl, n-propyl, isopropyl, phenyl, halophenyl and alkylphenyl; in some embodiments, Y is methyl.
如前所述，式(308)所示的siRNA缀合物的制备方法还包括以下步骤：合成siRNA的另一链(例如，当上述步骤合成了连接有缀合分子的siRNA正义链时，还包括按照固相合成方法合成siRNA的反义链，反之亦然)，分离正义链和反义链，以及退火。具体地，在分离步骤中，连接至核苷酸序列和/或缀合分子的固相载体被切割下来，同时必要的保护基团被脱除(此时，式(321)化合物中的各S1基团转化为对应的M1靶向基团)，获得连接有缀合分子的siRNA正义链(或反义链)以及对应的反义链(或正义链)，正义链与反义链退火形成双链RNA结构，获得式(308)所示的siRNA缀合物。As mentioned above, the method for preparing the siRNA conjugate shown in formula (308) further includes the following steps: synthesizing the other strand of siRNA (for example, when the above step synthesizes the siRNA sense strand connected to the conjugated molecule, it also includes synthesizing the antisense strand of siRNA according to the solid phase synthesis method, and vice versa), separating the sense strand and the antisense strand, and annealing. Specifically, in the separation step, the solid phase carrier connected to the nucleotide sequence and/or the conjugated molecule is cut off, and the necessary protective groups are removed (at this time, eachS1 group in the compound of formula (321) is converted into a correspondingM1 targeting group), and the siRNA sense strand (or antisense strand) connected to the conjugated molecule and the corresponding antisense strand (or sense strand) are obtained, and the sense strand and the antisense strand are annealed to form a double-stranded RNA structure to obtain the siRNA conjugate shown in formula (308).
在一些实施方式中，式(308)所示的siRNA缀合物的制备方法包含以下步骤：在偶联反应条件和偶联试剂存在下，将式(321)所示的化合物与正义链或反义链的3'端的第一个核苷单体接触，使式(321)所示的化合物连接上序列中第一个核苷酸，在亚磷酰胺固相合成的条件下，按照期望的正义链或反义链核苷酸种类和顺序，按照3'到5'的方向将核苷单体依次连接，合成siRNA的正义链或反义链；其中，式(321)所示化合物为R4中含有第1官能团和第2官能团，第1官能团含有被保护的羟基，第2官能团具有如式(C1')或(C3')所示结构的化合物，与第一个核苷单体连接前，式(321)化合物经过脱保护；每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应；得到连接有缀合基团的核酸的正义链或反义链；在亚磷酰胺固相合成的条件下，按照反义链或正义链核苷酸种类和顺序，按照3'到5'的方向将核苷单体依次连接，合成核酸的反义链或正义链；每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应；脱除保护基并与固相载体切割，分离纯化获得正义链和反义链，退火。In some embodiments, the method for preparing the siRNA conjugate represented by formula (308) comprises the following steps: contacting the compound represented by formula (321) with the first nucleoside monomer at the 3' end of the sense chain or antisense chain under coupling reaction conditions and in the presence of a coupling reagent, so that the compound represented by formula (321) is connected to the first nucleotide in the sequence, and under phosphoramidite solid phase synthesis conditions, the nucleoside monomers are sequentially connected in the direction from 3' to 5' according to the desired sense chain or antisense chain nucleotide type and sequence to synthesize the sense chain or antisense chain of siRNA; wherein the compound represented by formula (321) is R4 contains a first functional group and a second functional group, the first functional group contains a protected hydroxyl group, and the second functional group has a compound with a structure as shown in formula (C1') or (C3'); before connecting with the first nucleoside monomer, the compound of formula (321) is deprotected; the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization; the sense chain or antisense chain of the nucleic acid connected with the conjugated group is obtained; under the conditions of phosphoramidite solid phase synthesis, the nucleoside monomers are sequentially connected in the direction of 3' to 5' according to the type and sequence of nucleotides in the antisense chain or sense chain to synthesize the antisense chain or sense chain of the nucleic acid; the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization; the protecting group is removed and cut with the solid phase carrier, the sense chain and the antisense chain are separated and purified, and annealed.
在一些实施方式中，式(308)所示的siRNA缀合物的制备方法包含以下步骤：按照该双链siRNA中正义链或反义链的核苷酸种类和顺序，按照3'到5'的方向将核苷单体依次连接，合成正义链和反义链，每个核苷单体的连接包括脱保护、偶联、盖帽、氧化或硫化四步反应，得到连接在固相载体上的正义链和连接在固相载体上的反义链；在偶联反应条件和偶联试剂存在下，将式(321)所示的化合物与连接在固相载体上的正义链或连接在固相载体上的反义链接触，将式(321)化合物连接至正义链或反义链，其中，式(321)化合物是R4中含有第1官能团，第1官能团为亚磷酰胺基团的式(321)化合物；脱除保护基并与固相载体切割，分别分离纯化，获得siRNA的正义链或反义链，退火，其中，所述siRNA的正义链或反义链上连接有缀合基团。In some embodiments, the method for preparing the siRNA conjugate shown in formula (308) comprises the following steps: according to the nucleotide type and sequence of the sense strand or antisense strand in the double-stranded siRNA, the nucleoside monomers are sequentially connected in the 3' to 5' direction to synthesize the sense strand and the antisense strand, and the connection of each nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization to obtain the sense strand connected to the solid phase carrier and the antisense strand connected to the solid phase carrier; under coupling reaction conditions and in the presence of a coupling reagent, the compound shown in formula (321) is contacted with the sense strand connected to the solid phase carrier or the antisense strand connected to the solid phase carrier, and the compound of formula (321) is connected to the sense strand or the antisense strand, wherein the compound of formula (321) is R4 contains a first functional group, wherein the first functional group is a compound of formula (321) which is a phosphoramidite group; removing the protecting group and cutting with the solid phase carrier, separating and purifying respectively to obtain the sense chain or antisense chain of siRNA, and annealing, wherein the sense chain or antisense chain of the siRNA is connected with a conjugation group.
在一些实施方式中，式A59中的P原子连接至siRNA中的正义链的3'末端，式(308)所示的siRNA缀合物的制备方法包括：In some embodiments, the P atom in formula A59 is connected to the 3' end of the sense strand in the siRNA, and the method for preparing the siRNA conjugate shown in formula (308) comprises:
(1)脱除式(321)化合物(其中，式(321)化合物为R4中含有第1官能团和第2官能团，第1官能团含有被保护的羟基ORk，第2官能团具有如式(C1')或(C3')所示结构的化合物)中的羟基保护基团Rk；在偶联反应条件和偶联试剂存在下，将脱保护得到的产物与核苷单体接触，得到通过缀合分子连接至固相载体的核苷单体；(1) removing the hydroxyl protecting group Rk in the compound of formula (321) (wherein the compound of formula (321) is a compound wherein R4 contains a first functional group and a second functional group, the first functional group contains a protected hydroxyl group OR k , and the second functional group has a structure as shown in formula (C1′) or (C3′)) ; contacting the deprotected product with a nucleoside monomer under coupling reaction conditions and in the presence of a coupling reagent to obtain a nucleoside monomer connected to a solid phase carrier via a conjugated molecule;
(2)以该通过缀合分子连接至固相载体的核苷单体起始，按照3'-5'的方向通过亚磷酰胺固相合成方法合成siRNA的正义链；(2) starting with the nucleoside monomer connected to the solid phase carrier via the conjugated molecule, synthesizing the sense strand of the siRNA via a phosphoramidite solid phase synthesis method in the 3'-5' direction;
(3)通过亚磷酰胺固相合成方法，合成siRNA的反义链；(3) synthesizing the antisense strand of siRNA by phosphoramidite solid phase synthesis method;
(4)分离出siRNA的正义链和反义链并退火，获得式(308)所示的siRNA缀合物。(4) Separate the sense strand and antisense strand of siRNA and anneal them to obtain the siRNA conjugate represented by formula (308).
其中，在步骤(1)中，脱除上述式(321)化合物中的保护基团Rk的方法包括在脱保护条件下，将式(321)化合物与脱保护试剂接触。脱保护条件包括温度为0-50℃，在一些实施方式中为15-35℃，反应时间为30-300秒，在一些实施方式中为50-150秒，脱保护试剂可以选自三氟乙酸、三氯乙酸、二氯乙酸、一氯乙酸中的一种或多种，在一些实施方式中为二氯乙酸。脱保护试剂与式(321)化合物的摩尔比为10:1-1000:1，在一些实施方式中为50:1-500:1。Wherein, in step (1), the method for removing the protecting groupRk in the compound of formula (321) comprises contacting the compound of formula (321) with a deprotecting agent under deprotecting conditions. The deprotecting conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 30-300 seconds, in some embodiments 50-150 seconds, and the deprotecting agent can be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid, in some embodiments dichloroacetic acid. The molar ratio of the deprotecting agent to the compound of formula (321) is 10:1-1000:1, in some embodiments 50:1-500:1.
所述偶联反应条件和偶联试剂可使用任何适合于上述偶联反应的条件和试剂。在一些实施方式中，可使用与所采用的固相合成方法中的偶联反应相同的条件与试剂。The coupling reaction conditions and coupling reagents may use any conditions and reagents suitable for the above coupling reaction. In some embodiments, the same conditions and reagents as those used in the coupling reaction in the solid phase synthesis method may be used.
在一些实施方式中，所述偶联反应的条件包括反应温度为0-50℃，在一些实施方式中为15-35℃。式(321)化合物与核苷单体的摩尔比为1:1-1:50，在一些实施方式中为1:2-1:5；式(321)化合物和偶联试剂的摩尔比可以为1:1-1:50，在一些实施方式中为1:3-1:10，反应时间为200-3000秒，在一些实施方式中为500-1500秒。偶联试剂选自1H-四氮唑、5-乙硫基1H-四氮唑、5-苄硫基1H-四氮唑中的一种或多种，在一些实施方式中为5-乙硫基1H-四氮唑。所述偶联反应可在有机溶剂中进行，所述有机溶剂选自无水乙腈、无水DMF、无水二氯甲烷中的一种或多种，在一些实施方式中为无水乙腈。相对于式(321)化合物，所述有机溶剂的用量为3-50L/mol，在一些实施方式中为5-20L/mol。In some embodiments, the conditions of the coupling reaction include a reaction temperature of 0-50°C, in some embodiments 15-35°C. The molar ratio of the compound of formula (321) to the nucleoside monomer is 1:1-1:50, in some embodiments 1:2-1:5; the molar ratio of the compound of formula (321) to the coupling reagent can be 1:1-1:50, in some embodiments 1:3-1:10, and the reaction time is 200-3000 seconds, in some embodiments 500-1500 seconds. The coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, 5-benzylthio 1H-tetrazole, in some embodiments 5-ethylthio 1H-tetrazole. The coupling reaction can be carried out in an organic solvent, and the organic solvent is selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, in some embodiments anhydrous acetonitrile. Relative to the compound of formula (321), the amount of the organic solvent used is 3-50 L/mol, and in some embodiments, 5-20 L/mol.
在步骤(2)中，通过亚磷酰胺核酸固相合成的方法，利用上述步骤制备的通过缀合分子连接至固相载体的核苷单体起始，按照3'-5'的方向合成第二种siRNA缀合物的正义链S。此时，缀合基团连接至所得到的正义链的3'末端。In step (2), the sense strand S of the second siRNA conjugate is synthesized in the 3'-5' direction by phosphoramidite nucleic acid solid phase synthesis, starting from the nucleoside monomers prepared in the above steps and connected to the solid phase carrier through the conjugation molecule. At this time, the conjugation group is connected to the 3' end of the obtained sense strand.
步骤(2)和(3)中所述固相合成的其它条件，包括核苷单体脱保护条件，脱保护试剂种类和用量，偶联反应条件，偶联试剂的种类和用量，盖帽反应的条件，盖帽试剂的种类和用量，氧化反应条件，氧化试剂种类和用量，硫化反应条件，硫化试剂种类和用量采用本领域中常规使用的各种试剂、用量和条件。Other conditions of the solid phase synthesis described in steps (2) and (3), including nucleoside monomer deprotection conditions, deprotection reagent type and amount, coupling reaction conditions, coupling reagent type and amount, capping reaction conditions, capping reagent type and amount, oxidation reaction conditions, oxidation reagent type and amount, sulfurization reaction conditions, sulfurization reagent type and amount adopt various reagents, amounts and conditions conventionally used in the art.
例如，在一些实施方式中，步骤(2)和(3)中所述固相合成可使用如下条件：For example, in some embodiments, the solid phase synthesis in steps (2) and (3) may use the following conditions:
核苷单体脱保护条件包括温度为0-50℃，在一些实施方式中为15-35℃，反应时间为30-300秒，在一些实施方式中为50-150秒，脱保护试剂可以选自三氟乙酸、三氯乙酸、二氯乙酸、一氯乙酸、中的一种或多种，在一些实施方式中为二氯乙酸。脱保护试剂与固相载体上4,4'-二甲氧基三苯甲基保护基的的摩尔比可以为2:1-100:1，在一些实施方式中为3:1-50:1。The nucleoside monomer deprotection conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 30-300 seconds, in some embodiments 50-150 seconds, a deprotection agent can be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, and in some embodiments dichloroacetic acid. The molar ratio of the deprotection agent to the 4,4'-dimethoxytrityl protecting group on the solid phase carrier can be 2:1-100:1, in some embodiments 3:1-50:1.
偶联反应条件包括温度为0-50℃，在一些实施方式中为15-35℃，固相载体上连接的核酸序列与核苷单体的摩尔比可以为1:1-1:50，在一些实施方式中为1:5-1:15；固相载体上连接的核酸序列和偶联试剂的摩尔比为1:1-1:100，在一些实施方式中为1:50-1:80，反应时间和偶联试剂的选择与前述相同。The coupling reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, the molar ratio of the nucleic acid sequence connected to the solid phase support to the nucleoside monomer can be 1:1-1:50, in some embodiments 1:5-1:15; the molar ratio of the nucleic acid sequence connected to the solid phase support to the coupling reagent is 1:1-1:100, in some embodiments 1:50-1:80, and the reaction time and the selection of the coupling reagent are the same as above.
盖帽反应条件包括温度为0-50℃，在一些实施方式中为15-35℃，反应时间为5-500秒，在一些实施方式中为10-100秒，盖帽试剂的选择与前述相同。盖帽试剂的总量与固相载体上连接的核酸序列的摩尔比为1:100-100:1，在一些实施方式中为1:10-10:1。在盖帽试剂使用等摩尔量的乙酸酐与N-甲基咪唑的情况下，乙酸酐、N-甲基咪唑以及固相载体上连接的核酸序列的摩尔比可以为1:1:10-10:10:1，在一些实施方式中为1:1:2-2:2:1。The capping reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 5-500 seconds, in some embodiments 10-100 seconds, and the selection of the capping reagent is the same as above. The molar ratio of the total amount of the capping reagent to the nucleic acid sequence connected to the solid phase carrier is 1:100-100:1, in some embodiments 1:10-10:1. In the case where the capping reagent uses equimolar amounts of acetic anhydride and N-methylimidazole, the molar ratio of acetic anhydride, N-methylimidazole and the nucleic acid sequence connected to the solid phase carrier can be 1:1:10-10:10:1, in some embodiments 1:1:2-2:2:1.
氧化反应条件包括温度为0-50℃，在一些实施方式中为15-35℃，反应时间为1-100秒，在一些实施方式中为5-50秒，氧化试剂在一些实施方式中为碘(在一些实施方式中，以碘水的形式提供)。氧化试剂与偶联步骤中固相载体上连接的核酸序列的摩尔比可以为1:1-100:1，在一些实施方式中为5:1-50:1。在一些实施方式中，所述氧化反应在四氢呋喃:水:吡啶＝3:1:1-1:1:3的混合溶剂中进行。硫化反应条件包括温度为0-50℃，在一些实施方式中为15-35℃，反应时间为50-2000秒，在一些实施方式中为100-1000秒，硫化试剂在一些实施方式中为氢化黄原素。硫化试剂与偶联步骤中固相载体上连接的核酸序列的摩尔比为10:1-1000:1，在一些实施方式中为10:1-500:1。在一些实施方式中，所述硫化反应在乙腈:吡啶＝1:3-3:1的混合溶剂中进行。The oxidation reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 1-100 seconds, in some embodiments 5-50 seconds, and the oxidizing agent is iodine in some embodiments (in some embodiments, provided in the form of iodine water). The molar ratio of the oxidizing agent to the nucleic acid sequence connected to the solid phase carrier in the coupling step can be 1:1-100:1, in some embodiments 5:1-50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran: water: pyridine = 3:1:1-1:1:3. The sulfidation reaction conditions include a temperature of 0-50°C, in some embodiments 15-35°C, a reaction time of 50-2000 seconds, in some embodiments 100-1000 seconds, and the sulfiding agent is hydrogenated xanthan in some embodiments. The molar ratio of the sulfiding agent to the nucleic acid sequence connected to the solid phase carrier in the coupling step is 10:1-1000:1, in some embodiments 10:1-500:1. In some embodiments, the sulfurization reaction is carried out in a mixed solvent of acetonitrile:pyridine=1:3-3:1.
在将所有核苷单体连接之后，退火之前，该方法还包括分离出siRNA的正义链和反义链。分离的方法为本领域技术人员所公知，一般包括将合成得到的核苷酸序列从固相载体上切割下来，脱除碱基上、磷酸基上和配体上的保护基团，纯化和脱盐。After all nucleoside monomers are connected and before annealing, the method further includes separating the sense strand and the antisense strand of the siRNA. The separation method is well known to those skilled in the art and generally includes cutting the synthesized nucleotide sequence from the solid phase support, removing the protecting groups on the base, the phosphate group and the ligand, and purifying and desalting.
将合成得到的核苷酸序列从固相载体上切割下来，并脱除碱基上、磷酸基上和配体上的保护基团可按照siRNA合成中常规的切割和脱保护方法进行。例如，将得到的连接有固相载体的核苷酸序列与浓氨水接触；在脱保护的过程中，A46-A54基团的保护基团YCOO-转化为羟基，S1基团转化为相应的M1基团，生成式(308)所示的缀合物。其中，所述浓氨水可以是25-30重量％的氨水，浓氨水的用量与目标siRNA序列相比可以为0.2ml/μmol-0.8ml/μmol。The synthesized nucleotide sequence is cut from the solid phase support, and the protective groups on the base, phosphate group and ligand are removed according to the conventional cutting and deprotection methods in siRNA synthesis. For example, the obtained nucleotide sequence connected to the solid phase support is contacted with concentrated ammonia water; during the deprotection process, the protective group YCOO- of the A46-A54 group is converted into a hydroxyl group, and theS1 group is converted into the correspondingM1 group, generating a conjugate shown in formula (308). The concentrated ammonia water can be 25-30% by weight of ammonia water, and the amount of concentrated ammonia water used can be 0.2ml/μmol-0.8ml/μmol compared with the target siRNA sequence.
在所合成的核苷酸序列上存在至少一个2'-TBDMS保护时，所述方法还包括将脱除了固相载体的核苷酸序列与三乙胺三氢氟酸盐接触，以脱除该2'-TBDMS保护。此时，所得到的目标siRNA序列中的相应核苷酸具有游离的2'-羟基。三乙胺三氢氟酸盐纯品的用量与目标siRNA序列相比可以为0.4ml/μmol-1.0ml/μmol。这样即可得到式(308)所示的siRNA缀合物。When there is at least one 2'-TBDMS protection on the synthesized nucleotide sequence, the method further comprises contacting the nucleotide sequence from which the solid phase carrier has been removed with triethylamine trihydrofluoride to remove the 2'-TBDMS protection. At this time, the corresponding nucleotides in the obtained target siRNA sequence have free 2'-hydroxyl groups. The amount of pure triethylamine trihydrofluoride used can be 0.4 ml/μmol-1.0 ml/μmol compared to the target siRNA sequence. In this way, the siRNA conjugate shown in formula (308) can be obtained.
纯化和脱盐的方法是本领域技术人员熟知的。例如，可利用制备型离子色谱纯化柱，通过NaBr或NaCl的梯度洗脱，完成核酸的纯化；产品收集合并后，可采用反相色谱纯化柱进行脱盐。The methods of purification and desalting are well known to those skilled in the art. For example, nucleic acid purification can be completed by using a preparative ion chromatography purification column through gradient elution with NaBr or NaCl; after the products are collected and combined, a reverse phase chromatography purification column can be used for desalting.
这样得到的式(308)所示的siRNA缀合物中，核苷酸之间的磷酸二酯键或硫代磷酸二酯键中的非桥接氧原子或硫原子基本与钠离子结合，式(308)所示的siRNA缀合物基本以钠盐形式存在。可以采用熟知的离子交换方法，用氢离子和/或其他阳离子取代所述钠离子，得到其他形式的式(308)所示的siRNA缀合物。所述阳离子如前所述。In the siRNA conjugate of formula (308) thus obtained, the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or thiophosphodiester bond between nucleotides is basically bound to the sodium ion, and the siRNA conjugate of formula (308) is basically present in the form of a sodium salt. The well-known ion exchange method can be used to replace the sodium ion with hydrogen ions and/or other cations to obtain other forms of siRNA conjugates of formula (308). The cation is as described above.
在合成过程中，可随时对核酸序列的纯度和分子量进行检测，更好地把控合成质量，此类检测的方法为本领域技术人员所公知。例如，可通过离子交换色谱检测核酸纯度，并通过液质联用色谱(LC-MS)测定分子量。During the synthesis process, the purity and molecular weight of the nucleic acid sequence can be tested at any time to better control the synthesis quality. Such testing methods are well known to those skilled in the art. For example, the purity of the nucleic acid can be tested by ion exchange chromatography, and the molecular weight can be determined by liquid chromatography-mass spectrometry (LC-MS).
退火的方法也是本领域技术人员熟知的。例如，可简单地将所合成的正义链(S链)与反义链(AS链)以等摩尔比混合在注射用水中加热至70-95℃，随后室温冷却，使其通过氢键形成双链结构。这样即可得到式(308)所示的siRNA缀合物。Annealing methods are also well known to those skilled in the art. For example, the synthesized sense strand (S strand) and antisense strand (AS strand) can be simply mixed in an equal molar ratio in water for injection and heated to 70-95° C., and then cooled to room temperature to form a double-stranded structure through hydrogen bonding. In this way, the siRNA conjugate shown in formula (308) can be obtained.
在获得所述缀合物后，在一些实施方式中，还可利用例如液质联用色谱等方法，通过分子量检测等方式对所合成的式(308)所示的siRNA缀合物进行表征，确定所合成的siRNA缀合物为目标设计的式(308)所示的siRNA缀合物，且所合成的siRNA的序列为期望的siRNA的序列，例如为表1a、1b、1d、1e和1f中所列的序列之一。After obtaining the conjugate, in some embodiments, the synthesized siRNA conjugate of formula (308) can be characterized by molecular weight detection and the like using methods such as liquid chromatography-mass spectrometry to determine that the synthesized siRNA conjugate is the target designed siRNA conjugate of formula (308), and the sequence of the synthesized siRNA is the desired siRNA sequence, such as one of the sequences listed in Tables 1a, 1b, 1d, 1e and 1f.
式(321)所示化合物可以通过以下制备方法得到：该方法包括在有机溶剂中，在酯化反应条件下，以及在碱和酯化催化剂存在下，将式(313)所示化合物与环状酸酐接触，离子交换，分离得到式(321)所示化合物：The compound represented by formula (321) can be obtained by the following preparation method: the method comprises contacting the compound represented by formula (313) with a cyclic acid anhydride in an organic solvent under esterification reaction conditions and in the presence of a base and an esterification catalyst, ion exchange, and separation to obtain the compound represented by formula (321):
其中，n1、n3、m1、m2、m3、R10、R11、R12、R13、R14、R15、L1、S1各自的定义和可选择的范围如前所述；Wherein, the definitions and selectable ranges of n1, n3, m1, m2, m3, R10 , R11 , R12 , R13 , R14 , R15 , L1 and S1 are as described above;
R6为提供式(321)中R4的基团；在一些实施方式中，R6具有式(A61)所示的结构：R6 is a group that provides R4 in formula (321); in some embodiments, R6 has a structure shown in formula (A61):
其中，Ri为能够实现与含氮骨架上的N原子连接、与RkO连接并且连接有一个游离羟基的任意基团，Rk为羟基保护基团。此时，所获得的是R4中含有作为羟基保护基团的第1官能团和第2官能团，所述第2官能团含有如式(C1)或(C2)所示结构的式(321)化合物。Wherein, Ri is any group that can achieve connection with the N atom on the nitrogen-containing skeleton, connects with Rk O and connects with a free hydroxyl group, and Rk is a hydroxyl protecting group. In this case, what is obtained is a compound of formula (321) containing the first functional group and the second functional group as a hydroxyl protecting group in R4 , wherein the second functional group contains the structure shown in formula (C1) or (C2).
所述酯化反应条件包括反应温度为0-100℃，反应时间为8-48小时，在一些实施方式中，所述酯化反应条件为反应温度为10-40℃，反应时间为20-30小时。The esterification reaction conditions include a reaction temperature of 0-100° C. and a reaction time of 8-48 hours. In some embodiments, the esterification reaction conditions include a reaction temperature of 10-40° C. and a reaction time of 20-30 hours.
在一些实施方式中，所述有机溶剂包含环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N,N-二甲基甲酰胺和N,N-二异丙基乙胺中的一种或多种。在一些实施方式中，所述环氧类溶剂为二氧六环和/或四氢呋喃，所述醚类溶剂为乙醚和/或甲基叔丁基醚，所述卤代烷类溶剂为二氯甲烷、三氯甲烷和1,2-二氯乙烷中的一种或多种。在一些实施方式中，所述有机溶剂为二氯甲烷。相对于所述式(313)所示化合物，所述有机溶剂的用量为3-50L/mol，在一些实施方式中为5-20L/mol。In some embodiments, the organic solvent comprises one or more of an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and/or tetrahydrofuran, the ether solvent is ether and/or methyl tert-butyl ether, and the halogenated alkane solvent is one or more of dichloromethane, chloroform and 1,2-dichloroethane. In some embodiments, the organic solvent is dichloromethane. Relative to the compound shown in formula (313), the amount of the organic solvent is 3-50L/mol, and in some embodiments, 5-20L/mol.
在一些实施方式中，所述环状酸酐为丁二酸酐、戊二酸酐、己二酸酐或庚二酸酐中的一种，在一些实施方式中为丁二酸酐。所述环状酸酐与所述式(313)所示化合物的摩尔比为1:1-10:1，在一些实施方式中为2:1-5:1。In some embodiments, the cyclic anhydride is one of succinic anhydride, glutaric anhydride, adipic anhydride or pimelic anhydride, and in some embodiments, succinic anhydride. The molar ratio of the cyclic anhydride to the compound represented by formula (313) is 1:1-10:1, and in some embodiments, 2:1-5:1.
所述酯化催化剂可以是任何对该酯化反应起到催化作用的催化剂，例如该催化剂可以是4-二甲氨基吡啶。所述催化剂与式(313)所示化合物的摩尔比为1:1-10:1，在一些实施方式中为2:1-5:1。The esterification catalyst can be any catalyst that catalyzes the esterification reaction, for example, the catalyst can be 4-dimethylaminopyridine. The molar ratio of the catalyst to the compound represented by formula (313) is 1:1-10:1, and in some embodiments, 2:1-5:1.
在一些实施方式中，所述碱可以是任意的无机碱，有机碱或者它们的结合。考虑溶解性和产物稳定性，所述碱可以是例如三级胺。在一些实施方式中，所述三级胺为三乙胺或N,N-二异丙基乙胺。所述三级胺与式(313)所示化合物的摩尔比为1:1-20:1，在一些实施方式中为3:1-10:1。In some embodiments, the base can be any inorganic base, organic base or a combination thereof. Considering solubility and product stability, the base can be, for example, a tertiary amine. In some embodiments, the tertiary amine is triethylamine or N,N-diisopropylethylamine. The molar ratio of the tertiary amine to the compound shown in formula (313) is 1:1-20:1, and in some embodiments is 3:1-10:1.
所述离子交换作用是将式(321)化合物转化为期望的羧酸或羧酸盐的形式，离子交换的方法为本领域技术人员所公知，可以使用合适的离子交换溶液和交换条件，得到具有M+阳离子的缀合分子，在此不做详述。在一些实施方式中，所述离子交换反应使用三乙胺磷酸盐溶液进行，所述三乙胺磷酸盐溶液的浓度为0.2-0.8M，在一些实施方式中，所述三乙胺磷酸盐溶液的浓度为0.4-0.6M，相对于式(313)化合物，所述三乙胺磷酸盐溶液的用量为3-6L/mol，在进一步的实施方式中为4-5L/mol。The ion exchange reaction is to convert the compound of formula (321) into the desired carboxylic acid or carboxylate form. The method of ion exchange is well known to those skilled in the art. Suitable ion exchange solutions and exchange conditions can be used to obtain conjugated molecules with M+ cations, which will not be described in detail here. In some embodiments, the ion exchange reaction is carried out using a triethylamine phosphate solution, the concentration of the triethylamine phosphate solution is 0.2-0.8M, in some embodiments, the concentration of the triethylamine phosphate solution is 0.4-0.6M, relative to the compound of formula (313), the amount of the triethylamine phosphate solution is 3-6L/mol, and in further embodiments it is 4-5L/mol.
可使用任何合适的分离方法从反应混合物中分离式(321)化合物。在一些实施方式中，可通过蒸发除去溶剂、随后通过色谱方法分离式(321)化合物，例如，可使用如下两种色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用含1wt‰三乙胺的二氯甲烷:甲醇＝100:18-100:20梯度洗脱；或者(2)反相纯化：C18、C8反相填料，使用甲醇:乙腈＝0.1:1-1:0.1梯度洗脱。在一些实施方式中，可以直接除去溶剂得到式(321)化合物粗产品，该粗产品可以直接用于后续反应。Any suitable separation method can be used to separate the compound of formula (321) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then separated by chromatography. For example, the following two chromatographic conditions can be used for separation: (1) normal phase purification silica gel: 200-300 mesh silica gel filler, using dichloromethane containing 1wt‰ triethylamine: methanol = 100:18-100:20 gradient elution; or (2) reverse phase purification: C18, C8 reverse phase filler, using methanol: acetonitrile = 0.1:1-1:0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (321), which can be directly used in subsequent reactions.
在一些实施方式中，式(321)化合物的制备方法还进一步包括在缩合反应条件下，在有机溶剂中，在缩合剂、缩合催化剂和三级胺存在下，将上述离子交换反应得到的产物进一步与含有氨基或羟基的固相载体进行接触。此时，所获得的是R4中含有第1官能团和第2官能团，第1官能团含有羟基保护基团，第2官能团含有如式(C1')所示结构的式(321)化合物。In some embodiments, the preparation method of the compound of formula (321) further comprises contacting the product obtained by the above ion exchange reaction with a solid phase carrier containing an amino group or a hydroxyl group in an organic solvent under condensation reaction conditions in the presence of a condensation agent, a condensation catalyst and a tertiary amine. At this time, what is obtained is a compound of formula (321) in whichR4 contains a first functional group and a second functional group, the first functional group contains a hydroxyl protecting group, and the second functional group contains a structure as shown in formula (C1').
所述固相载体为固相合成siRNA中所用的载体中的一种，其中的一些为本领域技术人员所公知。例如,所述固相载体可以选自含有活性羟基或氨基官能团的固相载体，在一些实施方式中，所述固相载体为氨基树脂或羟基树脂。在一些实施方式中，所述氨基或羟基树脂具有如下参数：粒径100-400目(mesh)，表面氨基或羟基载量为0.2-0.5mmol/g。所述式(321)所示化合物与固相载体的用量比为10-400μmol化合物/每克固相载体(μmol/g)。在一些实施方式中，所述式(321)所示化合物与固相载体的用量比为50-200μmol/g。The solid phase carrier is one of the carriers used in the solid phase synthesis of siRNA, some of which are well known to those skilled in the art. For example, the solid phase carrier can be selected from a solid phase carrier containing an active hydroxyl or amino functional group. In some embodiments, the solid phase carrier is an amino resin or a hydroxyl resin. In some embodiments, the amino or hydroxyl resin has the following parameters: a particle size of 100-400 mesh, and a surface amino or hydroxyl loading of 0.2-0.5 mmol/g. The amount ratio of the compound represented by the formula (321) to the solid phase carrier is 10-400 μmol compound/gram of solid phase carrier (μmol/g). In some embodiments, the amount ratio of the compound represented by the formula (321) to the solid phase carrier is 50-200 μmol/g.
所述有机溶剂可以是本领域技术人员已知的任何合适的溶剂或混合溶剂。在一些实施方式中，所述有机溶剂为乙腈、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N,N-二甲基甲酰胺和N,N-二异丙基乙胺中的一种或多种。在一些实施方式中，所述环氧类溶剂为二氧六环和/或四氢呋喃，所述醚类溶剂为乙醚和/或甲基叔丁基醚，所述卤代烷类溶剂为二氯甲烷、三氯甲烷和1,2-二氯乙烷中的一种或多种。在一些实施方式中，所述有机溶剂为乙腈。相对于式(321)化合物，所述有机溶剂的用量为20-200L/mol，在一些实施方式中为50-100L/mol。The organic solvent can be any suitable solvent or mixed solvent known to those skilled in the art. In some embodiments, the organic solvent is acetonitrile, epoxy solvents, ether solvents, halogenated alkane solvents, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. In some embodiments, the epoxy solvent is dioxane and/or tetrahydrofuran, the ether solvent is ether and/or methyl tert-butyl ether, and the halogenated alkane solvent is one or more of dichloromethane, trichloromethane and 1,2-dichloroethane. In some embodiments, the organic solvent is acetonitrile. Relative to the compound of formula (321), the amount of the organic solvent is 20-200L/mol, and in some embodiments is 50-100L/mol.
在一些实施方式中，所述缩合剂可以是苯并三唑-1-基-氧基三吡咯烷基鏻六氟磷酸酯/盐(benzotriazol-1-yl-oxytripyrrolidino phosphonium hexafluorophosphate，PyBop)、3-二乙氧基磷酰基-1,2,3-苯唑4(3H)-酮和/或O-苯并三唑-四甲基脲六氟磷酸酯，在一些实施方式中，所述缩合剂为O-苯并三唑-四甲基脲六氟磷酸酯。所述缩合剂与式(321)所示化合物的摩尔比为1:1-20:1，在一些实施方式中为1:1-5:1。In some embodiments, the condensing agent can be benzotriazol-1-yl-oxytripyrrolidino phosphonium hexafluorophosphate/salt (benzotriazol-1-yl-oxytripyrrolidino phosphonium hexafluorophosphate, PyBop), 3-diethoxyphosphoryl-1,2,3-benzoxazole 4 (3H)-one and/or O-benzotriazole-tetramethyluronium hexafluorophosphate. In some embodiments, the condensing agent is O-benzotriazole-tetramethyluronium hexafluorophosphate. The molar ratio of the condensing agent to the compound represented by formula (321) is 1:1-20:1, and in some embodiments, it is 1:1-5:1.
在一些实施方式中，所述三级胺为三乙胺和/或N,N-二异丙基乙胺，在一些实施方式中为N,N-二异丙基乙胺；所述三级胺与式(321)所示化合物的摩尔比为1:1-20:1，在一些实施方式中为1:1-5:1。In some embodiments, the tertiary amine is triethylamine and/or N,N-diisopropylethylamine, and in some embodiments it is N,N-diisopropylethylamine; the molar ratio of the tertiary amine to the compound represented by formula (321) is 1:1-20:1, and in some embodiments it is 1:1-5:1.
在一些实施方式中，式(321)化合物的制备方法还可以包括在盖帽反应条件下，在有机溶剂中，将得到的缩合产物与盖帽试剂和酰化催化剂接触，分离得到式(321)所示化合物。所述盖帽反应的作用在于除去任何尚未反应完全的活性反应官能团，以避免在后续反应中产生不必要的副产物。所述盖帽反应的条件包括反应温度为0-50℃，在一些实施方式中为15-35℃，反应的时间为1-10h，在一些实施方式中为3-6h。盖帽试剂可以使用siRNA固相合成中所使用的盖帽试剂，siRNA固相合成中所使用的盖帽试剂为本领域技术人员所公知。In some embodiments, the preparation method of the compound of formula (321) may also include contacting the obtained condensation product with a capping agent and an acylation catalyst in an organic solvent under capping reaction conditions, and separating to obtain the compound of formula (321). The function of the capping reaction is to remove any active reaction functional groups that have not yet reacted completely to avoid the generation of unnecessary by-products in subsequent reactions. The conditions of the capping reaction include a reaction temperature of 0-50°C, in some embodiments 15-35°C, and a reaction time of 1-10h, in some embodiments 3-6h. The capping agent can use the capping agent used in siRNA solid phase synthesis, and the capping agent used in siRNA solid phase synthesis is well known to those skilled in the art.
在一些实施方式中，所述盖帽试剂由盖帽试剂1(cap1)和盖帽试剂2(cap2)组成，其中，盖帽试剂1为N-基甲基咪唑，在一些实施方式中以N-甲基咪唑的吡啶/乙腈混合溶液形式提供，其中，吡啶与乙腈的体积比为1:10-1:1，在一些实施方式中为1:3-1:1，吡啶与乙腈的总体积与N-甲基咪唑的体积比为1:1-10:1，在一些实施方式中为3:1-7:1。所述盖帽试剂2为乙酸酐。在一些实施方式中，所述盖帽试剂2以乙酸酐的乙腈溶液形式提供，其中，乙酸酐和乙腈的体积为1:1-1:10，在进一步的实施方式中为1:2-1:6。In some embodiments, the capping reagent is composed of capping reagent 1 (cap1) and capping reagent 2 (cap2), wherein capping reagent 1 is N-methylimidazole, and in some embodiments, it is provided in the form of a pyridine/acetonitrile mixed solution of N-methylimidazole, wherein the volume ratio of pyridine to acetonitrile is 1:10-1:1, in some embodiments, 1:3-1:1, and the volume ratio of the total volume of pyridine and acetonitrile to N-methylimidazole is 1:1-10:1, in some embodiments, 3:1-7:1. Capping reagent 2 is acetic anhydride. In some embodiments, capping reagent 2 is provided in the form of an acetonitrile solution of acetic anhydride, wherein the volume of acetic anhydride and acetonitrile is 1:1-1:10, and in further embodiments, 1:2-1:6.
在一些实施方式中，所述N-甲基咪唑的吡啶/乙腈混合溶液的体积与式(321)化合物的质量之比为5ml/g-50ml/g，在一些实施方式中为15ml/g-30ml/g。所述乙酸酐的乙腈溶液的体积与式(321)化合物的质量之比为0.5ml/g-10ml/g，在一些实施方式中为1ml/g-5ml/g。In some embodiments, the volume ratio of the pyridine/acetonitrile mixed solution of N-methylimidazole to the mass ratio of the compound of formula (321) is 5ml/g-50ml/g, and in some embodiments, 15ml/g-30ml/g. The volume ratio of the acetic anhydride acetonitrile solution to the mass ratio of the compound of formula (321) is 0.5ml/g-10ml/g, and in some embodiments, 1ml/g-5ml/g.
在一些实施方式中，盖帽试剂使用等摩尔量的乙酸酐与N-甲基咪唑。在一些实施方式中，所述有机溶剂为乙腈、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N,N-二甲基甲酰胺和N,N-二异丙基乙胺中的一种或多种。在一些实施方式中，所述有机溶剂为乙腈。相对于式(321)化合物，所述有机溶剂的用量为10-50L/mol，在一些实施方式中为5-30L/mol。In some embodiments, the capping agent uses equimolar amounts of acetic anhydride and N-methylimidazole. In some embodiments, the organic solvent is one or more of acetonitrile, epoxy solvents, ether solvents, halogenated alkane solvents, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. In some embodiments, the organic solvent is acetonitrile. Relative to the compound of formula (321), the amount of the organic solvent is 10-50 L/mol, and in some embodiments, 5-30 L/mol.
在一些实施方式中，所述酰化催化剂可以选自任何可用于酯化缩合或酰胺化缩合的催化剂，例如碱性杂环化合物。在一些实施方式中，所述酰化催化剂为4-二甲氨基吡啶。所述催化剂与式(321)所示化合物的质量之比为0.001:1-1:1，在一些实施方式中为0.01:1-0.1:1。In some embodiments, the acylation catalyst can be selected from any catalyst that can be used for esterification condensation or amidation condensation, such as a basic heterocyclic compound. In some embodiments, the acylation catalyst is 4-dimethylaminopyridine. The mass ratio of the catalyst to the compound represented by formula (321) is 0.001:1-1:1, and in some embodiments, 0.01:1-0.1:1.
在一些实施方式中，可使用任何合适的分离方法从反应混合物中分离式(321)化合物。在一些实施方式中，可通过以有机溶剂充分洗涤，并过滤，去除未反应的反应物、过量的盖帽试剂及其它杂质，得到式(321)化合物，所述有机溶剂选自乙腈、二氯甲烷、甲醇，在一些实施方式中为乙腈。In some embodiments, any suitable separation method can be used to separate the compound of formula (321) from the reaction mixture. In some embodiments, the compound of formula (321) can be obtained by washing with an organic solvent and filtering to remove unreacted reactants, excess capping reagents and other impurities, wherein the organic solvent is selected from acetonitrile, dichloromethane, methanol, and in some embodiments, acetonitrile.
在一些实施方式中，式(321)所示缀合分子的制备方法包括在有机溶剂中，在偶联反应条件下，以及在偶联试剂存在下，将式(313)所示化合物与亚磷酰二胺接触，分离得到式(321)所示化合物。此时，所获得的是R4中含有第1官能团和第2官能团，第1官能团含有羟基保护基团，第2官能团含有如式(C3)所示结构的式(321)化合物。In some embodiments, the preparation method of the conjugated molecule represented by formula (321) includes contacting the compound represented by formula (313) with phosphorodiamidite in an organic solvent under coupling reaction conditions and in the presence of a coupling reagent, and separating to obtain the compound represented by formula (321). At this time, what is obtained is a compound represented by formula (321) in whichR4 contains the first functional group and the second functional group, the first functional group contains a hydroxyl protecting group, and the second functional group contains a structure represented by formula (C3).
在一些实施方式中，偶联反应条件包括温度可以为0-50℃，例如为15-35℃，式(313)化合物与亚磷酰二胺的摩尔比可以为1:1-1:50，例如为1:5-1:15；式(313)化合物和偶联试剂的摩尔比可以为1:1-1:100，例如为1:50-1:80；反应时间可以为200-3000秒，例如为500-1500秒。所述亚磷酰二胺例如可使用双(二异丙基氨基)(2-氰基乙氧基)膦，其可商购获得或按照本领域中公知的方法合成获得。偶联试剂选自1H-四氮唑、5-乙硫基1H-四氮唑、5-苄硫基1H-四氮唑中的一种或多种，例如为5-乙硫基1H-四氮唑。所述偶联反应可在有机溶剂中进行，所述有机溶剂选自无水乙腈、无水DMF、无水二氯甲烷中的一种或多种，例如为无水乙腈。在一些实施方式中，相对于式(313)化合物，所述有机溶剂的用量为3-50L/mol，例如可以为5-20L/mol。通过进行该偶联反应，式(313)化合物中的羟基与亚磷酰二胺反应形成亚磷酰胺基团。在一些实施方式中，可以直接除去溶剂得到式(321)化合物粗产品，该粗产品可以直接用于后续反应。In some embodiments, the coupling reaction conditions include a temperature of 0-50°C, such as 15-35°C, a molar ratio of the compound of formula (313) to the phosphorodiamidite of 1:1-1:50, such as 1:5-1:15; a molar ratio of the compound of formula (313) to the coupling reagent of 1:1-1:100, such as 1:50-1:80; and a reaction time of 200-3000 seconds, such as 500-1500 seconds. The phosphorodiamidite can be, for example, bis(diisopropylamino)(2-cyanoethoxy)phosphine, which can be commercially available or synthesized according to methods known in the art. The coupling reagent is selected from one or more of 1H-tetrazole, 5-ethylthio 1H-tetrazole, and 5-benzylthio 1H-tetrazole, such as 5-ethylthio 1H-tetrazole. The coupling reaction can be carried out in an organic solvent, and the organic solvent is selected from one or more of anhydrous acetonitrile, anhydrous DMF, and anhydrous dichloromethane, such as anhydrous acetonitrile. In some embodiments, the amount of the organic solvent relative to the compound of formula (313) is 3-50L/mol, for example, 5-20L/mol. By carrying out the coupling reaction, the hydroxyl group in the compound of formula (313) reacts with the phosphorodiamidite to form a phosphoramidite group. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (321), which can be directly used in subsequent reactions.
在一些实施方式中，式(321)化合物的制备方法还进一步包括以下步骤：在偶联反应条件下，在有机溶剂中，以及在偶联试剂存在下，将分离得到的产物进一步与含有羟基的固相载体进行接触。随后，经盖帽反应、氧化反应，分离得到式(321)化合物。此时，所获得的是R4中含有第1官能团和第2官能团，第1官能团含有羟基保护基团，第2官能团具有如式(C3')所示结构的式(321)化合物。In some embodiments, the preparation method of the compound of formula (321) further comprises the following steps: under coupling reaction conditions, in an organic solvent, and in the presence of a coupling reagent, the separated product is further contacted with a solid phase carrier containing a hydroxyl group. Subsequently, the compound of formula (321) is separated through a capping reaction and an oxidation reaction. At this time, what is obtained is a compound of formula (321) in whichR4 contains a first functional group and a second functional group, the first functional group contains a hydroxyl protecting group, and the second functional group has a structure as shown in formula (C3').
在一些实施方式中，所述固相载体为本领域中公知的可用于核酸固相合成的固相载体，例如，可以是经脱保护反应后的市售的通用固相载体(HLUnyLinkerTM300Oligonucleotide Synthesis Support，Kinovate Life Sciences公司，结构如式B80所示)：In some embodiments, the solid phase carrier is a solid phase carrier known in the art that can be used for solid phase synthesis of nucleic acids, for example, it can be a commercially available general solid phase carrier ( HLUnyLinkerTM 300 Oligonucleotide Synthesis Support, Kinovate Life Sciences, the structure is shown in formula B80):
脱保护反应为本领域技术人员所公知。在一些实施方式中，脱保护条件包括温度为0-50℃，例如为15-35℃；反应时间为30-300秒，例如为50-150秒。脱保护试剂可以选自三氟乙酸、三氯乙酸、二氯乙酸、一氯乙酸中的一种或多种，在一些实施方式中，脱保护试剂为二氯乙酸。脱保护试剂与固定相上的-DMTr(4,4'-二甲氧基三苯甲基)保护基的摩尔比为2:1-100:1，例如为3:1-50:1。通过进行所述脱保护，在所述固相载体表面上获得具有反应活性的游离羟基，便于进行后续的偶联反应。Deprotection reactions are well known to those skilled in the art. In some embodiments, the deprotection conditions include a temperature of 0-50°C, for example, 15-35°C; a reaction time of 30-300 seconds, for example, 50-150 seconds. The deprotection reagent can be selected from one or more of trifluoroacetic acid, trichloroacetic acid, dichloroacetic acid, and monochloroacetic acid. In some embodiments, the deprotection reagent is dichloroacetic acid. The molar ratio of the deprotection reagent to the -DMTr (4,4'-dimethoxytrityl) protecting group on the stationary phase is 2:1-100:1, for example, 3:1-50:1. By performing the deprotection, reactive free hydroxyl groups are obtained on the surface of the solid phase carrier, which is convenient for subsequent coupling reactions.
偶联反应条件以及偶联试剂的选择可如上所述。通过进行该偶联反应，脱保护反应中形成的游离羟基与亚磷酰胺基团反应形成亚磷酸酯连接。The coupling reaction conditions and the selection of coupling reagents can be as described above.By carrying out the coupling reaction, the free hydroxyl group formed in the deprotection reaction reacts with the phosphoramidite group to form a phosphite linkage.
在一些实施方式中，盖帽反应条件包括温度为0-50℃，例如为15-35℃，反应时间为5-500秒，例如为10-100秒，所述盖帽反应在盖帽试剂存在下进行。盖帽试剂的选择和用量可如上所述。In some embodiments, the capping reaction conditions include a temperature of 0-50°C, such as 15-35°C, a reaction time of 5-500 seconds, such as 10-100 seconds, and the capping reaction is performed in the presence of a capping agent. The selection and dosage of the capping agent can be as described above.
氧化反应条件包括温度为0-50℃，例如可以为15-35℃，反应时间为1-100秒，例如可以为5-50秒，氧化试剂例如可以为碘(在一些实施方式中，以碘水的形式提供)。在一些实施方式中，氧化试剂与连接至固相载体的核酸序列的摩尔比为1:1-100:1，例如可以为5:1-50:1。在一些实施方式中，所述氧化反应在四氢呋喃:水:吡啶＝3:1:1-1:1:3的混合溶剂中进行。The oxidation reaction conditions include a temperature of 0-50° C., for example, 15-35° C., a reaction time of 1-100 seconds, for example, 5-50 seconds, and an oxidizing agent, for example, iodine (in some embodiments, provided in the form of iodine water). In some embodiments, the molar ratio of the oxidizing agent to the nucleic acid sequence connected to the solid phase carrier is 1:1-100:1, for example, 5:1-50:1. In some embodiments, the oxidation reaction is carried out in a mixed solvent of tetrahydrofuran: water: pyridine = 3:1:1-1:1:3.
在一些实施方式中，R6为式B7或B8基团中的一种，In some embodiments,R6 is one of the groups of formula B7 or B8,
其中q2的定义如前所述，where q2 is defined as above,
此时，式(313)所示化合物可以通过以下制备方法得到：在有机溶剂中，在酰胺化反应条件下，以及在酰胺化反应缩合剂和三级胺存在下，将式(314)所示化合物与式(A-1)所示化合物或式(A-2)化合物接触，随后进行分离：At this time, the compound represented by formula (313) can be obtained by the following preparation method: in an organic solvent, under the conditions of amidation reaction, and in the presence of an amidation reaction condensation agent and a tertiary amine, the compound represented by formula (314) is contacted with the compound represented by formula (A-1) or the compound represented by formula (A-2), followed by separation:
其中，n1、n3、m1、m2、m3、R10、R11、R12、R13、R14、R15、L1、S1、q2和Rk各自的定义和可选择的范围如前所述。Wherein, the definitions and selectable ranges of n1, n3, m1, m2, m3,R10 ,R11 , R12,R13 ,R14 ,R15 ,L1,S1 ,q2 andRk are as described above.
所述酰胺化反应条件可包括反应温度为0-100℃，反应时间为1-48小时，在一些实施方式中，所述酰胺化反应条件为反应温度为10-40℃，反应时间为2-16小时。The amidation reaction conditions may include a reaction temperature of 0-100° C. and a reaction time of 1-48 hours. In some embodiments, the amidation reaction conditions include a reaction temperature of 10-40° C. and a reaction time of 2-16 hours.
在一些实施方式中，所述有机溶剂为醇类溶剂、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N,N-二甲基甲酰胺和N,N-二异丙基乙胺中的一种或多种。所述醇类溶剂在一些实施方式中为甲醇、乙醇、丙醇中的一种或多种，在一些实施方式中为乙醇。所述环氧类溶剂在一些实施方式中为为二氧六环和/或四氢呋喃。所述醚类溶剂在一些实施方式中为为乙醚和/或甲基叔丁基醚。所述卤代烷类溶剂在一些实施方式中为为二氯甲烷、三氯甲烷和1,2-二氯乙烷中的一种或多种。在一些实施方式中，所述有机溶剂为二氯甲烷。相对于式(314)化合物，有机溶剂用量为3-50L/mol，在进一步的实施方式中为3-20L/mol。In some embodiments, the organic solvent is one or more of an alcohol solvent, an epoxy solvent, an ether solvent, a halogenated alkane solvent, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine. The alcohol solvent is one or more of methanol, ethanol and propanol in some embodiments, and ethanol in some embodiments. The epoxy solvent is dioxane and/or tetrahydrofuran in some embodiments. The ether solvent is ether and/or methyl tert-butyl ether in some embodiments. The halogenated alkane solvent is one or more of dichloromethane, trichloromethane and 1,2-dichloroethane in some embodiments. In some embodiments, the organic solvent is dichloromethane. Relative to the compound of formula (314), the amount of organic solvent used is 3-50L/mol, and in further embodiments is 3-20L/mol.
在一些实施方式中，所述酰胺化反应缩合剂为六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷、3-二乙氧基磷酰基-1,2,3-苯唑4(3H)-酮、4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐、2-乙氧基-1-乙氧碳酰基-1,2-二氢喹啉(EEDQ)或O-苯并三唑-四甲基脲六氟磷酸酯，在进一步的实施方式中为3-二乙氧基磷酰基-1,2,3-苯唑4(3H)-酮。所述酰胺化反应缩合剂与式(314)所示化合物的摩尔比可以为1:1-10:1，在一些实施方式中为2.5:1-5:1。In some embodiments, the amidation reaction condensation agent is benzotriazole-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 3-diethoxyphosphoryl-1,2,3-benzoxazole 4 (3H)-one, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) or O-benzotriazole-tetramethyluronium hexafluorophosphate, and in a further embodiment, 3-diethoxyphosphoryl-1,2,3-benzoxazole 4 (3H)-one. The molar ratio of the amidation reaction condensation agent to the compound represented by formula (314) can be 1:1-10:1, and in some embodiments, 2.5:1-5:1.
在一些实施方式中，所述三级胺为三乙胺或N,N-二异丙基乙胺，在进一步的实施方式中为N,N-二异丙基乙胺。所述三级胺与式(314)所示化合物的摩尔比为3:1-20:1，在一些实施方式中为5:1-10:1。In some embodiments, the tertiary amine is triethylamine or N,N-diisopropylethylamine, and in further embodiments, N,N-diisopropylethylamine. The molar ratio of the tertiary amine to the compound of formula (314) is 3:1-20:1, and in some embodiments, 5:1-10:1.
在一些实施方式中，式(A-1)和式(A-2)化合物可通过任何适当的方式制备。例如，当Rk为DMTr基团时，可通过甘油酸钙与DMTrCl反应制备式(A-1)化合物；类似地，可先将3-氨基-1,2-丙二醇与环状酸酐接触，随后再与DMTrCl反应制备式(A-2)化合物，所述环状酸酐可以是碳原子数为4-13、在一些实施方式中为4-8的环状酸酐。本领域技术人员容易理解的是，所述环状酸酐的选择对应于(A-2)化合物中q2的不同值，例如，当所述环状酸酐为丁二酸酐时，q2＝1，当所述环状酸酐为戊二酸酐时，q2＝2，以此类推。In some embodiments, the compounds of formula (A-1) and formula (A-2) can be prepared by any appropriate method. For example, when Rk is a DMTr group, the compound of formula (A-1) can be prepared by reacting calcium glycerate with DMTrCl; similarly, 3-amino-1,2-propanediol can be first contacted with a cyclic anhydride, and then reacted with DMTrCl to prepare the compound of formula (A-2), wherein the cyclic anhydride can be a cyclic anhydride having 4-13 carbon atoms, and in some embodiments, 4-8 carbon atoms. It is easy for a person skilled in the art to understand that the selection of the cyclic anhydride corresponds to different values of q2 in the compound (A-2), for example, when the cyclic anhydride is succinic anhydride, q2 =1, when the cyclic anhydride is glutaric anhydride, q2 =2, and so on.
在一些变型中，也可通过使式(314)所示化合物依次与所述环状酸酐、3-氨基-1,2-丙二醇和DMTrCl反应，制备式(313)化合物。本领域技术人员容易理解的是，这些变型不会影响式(313)化合物的结构与功能，并且这些变型是本领域技术人员在上述方法的基础上容易实现的。In some variations, the compound of formula (313) can also be prepared by reacting the compound of formula (314) with the cyclic anhydride, 3-amino-1,2-propanediol and DMTrCl in sequence. It is easy for a person skilled in the art to understand that these variations will not affect the structure and function of the compound of formula (313), and these variations are easy to implement by a person skilled in the art based on the above method.
与上述类似地，可使用任何合适的分离方法从反应混合物中分离式(313)化合物。在一些实施方式中，可通过蒸发除去溶剂、随后通过色谱方法分离式(313)化合物，例如，可使用如下两种色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用石油醚:乙酸乙酯:二氯甲烷:N,N-二甲基甲酰胺＝1:1:1:0.5-1:1:1:0.6梯度洗脱；以及(2)反相纯化：C18、C8反相填料，使用甲醇:乙腈＝0.1:1-1:0.1梯度洗脱。在一些实施方式中，可以直接除去溶剂得到式(313)化合物粗产品，该粗产品可以直接用于后续反应。Similar to the above, any suitable separation method can be used to separate the compound of formula (313) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then separated by chromatography. For example, the following two chromatographic conditions can be used for separation: (1) normal phase purification silica gel: 200-300 mesh silica gel filler, using petroleum ether: ethyl acetate: dichloromethane: N, N-dimethylformamide = 1:1:1:0.5-1:1:1:0.6 gradient elution; and (2) reverse phase purification: C18, C8 reverse phase filler, using methanol: acetonitrile = 0.1:1-1:0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (313), which can be directly used in subsequent reactions.
在一些实施方式中，式(314)所示化合物可以通过以下制备方法得到：该方法包括在有机溶剂中，在酰胺化反应缩合剂和三级胺存在下，在缩合反应条件下，将式(320)所示化合物与式(316)所示化合物接触，随后进行分离：In some embodiments, the compound represented by formula (314) can be obtained by the following preparation method: the method comprises contacting the compound represented by formula (320) with the compound represented by formula (316) in an organic solvent in the presence of an amidation reaction condensation agent and a tertiary amine under condensation reaction conditions, and then separating:
S1-L1-OHS1 -L1 -OH
式(316)Formula (316)
其中，n1、n3、m1、m2、m3、R10、R11、R12、R13、R14、R15各自的定义和可选择的范围如前所述。Here, the definitions and selectable ranges of n1, n3, m1, m2, m3, R10 , R11 , R12 , R13 , R14 and R15 are as described above.
式(316)化合物可使用例如J.Am.Chem.Soc.2014,136,16958-16961中所公开的化合物，或者，式(316)化合物可由本领域技术人员通过各种方法制备，例如，可参照美国专利US 8,106,022B2实施例1中所公开的方法制备某些式(316)化合物，以引用的方式将以上文献的全部内容整体并入本文。The compound of formula (316) can be prepared, for example, using the compounds disclosed in J.Am.Chem.Soc.2014,136,16958-16961, or the compound of formula (316) can be prepared by a person skilled in the art by various methods. For example, certain compounds of formula (316) can be prepared by referring to the method disclosed in Example 1 of U.S. Patent No. 8,106,022B2. The entire contents of the above documents are incorporated herein by reference in their entirety.
在一些实施方式中，所述缩合反应条件包括反应温度为0-100℃，反应时间为0.1-24小时，在一些实施方式中为反应温度为10-40℃，反应时间为0.5-16小时。In some embodiments, the condensation reaction conditions include a reaction temperature of 0-100° C. and a reaction time of 0.1-24 hours. In some embodiments, the reaction temperature is 10-40° C. and the reaction time is 0.5-16 hours.
考虑到期望产物式(314)化合物的结构，所述式(316)所示化合物与所述式(320)所示化合物的摩尔比应当基于与式(320)中n1与n3的和而确定。在一些实施方式中，例如，当n1+n3＝3时，为了保证反应完全而不过度，式(316)所示化合物与所述式(320)所示化合物的摩尔比可以为3:1-3.5:1，在一些实施方式中为3.01:1-3.15:1。Considering the structure of the desired product compound of formula (314), the molar ratio of the compound of formula (316) to the compound of formula (320) should be determined based on the sum of n1 and n3 in formula (320). In some embodiments, for example, when n1+n3=3, in order to ensure that the reaction is complete and not excessive, the molar ratio of the compound of formula (316) to the compound of formula (320) can be 3:1-3.5:1, and in some embodiments, 3.01:1-3.15:1.
在一些实施方式中，所述有机溶剂为乙腈、环氧类溶剂、醚类溶剂、卤代烷类溶剂、二甲基亚砜、N,N-二甲基甲酰胺和N,N-二异丙基乙胺中的一种或多种，所述环氧类溶剂在一些实施方式中为二氧六环和/或四氢呋喃，所述醚类溶剂在一些实施方式中为乙醚和/或甲基叔丁基醚，所述卤代烷类溶剂在一些实施方式中为二氯甲烷、三氯甲烷和1,2-二氯乙烷中的一种或多种，在一些实施方式中，所述有机溶剂为二氯甲烷。相对于式(320)化合物，所述有机溶剂的用量为3-50L/mol，在一些实施方式中为5-20L/mol。In some embodiments, the organic solvent is one or more of acetonitrile, epoxy solvents, ether solvents, halogenated alkane solvents, dimethyl sulfoxide, N,N-dimethylformamide and N,N-diisopropylethylamine, the epoxy solvent is dioxane and/or tetrahydrofuran in some embodiments, the ether solvent is ether and/or methyl tert-butyl ether in some embodiments, the halogenated alkane solvent is one or more of dichloromethane, chloroform and 1,2-dichloroethane in some embodiments, and the organic solvent is dichloromethane. Relative to the compound of formula (320), the amount of the organic solvent is 3-50 L/mol, and in some embodiments, 5-20 L/mol.
在一些实施方式中，所述酰胺化反应缩合剂为苯并三唑-1-基-氧基三吡咯烷基鏻六氟磷酸盐/酯、3-二乙氧基磷酰基-1,2,3-苯唑4(3H)-酮(DEPBT)、O-苯并三唑-四甲基脲六氟磷酸盐/酯、4-(4,6-二甲氧基三嗪-2-基)-4-甲基吗啉盐酸盐或1-羟基苯并三唑中的一种或多种，在进一步的实施方式中为苯并三唑-1-基-氧基三吡咯烷基鏻六氟磷酸盐/酯和1-羟基苯并三唑的混合物，其中苯并三唑-1-基-氧基三吡咯烷基鏻六氟磷酸盐/酯和1-羟基苯并三唑为等摩尔用量。所述总的酰胺化反应缩合剂与式(316)所示化合物的摩尔比可以为1:1-3:1，在一些实施方式中为1.05:1-1.5:1。In some embodiments, the amidation reaction condensation agent is one or more of benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 3-diethoxyphosphoryl-1,2,3-benzoxazole 4(3H)-one (DEPBT), O-benzotriazole-tetramethyluronium hexafluorophosphate, 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride or 1-hydroxybenzotriazole, and in a further embodiment, it is a mixture of benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate and 1-hydroxybenzotriazole, wherein benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate and 1-hydroxybenzotriazole are used in equimolar amounts. The molar ratio of the total amidation reaction condensation agent to the compound represented by formula (316) can be 1:1-3:1, and in some embodiments, it is 1.05:1-1.5:1.
所述三级胺可以为N-甲基吗啉、三乙胺或N,N-二异丙基乙胺，在一些实施方式中为N-甲基吗啉；所述三级胺与式(316)所示化合物的摩尔比可以为2:1-10:1，在一些实施方式中为2:1-5:1。The tertiary amine can be N-methylmorpholine, triethylamine or N,N-diisopropylethylamine, and in some embodiments it is N-methylmorpholine; the molar ratio of the tertiary amine to the compound represented by formula (316) can be 2:1-10:1, and in some embodiments it is 2:1-5:1.
与上述类似地，可使用任何合适的分离方法从反应混合物中分离式(314)化合物。在一些实施方式中，可通过蒸发除去溶剂、随后通过色谱方法分离式(314)化合物例如，可使用如下两种色谱条件进行分离：(1)正相纯化硅胶：200-300目硅胶填料，使用二氯甲烷:甲醇＝100:5-100:7梯度洗脱；以及(2)反相纯化：C18、C8反相填料，使用甲醇:乙腈＝0.1:1-1:0.1梯度洗脱。在一些实施方式中，可以直接除去溶剂得到式(314)化合物粗产品，该粗产品可以直接用于后续反应。Similar to the above, any suitable separation method can be used to separate the compound of formula (314) from the reaction mixture. In some embodiments, the solvent can be removed by evaporation and then separated by chromatography. For example, the following two chromatographic conditions can be used for separation: (1) normal phase purification silica gel: 200-300 mesh silica gel filler, using dichloromethane: methanol = 100:5-100:7 gradient elution; and (2) reverse phase purification: C18, C8 reverse phase filler, using methanol: acetonitrile = 0.1:1-1:0.1 gradient elution. In some embodiments, the solvent can be directly removed to obtain a crude product of the compound of formula (314), which can be directly used in subsequent reactions.
式(320)化合物可商购获得，或者由本领域技术人员使用已知的方法获得。例如，当m1＝m2＝m3＝3，n1＝1，n3＝2，且每个R10、R11、R12、R13、R14、R15均为H时，式(320)化合物可自阿法埃莎公司商购获得。The compound of formula (320) is commercially available or can be obtained by a person skilled in the art using known methods. For example, when m1=m2=m3=3, n1=1, n3=2, and each ofR10 ,R11 ,R12 ,R13 ,R14 , andR15 is H, the compound of formula (320) can be commercially obtained from Alfa Aesar.
本公开的siRNA缀合物也可以与药学上可接受的其它辅料联用，该辅料可以为本领域常规采用的各种制剂或化合物的一种或多种，详情可参见上文关于本公开的药物组合物的描述。The siRNA conjugates disclosed herein may also be used in combination with other pharmaceutically acceptable excipients, which may be one or more of various preparations or compounds conventionally used in the art. For details, see the above description of the pharmaceutical composition disclosed herein.
本公开的siRNA、含该siRNA的药物组合物及缀合物的应用Application of siRNA, pharmaceutical composition containing the siRNA and conjugate disclosed herein
在一些实施方式中，本公开提供了本公开的siRNA和/或药物组合物和/或siRNA缀合物在制备用于治疗和/或预防HAE和/或血栓形成的药物中的用途。In some embodiments, the present disclosure provides use of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure in the preparation of a medicament for treating and/or preventing HAE and/or thrombosis.
在一些实施方式中，本公开提供了一种预防和/或治疗HAE和/或血栓形成的方法，该方法包括将有效量的本公开的siRNA和/或药物组合物和/或siRNA缀合物给予有需要的受试者。In some embodiments, the present disclosure provides a method for preventing and/or treating HAE and/or thrombosis, the method comprising administering an effective amount of the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure to a subject in need thereof.
通过将本公开的siRNA活性成分给予有需要的受试者，可以通过RNA干扰的机制达到预防和/或治疗HAE和/或血栓形成的目的。因此，本公开的siRNA和/或药物组合物和/或siRNA缀合物可用于预防和/或治疗HAE和/或血栓形成，或用于制备用于预防和/或治疗HAE和/或血栓形成的药物。By administering the siRNA active ingredient disclosed herein to a subject in need, the purpose of preventing and/or treating HAE and/or thrombosis can be achieved through the mechanism of RNA interference. Therefore, the siRNA and/or pharmaceutical composition and/or siRNA conjugate disclosed herein can be used to prevent and/or treat HAE and/or thrombosis, or to prepare a drug for preventing and/or treating HAE and/or thrombosis.
本文所使用的术语“给药/给予”是指通过使得至少部分地将本公开的siRNA、药物组合物和/或siRNA缀合物定位于期望的位点以产生期望效果的方法或途径，将本公开的siRNA、药物组合物和/或siRNA缀合物放置入受试者体内。适于本公开方法的给药途径包括局部给药和全身给药。一般而言，局部给药导致与受试者体循环相比将更多siRNA缀合物递送至特定位点；而全身给药导致将本公开的siRNA、药物组合物和/或siRNA缀合物递送至受试者的基本体循环。考虑到本公开旨在提供预防和/或治疗HAE的手段，在一些实施方式中采用能够将药物递送至肝脏的给药方式。The term "administration/administration" as used herein refers to placing the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure into the body of a subject by a method or approach that at least partially localizes the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure to a desired site to produce a desired effect. Routes of administration suitable for the methods of the present disclosure include local administration and systemic administration. In general, local administration results in more siRNA conjugates being delivered to a specific site compared to the subject's systemic circulation; while systemic administration results in the delivery of the siRNA, pharmaceutical composition and/or siRNA conjugate of the present disclosure to the subject's basic systemic circulation. Considering that the present disclosure is intended to provide a means for preventing and/or treating HAE, in some embodiments, an administration method that is capable of delivering the drug to the liver is used.
可通过本领域已知的任何合适途径向受试者给药，所述途径包括但不仅限于：口服或胃肠外途径，如静脉内给药、肌肉内给药、皮下给药、经皮给药、气道给药(气雾剂)、肺部给药、鼻部给药、直肠给药和局部给药(包括口腔含化给药和舌下给药)。给药频率可以是每天、每周、每两周、每三周、每个月、每两个月、每季度、每半年或每年1次或多次。The drug may be administered to a subject by any suitable route known in the art, including but not limited to oral or parenteral routes, such as intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, nasal administration, rectal administration, and topical administration (including oral administration and sublingual administration). The administration frequency may be once or more every day, every week, every two weeks, every three weeks, every month, every two months, every quarter, every six months, or every year.
本公开所述的siRNA、药物组合物或siRNA缀合物的使用剂量可为本领域常规的剂量，所述剂量可以根据各种参数、尤其是受试者的年龄、体重和性别来确定。可在细胞培养或实验动物中通过标准药学程序测定毒性和疗效，例如测定LD50(使50％的群体死亡的致死剂量)和ED50(在量反应中指能引起50％最大反应强度的剂量，在质反应中指引起50％实验对象出现阳性反应时的剂量)。可基于由细胞培养分析和动物研究得到的数据得出人用剂量的范围。The dosage of the siRNA, pharmaceutical composition or siRNA conjugate disclosed in the present invention can be a conventional dosage in the art, which can be determined according to various parameters, especially the age, weight and sex of the subject. Toxicity and efficacy can be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as determiningLD50 (lethal dose that causes 50% of the population to die) andED50 (in quantitative response, it refers to the dose that can cause 50% of the maximum response intensity, and in qualitative response, it refers to the dose that causes 50% of the experimental subjects to have a positive reaction). The range of human dosage can be derived based on the data obtained from cell culture analysis and animal studies.
在给予本公开所述的siRNA、药物组合物、和/或siRNA缀合物时，例如，对于雄性或雌性、6-12周龄、体重18-25g的C57BL/6J或30-45g的ob/ob小鼠，以siRNA的量计：(i)对于siRNA缀合物，其siRNA用量可以为0.001-100mg/kg体重，在一些实施方式中为0.01-50mg/kg体重，在一些实施方式中为0.05-20mg/kg体重，在一些实施方式中为0.1-15mg/kg体重，另一些实施方式中为0.1-10mg/kg体重；(ii)对于siRNA与药学上可接受的载体形成的药物组合物，其siRNA用量可以为0.001-50mg/kg体重，在一些实施方式中为0.01-10mg/kg体重，在一些实施方式中为0.05-5mg/kg体重，在一些实施方式中为0.1-3mg/kg体重。When administering the siRNA, pharmaceutical composition, and/or siRNA conjugate described in the present disclosure, for example, for male or female, 6-12 week old, 18-25 g body weight C57BL/6J or 30-45 g ob/ob mice, based on the amount of siRNA: (i) for siRNA conjugates, the amount of siRNA can be 0.001-100 mg/kg body weight, in some embodiments, 0.01-50 mg/kg body weight, in some embodiments, 0.05-20 mg/kg body weight, in some embodiments, 0.1-15 mg/kg body weight, and in other embodiments, 0.1-10 mg/kg body weight; (ii) for pharmaceutical compositions formed by siRNA and a pharmaceutically acceptable carrier, the amount of siRNA can be 0.001-50 mg/kg body weight, in some embodiments, 0.01-10 mg/kg body weight, in some embodiments, 0.05-5 mg/kg body weight, and in some embodiments, 0.1-3 mg/kg body weight.
在一些实施方式中，本公开提供了一种抑制肝细胞中FXII基因表达的方法，该方法包括将有效量的本公开的siRNA和/或药物组合物和/或siRNA缀合物与所述肝细胞接触，将本公开的siRNA和/或药物组合物和/或siRNA缀合物导入所述肝细胞，通过RNA干扰的机制达到抑制肝细胞中FXII基因表达的目的。所述肝细胞可以选自SMMC-7721、HepG2、Huh7等肝癌细胞系或分离的肝原代细胞。在一些实施方式中，所述细胞为人肝原代细胞。In some embodiments, the present disclosure provides a method for inhibiting FXII gene expression in hepatocytes, the method comprising contacting an effective amount of siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure with the hepatocytes, introducing the siRNA and/or pharmaceutical composition and/or siRNA conjugate of the present disclosure into the hepatocytes, and achieving the purpose of inhibiting FXII gene expression in hepatocytes through the mechanism of RNA interference. The hepatocytes can be selected from liver cancer cell lines such as SMMC-7721, HepG2, Huh7, or isolated primary liver cells. In some embodiments, the cells are primary human liver cells.
采用本公开提供的方法抑制FXII基因在细胞中表达，所提供的修饰的siRNA、药物组合物和/或siRNA缀合物中的siRNA用量一般是这样的量：其足以减少靶基因的表达，并导致在靶细胞表面处1pM至1μM、或0.01nM至100nM、或0.05nM至50nM或0.05nM至约5nM的细胞外浓度。达到该局部浓度所需的量将随各种因素而变化，所述因素包括递送方法、递送部位、在递送部位和靶细胞或组织之间的细胞层的数目、递送途径(局部还是全身)等。在递送部位处的浓度可以显著高于在靶细胞或组织的表面处的浓度。The method provided by the present disclosure is used to inhibit the expression of FXII gene in cells. The amount of siRNA used in the modified siRNA, pharmaceutical composition and/or siRNA conjugate provided is generally such an amount: it is sufficient to reduce the expression of the target gene and result in an extracellular concentration of 1pM to 1μM, or 0.01nM to 100nM, or 0.05nM to 50nM or 0.05nM to about 5nM at the surface of the target cell. The amount required to reach this local concentration will vary with various factors, including the delivery method, delivery site, the number of cell layers between the delivery site and the target cell or tissue, the delivery route (local or systemic), etc. The concentration at the delivery site can be significantly higher than the concentration at the surface of the target cell or tissue.
试剂盒Reagent test kit
本公开提供了一种试剂盒，所述试剂盒包含有效量的本公开的修饰的siRNA、药物组合物和siRNA缀合物的至少一种。The present disclosure provides a kit comprising an effective amount of at least one of the modified siRNA, pharmaceutical composition and siRNA conjugate of the present disclosure.
在一些实施方式中，本文所述的试剂盒可在一个容器中提供修饰的siRNA。在一些实施方式中，本文所述的试剂盒可包含一个提供药学上可接受的赋形剂的容器。在一些实施方式中，所述试剂盒中还可包含其它成分，如稳定剂或防腐剂等。在一些实施方式中，本文所述的试剂盒可在不同于提供本文所述修饰的siRNA的容器以外的其它容器中包含至少一种其它治疗剂。在一些实施方式中，所述试剂盒可包含用于将修饰的siRNA与药学上可接受的载体和/或辅料或其它成分(若有的话)进行混合的说明书。In some embodiments, the kit described herein may provide the modified siRNA in one container. In some embodiments, the kit described herein may include a container providing a pharmaceutically acceptable excipient. In some embodiments, the kit may also include other ingredients, such as stabilizers or preservatives. In some embodiments, the kit described herein may include at least one other therapeutic agent in a container other than the container providing the modified siRNA described herein. In some embodiments, the kit may include instructions for mixing the modified siRNA with a pharmaceutically acceptable carrier and/or excipient or other ingredients (if any).
在本公开的试剂盒中，所述修饰的siRNA和药学上可接受的载体和/或辅料以及所述修饰的siRNA、药物组合物和/或siRNA缀合物和/或缀合物，和/或药学上可接受的辅料可以任何形式提供，例如液体形式、干燥形式或冻干形式。在一些实施方式中，所述修饰的siRNA和药学上可接受的载体和/或辅料以及所述药物组合物和/或缀合物和任选的药学上可接受的辅料基本上纯净和/或无菌。在一些实施方式中，可在本公开的试剂盒中提供无菌水。In the kit of the present disclosure, the modified siRNA and pharmaceutically acceptable carrier and/or adjuvant and the modified siRNA, pharmaceutical composition and/or siRNA conjugate and/or conjugate, and/or pharmaceutically acceptable adjuvant can be provided in any form, such as liquid form, dry form or lyophilized form. In some embodiments, the modified siRNA and pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and/or conjugate and optional pharmaceutically acceptable adjuvant are substantially pure and/or sterile. In some embodiments, sterile water can be provided in the kit of the present disclosure.
下面将通过实施例来进一步说明本公开，但是本公开并不因此而受到任何限制。The present disclosure will be further described below by way of examples, but the present disclosure is not limited thereby.
实施例Example
除非特别说明，以下实施例中所用到的试剂、培养基均为市售商品，所用到的核酸电泳、real-time PCR等操作均参照Molecular Cloning(Cold Spring Harbor LaboratoryPress(1989))所记载的方法进行。Unless otherwise specified, the reagents and culture media used in the following examples are all commercially available products, and the nucleic acid electrophoresis, real-time PCR and other operations used are all performed according to the methods described in Molecular Cloning (Cold Spring Harbor Laboratory Press (1989)).
人肝原代细胞由北京大学分子医学研究所核酸技术实验室提供，使用前在37℃水浴中解冻，随后在I型胶原蛋白包被的玻璃或塑料盖玻片或组织培养皿中接种适当密度的细胞，用含有1×双抗和10％FBS的RPMI 1460培养基培养细胞，于37℃在含5％ CO2/95％空气的培养箱中培养细胞。Human primary liver cells were provided by the Nucleic Acid Technology Laboratory, Institute of Molecular Medicine, Peking University. They were thawed in a 37°C water bath before use, and then seeded at an appropriate density on glass or plastic coverslips or tissue culture dishes coated with type I collagen. The cells were cultured in RPMI 1460 medium containing 1× double antibody and 10% FBS, and the cells were cultured at 37°C in an incubator containing 5% CO2 /95% air.
C57小鼠肝原代细胞由北京大学分子医学研究所核酸技术实验室提供，获取自C57小鼠(购自昭衍公司)。在I型胶原蛋白包被的玻璃或塑料盖玻片或组织培养皿中接种适当密度的细胞，用含有1×双抗和10％FBS的RPMI 1460培养基培养细胞，于37℃在含5％ CO2/95％空气的培养箱中培养细胞15-30min。C57 mouse liver primary cells were provided by the Nucleic Acid Technology Laboratory, Institute of Molecular Medicine, Peking University, and obtained from C57 mice (purchased from Zhaoyan Company). Cells were seeded at an appropriate density on glass or plastic coverslips or tissue culture dishes coated with type I collagen, cultured with RPMI 1460 medium containing 1× double antibody and 10% FBS, and cultured at 37°C in an incubator containing 5% CO2 /95% air for 15-30 minutes.
所使用的C57小鼠为6-8周龄小鼠，购于北京维通利华实验动物技术有限公司。The C57 mice used were 6-8 weeks old and purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.
本公开合成的针对FXII基因的siRNA、siRNA缀合物或作为阴性对照的siRNA、siRNA缀合物转染细胞时，使用LipofectamineTM2000(Invitrogen)作为转染试剂，具体操作参照制造商提供的说明书。When the siRNA or siRNA conjugate synthesized against FXII gene or the siRNA or siRNA conjugate used as negative control is transfected into cells, Lipofectamine™ 2000 (Invitrogen) is used as a transfection reagent. The specific operation is referred to the instructions provided by the manufacturer.
若无其它说明，以下提供的试剂比例均按体积比(v/v)计算。Unless otherwise specified, the reagent ratios provided below are calculated by volume (v/v).
实验数据均以表示，数据分析采用Graphpad prism统计分析软件。The experimental data are Data analysis was performed using Graphpad prism statistical analysis software.
制备例1缀合物1的制备Preparation Example 1 Preparation of Conjugate 1
本制备例中，合成了缀合物1。该缀合物为L-9缀合分子与表3中列出编号为siFXIIa1M1SPsiRNA缀合后形成的缀合物。In this preparation example, conjugate 1 was synthesized. The conjugate was formed by conjugating the L-9 conjugated molecule with the siFXIIa1M1SP siRNA listed in Table 3.
(1-1)L-10化合物的合成(1-1) Synthesis of Compound L-10
按照以下方法，合成了L-10化合物：Compound L-10 was synthesized according to the following method:
(1-1-1)缀合末端段GAL-5的合成(1-1-1) Synthesis of conjugated terminal segment GAL-5
(1-1-1a)GAL-2的合成(1-1-1a) Synthesis of GAL-2
将100.0g GAL-1(N-乙酰-D-半乳糖胺盐酸盐，CAS号：1772-03-8，购自宁波弘翔生化公司，463.8mmol)溶于1000ml无水吡啶，冰水浴下加入540ml乙酸酐(购自Enox公司，5565.6mmol)，室温搅拌反应1.5小时。将反应液倒入10L冰水中，减压抽滤，滤饼用2L冰水洗涤后，加乙腈/甲苯混合溶剂(体积比乙腈:甲苯＝1:1)至完全溶解，蒸干溶剂，得到白色固体产品GAL-2 130.0g。100.0g GAL-1 (N-acetyl-D-galactosamine hydrochloride, CAS No.: 1772-03-8, purchased from Ningbo Hongxiang Biochemical Co., Ltd., 463.8mmol) was dissolved in 1000ml anhydrous pyridine, 540ml acetic anhydride (purchased from Enox, 5565.6mmol) was added under ice-water bath, and stirred at room temperature for 1.5 hours. The reaction solution was poured into 10L ice water, filtered under reduced pressure, the filter cake was washed with 2L ice water, and acetonitrile/toluene mixed solvent (volume ratio of acetonitrile: toluene = 1:1) was added until it was completely dissolved, and the solvent was evaporated to obtain 130.0g of white solid product GAL-2.
(1-1-1b)GAL-3的合成(1-1-1b) Synthesis of GAL-3
将步骤(1-1-1a)中获得的GAL-2(35.1g，90.0mmol)溶解于213ml无水1,2-二氯乙烷中，在冰水浴且氮气保护条件下，加入24.0g TMSOTf(CAS号：27607-77-8，购自麦克林公司，108.0mmol)，室温反应过夜。The GAL-2 (35.1 g, 90.0 mmol) obtained in step (1-1-1a) was dissolved in 213 ml of anhydrous 1,2-dichloroethane. 24.0 g of TMSOTf (CAS No.: 27607-77-8, purchased from MacLean, 108.0 mmol) was added in an ice-water bath under nitrogen protection, and the reaction was carried out at room temperature overnight.
在反应液中加入400ml二氯甲烷稀释，以硅藻土过滤，再加入1L饱和碳酸氢钠水溶液，搅拌均匀，分出有机相，水相用二氯乙烷萃取两次，每次300ml，合并有机相，分别用300ml饱和碳酸氢钠水溶液和300ml饱和食盐水洗涤，分出有机相，无水硫酸钠干燥，减压蒸干溶剂，得到浅黄色粘稠糖稀状产品GAL-3 26.9g。Add 400 ml of dichloromethane to the reaction solution to dilute it, filter it with diatomaceous earth, add 1 L of saturated sodium bicarbonate aqueous solution, stir it evenly, separate the organic phase, extract the aqueous phase twice with dichloroethane, 300 ml each time, combine the organic phases, wash them with 300 ml of saturated sodium bicarbonate aqueous solution and 300 ml of saturated brine, separate the organic phase, dry it over anhydrous sodium sulfate, and evaporate the solvent under reduced pressure to obtain 26.9 g of light yellow viscous syrupy product GAL-3.
(1-1-1c)GAL-4的合成(1-1-1c) Synthesis of GAL-4
将步骤(1-1-1b)中获得的GAL-3(26.9g，81.7mmol)溶于136ml无水1,2-二氯乙烷中，加入干燥的分子筛粉末30g，再加入9.0g 5-己烯-1-醇(CAS号：821-41-0，购自Adamas-beta公司，89.9mmol)，室温下搅拌30分钟，冰浴和氮气保护下加入9.08g TMSOTf(40.9mmol)，室温下搅拌反应过夜。过滤除去分子筛粉末，滤液中加入300ml二氯甲烷稀释，以硅藻土过滤，再加入500ml饱和碳酸氢钠水溶液搅拌10分钟洗涤，分出有机相，水相用300ml二氯乙烷萃取一次，合并有机相并分别用300ml饱和碳酸氢钠水溶液和300ml饱和食盐水洗涤，分出有机相，无水硫酸钠干燥，减压蒸干溶剂，得到黄色糖稀状产品GAL-441.3g，不进行纯化直接进行下一步氧化反应。GAL-3 (26.9 g, 81.7 mmol) obtained in step (1-1-1b) was dissolved in 136 ml of anhydrous 1,2-dichloroethane and dried 30g molecular sieve powder, then add 9.0g 5-hexen-1-ol (CAS No.: 821-41-0, purchased from Adamas-beta, 89.9mmol), stir at room temperature for 30 minutes, add 9.08g TMSOTf (40.9mmol) under ice bath and nitrogen protection, stir and react at room temperature overnight. Filter to remove Molecular sieve powder, add 300ml of dichloromethane to the filtrate to dilute, filter with diatomaceous earth, add 500ml of saturated sodium bicarbonate aqueous solution, stir for 10 minutes to wash, separate the organic phase, extract the aqueous phase once with 300ml of dichloroethane, combine the organic phases and wash them with 300ml of saturated sodium bicarbonate aqueous solution and 300ml of saturated brine respectively, separate the organic phase, dry over anhydrous sodium sulfate, evaporate the solvent under reduced pressure to obtain 1.3g of yellow syrupy product GAL-44, which is directly carried out to the next oxidation reaction without purification.
(1-1-1d)GAL-5的合成(1-1-1d) Synthesis of GAL-5
将按照步骤(1-1-1c)中描述的方法得到的GAL-4(14.9g，34.7mmol，)溶于77ml二氯甲烷和77ml乙腈的混合溶剂中，分别加入103ml去离子水和29.7g高碘酸钠(CAS号：7790-28-5，购自阿拉丁公司，138.8mmol)，冰水浴下搅拌10分钟，加入三氯化钌(CAS号：14898-67-0，购自安耐吉公司，238mg，1.145mmol)，室温反应过夜。反应液加入300ml水稀释搅拌，加饱和碳酸氢钠调pH约为7.5，分出并弃去有机相，水相用二氯甲烷萃取三次，每次200ml，弃去有机相。水相用柠檬酸固体调节pH约为3，用二氯甲烷萃取三次，每次200ml，合并有机相，无水硫酸钠干燥，减压蒸干溶剂，得到白色泡沫状固体产品GAL-5 6.85g。1H NMR(400MHz,DMSO)δ12.01(br,1H),7.83(d,J＝9.2Hz,1H),5.21(d,J＝3.2Hz,1H),4.96(dd,J＝11.2,3.2Hz,1H),4.49(d,J＝8.4 Hz,1H),4.07–3.95(m,3H),3.92–3.85(m,1H),3.74–3.67(m,1H),3.48–3.39(m,1H),2.20(t,J＝6.8 Hz,2H),2.11(s,3H),2.00(s,3H),1.90(s,3H),1.77(s,3H),1.55–1.45(m,4H).GAL-4 (14.9 g, 34.7 mmol) obtained by the method described in step (1-1-1c) was dissolved in a mixed solvent of 77 ml of dichloromethane and 77 ml of acetonitrile, and 103 ml of deionized water and 29.7 g of sodium periodate (CAS No.: 7790-28-5, purchased from Aladdin, 138.8 mmol) were added respectively, stirred for 10 minutes under an ice-water bath, and ruthenium trichloride (CAS No.: 14898-67-0, purchased from Anergy, 238 mg, 1.145 mmol) was added, and the reaction was allowed to proceed overnight at room temperature. The reaction solution was diluted and stirred with 300 ml of water, saturated sodium bicarbonate was added to adjust the pH to about 7.5, the organic phase was separated and discarded, and the aqueous phase was extracted three times with dichloromethane, 200 ml each time, and the organic phase was discarded. The pH of the aqueous phase was adjusted to about 3 with solid citric acid, and extracted three times with dichloromethane, 200 ml each time. The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain 6.85 g of a white foamy solid product GAL-5.1 H NMR (400MHz, DMSO) δ12.01(br,1H),7.83(d,J＝9.2Hz,1H),5.21(d,J＝3.2Hz,1H),4.96(dd,J＝11.2,3.2Hz,1H),4.49(d,J＝8.4Hz,1H),4.07–3.95(m,3H),3. 92–3.85(m,1H),3.74–3.67(m,1H),3.48–3.39(m,1H),2.20(t,J＝6.8 Hz,2H),2.11(s,3H),2.00(s,3H),1.90(s,3H),1.77(s,3H),1.55–1.45(m,4H) .
(1-1-2)L-8的合成：(1-1-2) Synthesis of L-8:
将J-0(9.886g，52.5mmol，商购自阿法埃沙公司)和步骤(1-1-1)中得到的GAL-5(72.819g，162.75mmol，由多批次产物合并而得)溶于525ml二氯甲烷，加入二异丙基乙胺(DIEA，44.782g，346.50mmol)、苯并三唑-1-基-氧基三吡咯烷基鏻六氟磷酸盐/酯(PyBOP，90.158g，173.25mmol)和羟基苯并三唑(HOBt，23.410g，173.25mmol)，室温下反应4h，加入20ml饱和碳酸氢钠和200ml饱和食盐水进行洗涤，水相用二氯甲烷萃取2次，每次100ml，合并有机相，用无水硫酸钠干燥，过滤后减压蒸干溶剂得粗品。纯化使用200-300目正相硅胶，以10wt％三乙胺中和硅胶酸性，1wt‰三乙胺平衡柱子，以二氯甲烷:甲醇＝100:25-100:40梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品L-8 38.8g。1H NMR(400 MHz,DMSO)δ7.84(d,J＝9.0 Hz,3H),7.27–7.23(m,1H),7.13–7.18(m,1H),5.22(d,J＝3.1 Hz,3H),4.97(dd,J＝11.3,3.1 Hz,3H),4.48(d,J＝8.4 Hz,3H),4.09–3.98(m,9H),3.88(dd,J＝19.3,9.3 Hz,3H),3.75–3.66(m,3H),3.44–3.38(m,3H),3.17–3.30(m,4H),3.10–2.97(m,4H),2.35–2.20(m,6H),2.15–2.08(m,9H),2.07–1.98(m,13H),1.94–1.87(m,9H),1.81–1.74(m,9H),1.65–1.42(m,18H).MS m/z：C85H119N7O30，[M+H]+，理论：1477.59，实测：1477.23。J-0 (9.886 g, 52.5 mmol, commercially available from Alfa Aesar) and GAL-5 (72.819 g, 162.75 mmol, obtained by combining multiple batches of products) obtained in step (1-1-1) were dissolved in 525 ml of dichloromethane, and diisopropylethylamine (DIEA, 44.782 g, 346.50 mmol), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate/ester (PyBOP, 90.158 g, 173.25 mmol) and hydroxybenzotriazole (HOBt, 23.410 g, 173.25 mmol) were added. The mixture was reacted at room temperature for 4 h, and 20 ml of saturated sodium bicarbonate and 200 ml of saturated brine were added for washing. The aqueous phase was extracted with dichloromethane twice, 100 ml each time, and the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain a crude product. Purification was performed using 200-300 mesh normal phase silica gel, 10wt% triethylamine was used to neutralize the acidity of the silica gel, 1wt‰ triethylamine was used to balance the column, and dichloromethane: methanol = 100:25-100:40 gradient elution was performed, the product eluate was collected, and the solvent was evaporated under reduced pressure to obtain 38.8 g of pure product L-8.1 H NMR (400 MHz, DMSO) δ7.84 (d, J = 9.0 Hz, 3H), 7.27–7.23 (m, 1H), 7.13–7.18 (m, 1H), 5.22 (d, J = 3.1 Hz, 3H), 4.97 (dd, J = 11.3, 3.1 Hz, 3H), 4.48 (d, J = 8.4 Hz, 3H), 4.09–3.98 (m, 9H), 3.88 (dd, J = 19.3, 9.3 Hz, 3H), 3.75–3.66 (m, 3H), 3.44–3.38 (m, 3H), 3.17–3.30 (m, 4H), 3.10–2.97 (m, 4H), 2.35–2.20 (m, 6H), 2.15–2.08 (m, 9H), 2.07–1.98 (m, 13H), 1.94–1.87 (m, 9H), 1.81–1.74 (m, 9H), 1.65–1.42 (m, 18H). MS m/z: C85 H119 N7 O30 , [M+H]+ , theory: 1477.59, found: 1477.23.
(1-1-3a)A-1的合成(1-1-3a) Synthesis of A-1
将DMTrCl(4,4'-双甲氧基三苯甲基氯，101.65g，300mmol)溶于1000ml无水吡啶中，加入DL-甘油酸钙水合物(28.63g，100mmol)，在45℃反应20h，将反应液过滤，滤饼用200ml DCM淋洗，滤液减压浓缩至干，剩余物用500ml二氯甲烷重新溶解，0.5M三乙胺磷酸盐(pH＝7-8)洗涤2次，每次200ml，水相以二氯甲烷萃取2次，每次200ml，合并有机相，用无水硫酸钠干燥，过滤，减压蒸干溶剂，200-300目正相硅胶柱纯化，以石油醚:乙酸乙酯:二氯甲烷:甲醇＝1:1:1:0.35-1:1:1:0.55梯度洗脱，收集产物洗脱液，减压蒸干溶剂，600ml二氯甲烷重新溶解，以200ml 0.5M三乙胺磷酸盐洗涤1次，水相用200ml二氯甲烷萃取1次，合并有机相，无水硫酸钠干燥，过滤，减压蒸干溶剂，真空油泵减压下过夜，得到白色固体产品A-1 50.7g。1H NMR(400 MHz,DMSO-d6)δ7.46(ddd,J＝6.5,2.3,1.1 Hz,1H),7.40–7.28(m,7H),6.89–6.81(m,4H),4.84(d,J＝5.0 Hz,1H),4.36–4.24(m,1H),4.29(s,6H),3.92(dd,J＝12.4,7.0 Hz,1H),3.67(dd,J＝12.3,7.0 Hz,1H),2.52(q,J＝6.3 Hz,6H),1.03(t,J＝6.3 Hz,9H).MS m/z：C24H23O6，[M-H]-，理论：407.15，实测：406.92。Dissolve DMTrCl (4,4'-bismethoxytrityl chloride, 101.65 g, 300 mmol) in 1000 ml of anhydrous pyridine, add DL-calcium glycerate hydrate (28.63 g, 100 mmol), react at 45 °C for 20 h, filter the reaction solution, and purify the filter cake with 200 ml The residue was washed with DCM, and the filtrate was concentrated to dryness under reduced pressure. The residue was redissolved in 500 ml of dichloromethane, washed twice with 0.5 M triethylamine phosphate (pH = 7-8), 200 ml each time, and the aqueous phase was extracted twice with dichloromethane, 200 ml each time. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The product was purified on a 200-300 mesh normal phase silica gel column by gradient elution with petroleum ether: ethyl acetate: dichloromethane: methanol = 1:1:1:0.35-1:1:1:0.55. The product eluate was collected, and the solvent was evaporated under reduced pressure. The product was redissolved in 600 ml of dichloromethane, washed once with 200 ml of 0.5 M triethylamine phosphate, and the aqueous phase was extracted once with 200 ml of dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The product was left to dryness overnight under reduced pressure with a vacuum oil pump to obtain 50.7 g of a white solid product A-1.1 H NMR (400 MHz, DMSO-d6) δ7.46 (ddd, J＝6.5, 2.3, 1.1 Hz, 1H), 7.40–7.28 (m, 7H), 6.89–6.81 (m, 4H), 4.84 (d, J＝5.0 Hz, 1H), 4.36–4.24 (m, 1H), 4.29 (s, 6H), 3.92 (dd, J＝12.4, 7.0 Hz, 1H), 3.67 (dd, J＝12.3, 7.0 Hz, 1H), 2.52 (q, J＝6.3 Hz, 6H), 1.03 (t, J＝6.3 Hz, 9H). MS m/z: C24 H23 O6 , [MH]- , theory: 407.15, found: 406.92.
(1-1-3b)L-7的合成：(1-1-3b) Synthesis of L-7:
将步骤(1-1-2)中获得的L-8(40g，27.09mmol，由多批次产物合并而得)和步骤(1-1-3a)中获得的A-1(41.418g，81.27mmol)混合，溶于271ml二氯甲烷，加入3-二乙氧基磷酰基-1,2,3-苯唑4(3H)-酮(DEPBT)(24.318g，81.37mmol)，再加入二异丙基乙胺(21.007g，162.54mmol)，25℃下搅拌反应1.5h，用800ml饱和碳酸氢钠洗涤有机相，水相以二氯甲烷萃取3次，每次50ml，以150ml饱和食盐水洗涤有机相，水相以50ml二氯甲烷萃取1次，合并有机相并以无水硫酸钠干燥，过滤后减压蒸干溶剂，真空油泵发泡干燥过夜，得到粗品。柱纯化使用2kg 200-300目正相硅胶，以200ml三乙胺中和硅胶酸性，以含1wt％三乙胺的石油醚平衡柱子，以石油醚:乙酸乙酯:二氯甲烷:N,N-二甲基甲酰胺＝1:1:1:0.5-1:1:1:0.6梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品L-7 40.4g。1H NMR(400MHz,DMSO)δ7.90–7.78(m,4H),7.75–7.64(m,1H),7.38–7.18(m,9H),6.91–6.83(m,4H),5.25–5.10(m,4H),4.97(dd,J＝11.2,3.2Hz,3H),4.48–4.30(m,4H),4.02(s,9H),3.93–3.84(m,3H),3.76–3.66(m,9H),3.45–3.35(m,3H),3.24–2.98(m,10H),2.30–2.20(m,2H),2.11–1.88(m,31H),1.80–1.40(m,28H).MS m/z：C90H128N7O35，[M-DMTr]+，理论：1564.65，实测：1564.88。L-8 (40 g, 27.09 mmol, obtained by combining multiple batches of products) obtained in step (1-1-2) and A-1 (41.418 g, 81.27 mmol) obtained in step (1-1-3a) were mixed, dissolved in 271 ml of dichloromethane, 3-diethoxyphosphoryl-1,2,3-benzoxazol-4(3H)-one (DEPBT) (24.318 g, 81.37 mmol) was added, and then diisopropylethylamine (21.007 g, 162.54 mmol) was added. The reaction was stirred at 25°C for 1.5 h, and the organic phase was washed with 800 ml of saturated sodium bicarbonate. The aqueous phase was extracted with dichloromethane 3 times, 50 ml each time, and the organic phase was washed with 150 ml of saturated brine. The aqueous phase was extracted once with 50 ml of dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. After filtering, the solvent was evaporated under reduced pressure and dried overnight with a vacuum oil pump to obtain a crude product. Column purification was performed using 2 kg of 200-300 mesh normal phase silica gel. The acidity of the silica gel was neutralized with 200 ml of triethylamine. The column was equilibrated with petroleum ether containing 1 wt% of triethylamine. The column was eluted with a gradient of petroleum ether: ethyl acetate: dichloromethane: N,N-dimethylformamide = 1:1:1:0.5-1:1:1:0.6. The product eluate was collected and the solvent was evaporated under reduced pressure to obtain 40.4 g of pure L-7.1 H NMR (400MHz, DMSO) δ7.90–7.78(m,4H),7.75–7.64(m,1H),7.38–7.18(m,9H),6.91–6.83(m,4H),5.25–5.10(m,4H),4.97(dd,J＝11.2,3.2Hz,3H),4.4 8–4.30(m,4H),4.02(s,9H),3.93–3.84(m,3H),3.76–3.66(m,9H),3.45–3.35(m,3H),3.24–2.98(m,10H),2.30–2.20(m,2H),2.11–1.88(m,31H) ,1.80–1.40(m,28H).MS m/z: C90 H128 N7 O35 , [M-DMTr]+ , theory: 1564.65, found: 1564.88.
(1-1-4)L-9的合成：(1-1-4) Synthesis of L-9:
将步骤(1-1-3b)中获得的L-7(40g，21.4247mmol)、丁二酸酐(4.288g，42.8494mmol)和4-二甲氨基吡啶(DMAP，5.235g，42.8494mmol)混合溶于215ml二氯甲烷，再加入二异丙基乙胺(DIEA，13.845g，107.1235mmol)，25℃下搅拌24h，800ml 0.5M三乙胺磷酸盐洗涤反应液，水相以二氯甲烷萃取3次，每次5ml，合并有机相减压蒸干得到粗品。柱纯化使用1kg200-300目正相硅胶，以1wt％三乙胺中和硅胶酸性，以二氯甲烷平衡柱子，以含1wt‰三乙胺的二氯甲烷:甲醇＝100:18-100:20梯度洗脱，收集产物洗脱液，减压蒸干溶剂得到纯品L-9缀合分子31.0g。1H NMR(400MHz,DMSO)δ8.58(d,J＝4.2Hz,1H),7.94–7.82(m,3H),7.41–7.29(m,5H),7.22(d,J＝8.1Hz,5H),6.89(d,J＝8.3Hz,4H),5.49–5.37(m,1H),5.21(d,J＝3.0Hz,3H),4.97(d,J＝11.1Hz,3H),4.49(d,J＝8.2Hz,3H),4.02(s,9H),3.88(dd,J＝19.4,9.4Hz,3H),3.77–3.65(m,9H),3.50–3.39(m,6H),3.11–2.90(m,5H),2.61–2.54(m,4H),2.47–2.41(m,2H),2.26–2.17(m,2H),2.15–1.95(m,22H),1.92–1.84(m,9H),1.80–1.70(m,10H),1.65–1.35(m,17H),1.31–1.19(m,4H),0.96(t,J＝7.1Hz,9H).MS m/z：C94H132N7O38，[M-DMTr]+，理论：1664.72，实测：1665.03。The L-7 (40 g, 21.4247 mmol) obtained in step (1-1-3b), succinic anhydride (4.288 g, 42.8494 mmol) and 4-dimethylaminopyridine (DMAP, 5.235 g, 42.8494 mmol) were mixed and dissolved in 215 ml of dichloromethane, and diisopropylethylamine (DIEA, 13.845 g, 107.1235 mmol) was added. The mixture was stirred at 25°C for 24 h, and the reaction solution was washed with 800 ml of 0.5 M triethylamine phosphate. The aqueous phase was extracted with dichloromethane 3 times, 5 ml each time, and the organic phases were combined and evaporated to dryness under reduced pressure to obtain a crude product. Column purification used 1 kg 200-300 mesh normal phase silica gel, 1 wt% triethylamine was used to neutralize the acidity of the silica gel, the column was balanced with dichloromethane, and the gradient elution was carried out with dichloromethane containing 1 wt‰ triethylamine: methanol = 100:18-100:20, the product eluate was collected, and the solvent was evaporated under reduced pressure to obtain31.0 g of pure L-9 conjugated molecule. NMR (400MHz, DMSO) δ8.58(d,J＝4.2Hz,1H),7.94–7.82(m,3H),7.41–7.29(m,5H),7.22(d,J＝8.1Hz,5H),6.89(d,J＝8.3Hz,4H),5.49–5.37(m,1H),5.21(d,J＝ 3.0Hz,3H),4.97(d,J＝11.1Hz,3H),4.49(d,J＝8.2Hz,3H),4.02(s,9H),3.88(dd,J＝19.4,9. 4 Hz, 3H), 3.77–3.65 (m, 9H), 3.50–3.39 (m, 6H), 3.11–2.90 (m, 5H), 2.61–2.54 (m, 4H), 2.47–2.41 (m, 2H), 2.26–2.17 (m, 2H), 2.15–1.95 (m, 22H), 1.92–1.84 (m, 9H), 1.80–1.70 (m, 10H), 1.65–1.35 (m, 17H), 1.31–1.19 (m, 4H), 0.96 (t, J=7.1 Hz, 9H). MS m/z: C94 H132 N7 O38 , [M-DMTr]+ , theory: 1664.72, found: 1665.03.
(1-1-5)L-10化合物的合成：(1-1-5) Synthesis of compound L-10:
此步骤中，通过将L-9缀合分子连接至固相载体，制备了L-10化合物。In this step, the L-10 compound was prepared by linking the L-9 conjugate molecule to a solid support.
将步骤(1-1-4)中获得的L-9缀合分子(22.751g，11mmol)、O-苯并三唑-四甲基脲六氟磷酸盐/酯(HBTU，6.257g，16.5mmol)和二异丙基乙胺(DIEA，2.843g，22mmol)混合，溶于900ml乙腈，室温搅拌5分钟，向反应液中加入氨甲基树脂(88g，100-200目，氨基载量400μmol/g，购自南开和成公司)，25℃下进行摇床反应，转速150转/分钟，反应18h后过滤，滤饼以DCM淋洗2次，每次300ml，乙腈淋洗3次，每次300ml，真空油泵干燥18h，随后再按照表2中示出的投料配比加入原料(CapA、CapB、4-二甲氨基吡啶(DMAP)和乙腈)进行盖帽反应。25℃下置于摇床上，转速150转/分钟，反应5h，反应液过滤，滤饼用乙腈淋洗3次，每次300ml，减压蒸发溶剂至干，真空油泵减压下干燥过夜，得到L-10化合物(即，连接固相载体的L-9缀合分子)102g，载量90.8μmol/g。The L-9 conjugated molecule (22.751 g, 11 mmol) obtained in step (1-1-4), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU, 6.257 g, 16.5 mmol) and diisopropylethylamine (DIEA, 2.843 g, 22 mmol) were mixed and dissolved in 900 ml of acetonitrile. The mixture was stirred at room temperature for 5 minutes. Aminomethyl resin (88 g, 100-200 mesh, amino loading 400 μmol/g, purchased from Nankai Hecheng Company) was added to the reaction solution. The reaction was carried out on a shaking table at 25°C at a speed of 150 rpm. After 18 hours of reaction, the filter cake was washed with DCM twice, 300 ml each time, and acetonitrile three times, 300 ml each time. The filter cake was dried with a vacuum oil pump for 18 hours, and then the raw materials (CapA, CapB, 4-dimethylaminopyridine (DMAP) and acetonitrile) were added according to the feed ratio shown in Table 2 for capping reaction. The mixture was placed on a shaker at 25°C at a speed of 150 rpm for 5 h. The reaction solution was filtered and the filter cake was rinsed with acetonitrile three times, 300 ml each time. The solvent was evaporated to dryness under reduced pressure and dried overnight under reduced pressure with a vacuum oil pump to obtain 102 g of L-10 compound (i.e., L-9 conjugated molecule connected to a solid phase carrier) with a loading of 90.8 μmol/g.
表2盖帽反应投料配比Table 2 Capping reaction feed ratio
其中，CapA和CapB为盖帽试剂溶液，CapA为20体积％N-甲基咪唑的吡啶/乙腈混合溶液，吡啶与乙腈的体积比为3:5；CapB为20体积％乙酸酐的乙腈溶液。CapA and CapB are capping reagent solutions, CapA is a 20 volume % N-methylimidazole mixed solution in pyridine/acetonitrile, with a volume ratio of pyridine to acetonitrile of 3:5; CapB is a 20 volume % acetic anhydride solution in acetonitrile.
(1-2)合成缀合物1的正义链(1-2) Synthesis of the positive chain of conjugate 1
通过固相亚磷酰胺法，利用上述步骤制备的L-10化合物起始循环，按照正义链核苷酸排布顺序自3'-5'方向逐一连接核苷单体。每连接一个核苷单体都包括脱保护、偶联、盖帽、氧化或硫化四步反应。其中，两个核苷酸之间采用磷酸酯连接时，连接后一个核苷单体时，包括脱保护、偶联、盖帽、氧化四步反应。两个核苷酸之间采用硫代磷酸酯连接时，连接后一个核苷单体时，包括保护、偶联、盖帽、硫化四步反应。合成条件给定如下：By solid phase phosphoramidite method, the L-10 compound prepared in the above steps is used to start the cycle, and the nucleoside monomers are connected one by one from the 3'-5' direction according to the arrangement order of the positive chain nucleotides. Each connection of a nucleoside monomer includes four steps of deprotection, coupling, capping, oxidation or sulfurization. Among them, when phosphate is used to connect two nucleotides, the four steps of deprotection, coupling, capping and oxidation are included when the latter nucleoside monomer is connected. When thiophosphate is used to connect two nucleotides, the four steps of protection, coupling, capping and sulfurization are included when the latter nucleoside monomer is connected. The synthesis conditions are given as follows:
核苷单体以0.1M浓度的乙腈溶液提供，每一步的脱保护反应的条件相同，即温度为25℃，反应时间为70秒，脱保护试剂为二氯乙酸的二氯甲烷溶液(3％v/v)，二氯乙酸与固相载体上4,4'-二甲氧基三苯甲基保护基的摩尔比为5:1。The nucleoside monomer is provided in a 0.1 M acetonitrile solution. The conditions of the deprotection reaction in each step are the same, namely, the temperature is 25° C., the reaction time is 70 seconds, the deprotection reagent is a dichloroacetic acid dichloromethane solution (3% v/v), and the molar ratio of dichloroacetic acid to the 4,4'-dimethoxytrityl protecting group on the solid phase carrier is 5:1.
每一步偶联反应条件均相同，包括温度为25℃，固相载体上连接的核酸序列与核苷单体的摩尔比为1:10，固相载体上连接的核酸序列和偶联试剂的摩尔比为1:65，反应时间为600秒，偶联试剂为5-乙硫基-1H-四氮唑(5-(Ethylthio)-1H-tetrazole，ETT)的0.5M乙腈溶液。The coupling reaction conditions for each step were the same, including a temperature of 25° C., a molar ratio of the nucleic acid sequence connected to the solid phase support to the nucleoside monomer of 1:10, a molar ratio of the nucleic acid sequence connected to the solid phase support to the coupling reagent of 1:65, a reaction time of 600 seconds, and a coupling reagent of 0.5 M acetonitrile solution of 5-(Ethylthio)-1H-tetrazole (ETT).
每一步盖帽条件均相同，包括温度为25℃，反应时间为15秒。盖帽试剂溶液为摩尔比为1:1的CapA和CapB的混合溶液，盖帽试剂与固相载体上连接的核酸序列的摩尔比为乙酸酐:N-甲基咪唑:固相载体上连接的核酸序列＝1:1:1。The capping conditions in each step were the same, including a temperature of 25°C and a reaction time of 15 seconds. The capping reagent solution was a mixed solution of CapA and CapB in a molar ratio of 1:1, and the molar ratio of the capping reagent to the nucleic acid sequence connected to the solid phase carrier was acetic anhydride: N-methylimidazole: nucleic acid sequence connected to the solid phase carrier = 1:1:1.
每一步氧化反应条件相同，包括温度为25℃，反应时间为15秒，氧化试剂为浓度为0.05M的碘水。碘与偶联步骤中固相载体上连接的核酸序列的摩尔比为30:1。反应在四氢呋喃:水:吡啶＝3:1:1的混合溶剂中进行。The oxidation reaction conditions in each step were the same, including a temperature of 25°C, a reaction time of 15 seconds, and an oxidizing agent of 0.05 M iodine water. The molar ratio of iodine to the nucleic acid sequence connected to the solid phase support in the coupling step was 30:1. The reaction was carried out in a mixed solvent of tetrahydrofuran: water: pyridine = 3:1:1.
每一步硫化反应的条件相同，包括温度为25℃，反应时间为300秒，硫化试剂为氢化黄原素。硫化试剂与偶联步骤中固相载体上连接的核酸序列的摩尔比为120:1。反应在乙腈:吡啶＝1:1的混合溶剂中进行。The conditions of each step of the sulfurization reaction are the same, including a temperature of 25°C, a reaction time of 300 seconds, and a sulfurization reagent of hydrogenated xanthan. The molar ratio of the sulfurization reagent to the nucleic acid sequence connected to the solid phase support in the coupling step is 120:1. The reaction is carried out in a mixed solvent of acetonitrile:pyridine=1:1.
切割和脱保护条件如下：将合成的连接有载体的核苷酸序列加入浓度为25wt％的氨水中，氨水用量为0.5ml/μmol，在55℃反应16h，除去液体，将残余物真空浓缩至干。The cleavage and deprotection conditions are as follows: add the synthesized nucleotide sequence connected to the carrier into 25 wt% ammonia water, the amount of ammonia water used is 0.5 ml/μmol, react at 55° C. for 16 h, remove the liquid, and vacuum concentrate the residue to dryness.
纯化与脱盐：利用制备型离子色谱纯化柱(Source 15Q)，通过NaCl的梯度洗脱，实现核酸的纯化。具体而言为：洗脱剂A：20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9:1(体积比)；洗脱剂B：1.5M氯化钠，20mM磷酸钠(pH 8.1)，溶剂为水/乙腈＝9:1(体积比)；洗脱梯度：洗脱剂A:洗脱剂B＝100:0-50:50梯度洗脱。收集产品洗脱液后合并，采用反相色谱纯化柱进行脱盐，具体条件包括采用葡聚糖凝胶柱进行脱盐，填料为葡聚糖凝胶G25(SephadexG25)，以去离子水洗脱。Purification and desalting: Use a preparative ion chromatography purification column (Source 15Q) to purify nucleic acids through gradient elution with NaCl. Specifically: eluent A: 20mM sodium phosphate (pH 8.1), solvent water/acetonitrile = 9:1 (volume ratio); eluent B: 1.5M sodium chloride, 20mM sodium phosphate (pH 8.1), solvent water/acetonitrile = 9:1 (volume ratio); elution gradient: eluent A: eluent B = 100:0-50:50 gradient elution. The product eluates were collected and combined, and desalted using a reverse phase chromatography purification column. The specific conditions included using a dextran gel column for desalting, the filler was dextran gel G25 (SephadexG25), and eluted with deionized water.
检测：使用离子交换色谱(IEX-HPLC)检测纯度，使用液质联用(LC-MS)分析分子量。实测值与理论值相符，表明所合成的是3'末端缀合了L-9缀合分子的正义链S。Detection: Purity was detected by ion exchange chromatography (IEX-HPLC), and molecular weight was analyzed by liquid chromatography-mass spectrometry (LC-MS). The measured value was consistent with the theoretical value, indicating that the synthesized product was the positive chain S with the L-9 conjugated molecule conjugated to the 3' end.
(1-3)合成缀合物1的反义链(1-3) Synthesis of the antisense strand of conjugate 1
通过固相亚磷酰胺法，利用通用固相载体(UnyLinkerTMloadedHLSolid Supports，Kinovate Life Sciences公司)起始循环，合成缀合物1的反义链AS。固相合成方法中的脱保护、偶联、盖帽、氧化或硫化反应条件，切割和脱保护，纯化与脱盐条件与合成正义链相同。Through the solid phase phosphoramidite method, using a universal solid phase carrier (UnyLinkerTM loaded HLSolid Supports, Kinovate Life Sciences) were used as the starting cycle to synthesize the antisense chain AS of conjugate 1. The deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection, purification and desalting conditions in the solid phase synthesis method were the same as those for synthesizing the sense chain.
当目标序列中在反义链5'-末端第一个核苷酸处具有5-P修饰时，按照固相亚磷酰胺法制备反义链过程中，在连接反义链最后一个核苷单体后，再经脱保护、偶联、盖帽、氧化四步反应将CPR-I单体(苏州吉玛，货号Cat#13-2601-XX)连接至反义链5'末端，形成5'-磷酸酯修饰。When the target sequence has a 5-P modification at the first nucleotide at the 5'-end of the antisense chain, in the process of preparing the antisense chain according to the solid phase phosphoramidite method, after connecting the last nucleoside monomer of the antisense chain, the CPR-I monomer (Suzhou Jima, Cat#13-2601-XX) is connected to the 5' end of the antisense chain through four steps of deprotection, coupling, capping, and oxidation to form a 5'-phosphate modification.
该连接中，使用的通用固相载体，脱保护、偶联、盖帽、氧化或硫化反应条件，切割和脱保护，纯化与脱盐条件与合成正义链相同。In this connection, the universal solid phase support, deprotection, coupling, capping, oxidation or sulfurization reaction conditions, cleavage and deprotection, purification and desalting conditions used are the same as those for synthesizing the sense chain.
检测：采用离子交换色谱(IEX-HPLC)检测纯度；采用液质联用(LC-MS)分析所得到的产物的分子量。其结果，实测值与理论值相符，表明所合成的是具有目标序列的反义链AS。Detection: Ion exchange chromatography (IEX-HPLC) was used to detect purity; liquid chromatography-mass spectrometry (LC-MS) was used to analyze the molecular weight of the obtained product. As a result, the measured value was consistent with the theoretical value, indicating that the synthesized product was the antisense chain AS with the target sequence.
(1-4)合成缀合物1(1-4) Synthesis of Conjugate 1
对于缀合物1，将S链与AS链分别溶于注射用水中，得到40mg/mL的溶液，以等摩尔比混合，50℃加热15min，室温冷却后，得到退火后的产品，冻干，得到冻干粉。使用超纯水(Milli-Q超纯水仪，电阻率18.2MΩ*cm(25℃))将缀合物稀释至浓度为0.2mg/mL后，利用液质联用仪(LC-MS，Liquid Chromatography-Mass Spectrometry，购于Waters公司，型号：LCT Premier)进行分子量检测。实测值与理论值一致，说明所合成的缀合物1是目标设计的带有L-9缀合分子的双链核酸序列。其结构如式(403)所示。For conjugate 1, the S chain and the AS chain were dissolved in water for injection respectively to obtain a 40 mg/mL solution, mixed in an equimolar ratio, heated at 50°C for 15 min, cooled at room temperature, and then the annealed product was obtained, which was lyophilized to obtain a lyophilized powder. The conjugate was diluted to a concentration of 0.2 mg/mL using ultrapure water (Milli-Q ultrapure water instrument, resistivity 18.2 MΩ*cm (25°C)), and then the molecular weight was detected using a liquid chromatography-mass spectrometer (LC-MS, Liquid Chromatography-Mass Spectrometry, purchased from Waters, model: LCT Premier). The measured value was consistent with the theoretical value, indicating that the synthesized conjugate 1 was the target designed double-stranded nucleic acid sequence with an L-9 conjugated molecule. Its structure is shown in formula (403).
制备例2缀合物2-6的制备Preparation Example 2 Preparation of Conjugate 2-6
采用与制备例1相同的方法，分别合成了缀合物2、缀合物3、缀合物4、缀合物5和缀合物6，不同的是，所述缀合物中的siRNA分别具有表3中所示的对应于缀合物2、缀合物3、缀合物4、缀合物5和缀合物6的正义链和反义链，因此为了合成这些缀合物，在制备正义链与反义链时，分别按照表3中所示siRNA的正义链与反义链序列，合成相应的正义链与反义链。利用LC-MS分别检测得到的缀合物2-6的分子量，实测值与理论值一致，说明所合成的缀合物是目标设计的带有L-9缀合分子的双链核酸序列。缀合物2-6的结构如式(403)所示。The same method as in Preparation Example 1 was used to synthesize conjugates 2, 3, 4, 5 and 6, respectively. The difference was that the siRNA in the conjugates had the sense strand and antisense strand corresponding to conjugates 2, 3, 4, 5 and 6 shown in Table 3, respectively. Therefore, in order to synthesize these conjugates, when preparing the sense strand and antisense strand, the corresponding sense strand and antisense strand were synthesized according to the sense strand and antisense strand sequences of the siRNA shown in Table 3. The molecular weights of the obtained conjugates 2-6 were detected by LC-MS, and the measured values were consistent with the theoretical values, indicating that the synthesized conjugates were the double-stranded nucleic acid sequences with L-9 conjugated molecules of the target design. The structure of conjugate 2-6 is shown in formula (403).
表3siRNA缀合物Table 3 siRNA conjugates
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母s左右两个核苷酸之间为硫代磷酸酯基连接；大写字母P表示该字母P右侧相邻的一个核苷酸为5'-磷酸核苷酸修饰的核苷酸。Among them, capital letters C, G, U, and A represent the base composition of the nucleotide; lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; lowercase letter s indicates that the two nucleotides on the left and right of the letter s are connected by a thiophosphate group; capital letter P indicates that the nucleotide adjacent to the right of the letter P is a 5'-phosphate-modified nucleotide.
上述缀合物中，缀合物3与缀合物1中的siRNA之间，正义链和反义链分别包含一个核苷酸碱基差异(5'-3'方向，正义链第2个核苷酸处G-A差异、反义链第18个核苷酸处C-U差异)，缀合物1的siRNA反义链与人FXIImRNA完全反向互补，而缀合物3的siRNA反义链与小鼠FXIImRNA完全反向互补。其余缀合物2、4、5和6的siRNA反义链与人FXIImRNA和小鼠FXIImRNA均完全反向互补。Among the above conjugates, the sense strand and antisense strand of the siRNA in conjugate 3 and conjugate 1 each contain one nucleotide base difference (5'-3' direction, G-A difference at the second nucleotide of the sense strand, C-U difference at the 18th nucleotide of the antisense strand), the siRNA antisense strand of conjugate 1 is completely reverse complementary to human FXII mRNA, and the siRNA antisense strand of conjugate 3 is completely reverse complementary to mouse FXII mRNA. The siRNA antisense strands of the remaining conjugates 2, 4, 5 and 6 are completely reverse complementary to both human FXII mRNA and mouse FXII mRNA.
制备例3合成siRNA序列Preparation Example 3 Synthetic siRNA sequence
通过固相合成方法得到表4中所列的siRNA正义链和反义链，使用DEPC水溶解等摩尔的正义链和反义链混合物，随后退火以形成siRNA双链，冻干，以冻干粉形式获得表4中所列的siRNA。The siRNA sense and antisense chains listed in Table 4 were obtained by solid phase synthesis. Equimolar mixtures of the sense and antisense chains were dissolved in DEPC water, followed by annealing to form siRNA double strands, and lyophilized to obtain the siRNA listed in Table 4 in the form of lyophilized powder.
表4siRNA序列Table 4 siRNA sequences
其中，大写字母C、G、U、A表示核苷酸的碱基组成；小写字母m表示该字母m左侧相邻的一个核苷酸为甲氧基修饰的核苷酸；小写字母f表示该字母f左侧相邻的一个核苷酸为氟代修饰的核苷酸；小写字母s表示该字母s左右两个核苷酸之间为硫代磷酸酯基连接；dT表示胸腺嘧啶脱氧核苷酸。Among them, capital letters C, G, U, and A represent the base composition of the nucleotide; lowercase letter m indicates that the nucleotide adjacent to the left of the letter m is a methoxy-modified nucleotide; lowercase letter f indicates that the nucleotide adjacent to the left of the letter f is a fluorine-modified nucleotide; lowercase letter s indicates that the two nucleotides on the left and right of the letter s are connected by a thiophosphate group; dT represents thymidine deoxynucleotide.
上述序列的制备过程中，当目标序列中包含未修饰的核苷酸时，在切割与脱保护条件中，在氨水处理后，相对于单链核酸的量，用0.4ml/μmol N-甲基吡咯烷酮溶解产品，随后加入0.3ml/μmol三乙胺和0.6ml/μmol三乙胺三氢氟酸盐，以脱除核糖上的2'-TBDMS保护。In the preparation process of the above sequence, when the target sequence contains unmodified nucleotides, in the cleavage and deprotection conditions, after ammonia treatment, the product is dissolved with 0.4 ml/μmol N-methylpyrrolidone relative to the amount of single-stranded nucleic acid, and then 0.3 ml/μmol triethylamine and 0.6 ml/μmol triethylamine trihydrofluoride are added to remove the 2'-TBDMS protection on the ribose.
实验例4siRNA在人肝原代细胞中对FXII mRNA表达量的抑制效率检测。Experimental Example 4 Detection of the inhibitory efficiency of siRNA on FXII mRNA expression in human primary liver cells.
按照供应商提供的说明书，使用LipofectamineTM2000将待测siRNA(siRNA7、8、10、11和12以及对比序列NC的各溶液，其中NC是与FXII mRNA没有明显序列相关性的阴性对照序列，所述溶液是指实验前用DEPC化水将siRNA分别按所需浓度重新溶解而形成的水溶液)分别转染至人肝原代细胞中，每一siRNA终浓度分别为50nM，每个浓度2个复孔。According tothe instructions provided by the supplier, the siRNAs to be tested (solutions of siRNA 7, 8, 10, 11 and 12 and the comparison sequence NC, wherein NC is a negative control sequence with no obvious sequence correlation with FXII mRNA, and the solution refers to the aqueous solution formed by re-dissolving the siRNAs at the required concentrations in DEPC-treated water before the experiment) were transfected into human primary liver cells using Lipofectamine TM 2000. The final concentration of each siRNA was 50 nM, and each concentration was replicated in 2 wells.
通过实时荧光定量PCR(Quantitative Real-Time PCR)分别检测转染了各siRNA的人肝原代细胞中FXII mRNA的表达量。具体步骤为：培养转染的细胞24小时后，使用Trizol(Thermo Fisher公司)根据总RNA提取的标准操作步骤提取细胞中的总RNA；分别取1μg总RNA，使用反转录试剂盒(Promega公司，货号A3500)按其说明书的操作方法反转录得到cDNA。使用2×Ultra SYBR Mixture(with ROX)(北京康为世纪生物科技有限公司，货号CW0956)试剂盒，以cDNA为模板按照说明书的步骤进行FXII mRNA表达量的检测。其中，用于扩增FXII和作为内参基因的GAPDH的PCR引物如表5所示。The expression of FXII mRNA in human liver primary cells transfected with each siRNA was detected by real-time fluorescence quantitative PCR (Quantitative Real-Time PCR). The specific steps are: after culturing the transfected cells for 24 hours, the total RNA in the cells was extracted using Trizol (Thermo Fisher) according to the standard operating procedures for total RNA extraction; 1 μg of total RNA was taken respectively, and the reverse transcription kit (Promega, Catalog No. A3500) was used to reverse transcribe cDNA according to the operating method of its instructions. The 2×Ultra SYBR Mixture (with ROX) (Beijing Kangwei Century Biotechnology Co., Ltd., Catalog No. CW0956) kit was used to detect the expression of FXII mRNA using cDNA as a template according to the steps of the instructions. Among them, the PCR primers used to amplify FXII and GAPDH as an internal reference gene are shown in Table 5.
表5引物信息Table 5 Primer information
FXII mRNA表达量按如下等式计算：FXII mRNA表达量＝(测试组FXII mRNA的表达量/测试组GAPDH mRNA的表达量)/(对照组FXII mRNA的表达量/对照组GAPDH mRNA的表达量)×100％。The expression level of FXII mRNA was calculated according to the following equation: FXII mRNA expression level = (expression level of FXII mRNA in the test group/expression level of GAPDH mRNA in the test group)/(expression level of FXII mRNA in the control group/expression level of GAPDH mRNA in the control group)×100%.
mRNA抑制率＝(1-FXII mRNA表达量)×100％。其中，各测试组为分别经各浓度siRNA处理的人肝原代细胞，对照组为对比序列NC处理的细胞。结果如表6所示。mRNA inhibition rate = (1-FXII mRNA expression) × 100%. Wherein, each test group is human liver primary cells treated with siRNA of different concentrations, and the control group is cells treated with the comparison sequence NC. The results are shown in Table 6.
表6人肝原代细胞中FXII mRNA的抑制Table 6 Inhibition of FXII mRNA in primary human liver cells
由表6的结果可见，本公开提供的修饰的siRNA在人肝原代细胞中显示出较高的FXIImRNA抑制活性，其中50nM的siRNA12对FXII mRNA表达量的抑制率可达78.70％。From the results in Table 6, it can be seen that the modified siRNA provided by the present disclosure exhibits a high FXII mRNA inhibitory activity in human primary liver cells, wherein the inhibition rate of 50 nM siRNA12 on FXII mRNA expression can reach 78.70%.
实验例5siRNA在C57小鼠肝原代细胞中对FXII mRNA表达量的抑制效率检测。Experimental Example 5 Detection of the inhibitory efficiency of siRNA on FXII mRNA expression in C57 mouse primary liver cells.
按照与实验例4相同的方法进行检测，区别在于，在C57小鼠肝原代细胞中进行检测siRNA7、siRNA8、siRNA10、siRNA11和siRNA12的mRNA抑制率，同时使用表7所示的用于扩增FXII和作为内参基因的GAPDH的PCR引物代替表5所示引物。The detection was performed in the same manner as in Experimental Example 4, except that the mRNA inhibition rates of siRNA7, siRNA8, siRNA10, siRNA11, and siRNA12 were detected in C57 mouse liver primary cells, and the PCR primers for amplifying FXII and GAPDH as an internal reference gene shown in Table 7 were used instead of the primers shown in Table 5.
表7引物信息Table 7 Primer information
各siRNA在小鼠肝原代细胞中的抑制活性如表8所示。The inhibitory activity of each siRNA in mouse liver primary cells is shown in Table 8.
表8C57小鼠肝原代细胞中FXIImRNA的抑制Table 8 Inhibition of FXII mRNA in C57 mouse liver primary cells
由表8的结果可见，本公开提供的siRNA在C57小鼠肝原代细胞中显示出较高的FXIImRNA抑制活性，其中50nM的siRNA12对FXII mRNA表达量的抑制率可达70.09％。From the results in Table 8, it can be seen that the siRNA provided by the present disclosure exhibits high FXII mRNA inhibitory activity in C57 mouse liver primary cells, wherein the inhibition rate of 50 nM siRNA12 on FXII mRNA expression can reach 70.09%.
实验例6siRNA缀合物在C57小鼠体内对FXII mRNA表达量的抑制效率Experimental Example 6 Inhibitory Efficiency of siRNA Conjugates on FXII mRNA Expression in C57 Mice
本实验例中，通过两次实验分别考察了缀合物1-6在C57小鼠体内对肝脏组织中FXII mRNA的抑制率。In this experimental example, two experiments were conducted to investigate the inhibition rate of conjugates 1-6 on FXII mRNA in liver tissue of C57 mice.
(6-A)缀合物1、2和3在C57小鼠体内对肝脏组织中FXII mRNA的抑制率(6-A) Inhibition rate of FXII mRNA in liver tissue of C57 mice by conjugates 1, 2 and 3
将6-8周龄C57小鼠随机分为7组，每组5只，分别向每组小鼠给予缀合物1、2或3(每种缀合物对应2个不同剂量的组)或对比序列NC(1组)的溶液(所述溶液是指，在实验前用1×PBS(pH7.4)缓冲液将siRNA缀合物分别按所需浓度重新溶解而形成的溶液)，以及PBS空白对照组(15只小鼠，给予1×PBS)。所有动物根据体重计算药量，采用皮下注射方式单次给药，siRNA缀合物给药剂量(以siRNA的量计)分别为5mg/kg和1mg/kg两个剂量组，给药体积为10mL/kg，根据给药剂量和给药体积，换算出各siRNA缀合物应配制的药物浓度。6-8 week old C57 mice were randomly divided into 7 groups, 5 mice in each group, and each group of mice was given a solution of conjugate 1, 2 or 3 (each conjugate corresponds to 2 groups with different doses) or comparison sequence NC (1 group) (the solution refers to the solution formed by re-dissolving the siRNA conjugates at the required concentrations with 1×PBS (pH7.4) buffer before the experiment), and a PBS blank control group (15 mice, given 1×PBS). The drug dose of all animals was calculated according to body weight, and a single subcutaneous injection was used for administration. The siRNA conjugate dosage (in terms of siRNA amount) was 5 mg/kg and 1 mg/kg, respectively, and the administration volume was 10 mL/kg. According to the dosage and administration volume, the drug concentration of each siRNA conjugate to be prepared was converted.
给药后7天处死小鼠，收集肝脏，用RNA later(Sigma Aldrich公司)保存；随后用组织匀浆仪匀浆肝组织，再用Trizol(Thermo Fisher公司)根据总RNA提取的标准操作步骤提取得到肝组织总RNA。The mice were killed 7 days after administration, and the livers were collected and preserved with RNA later (Sigma Aldrich). The liver tissue was then homogenized with a tissue homogenizer, and total RNA from the liver tissue was extracted using Trizol (Thermo Fisher) according to the standard operating procedures for total RNA extraction.
采用实时荧光定量PCR检测肝组织中FXII mRNA的表达量，具体地：使用反转录试剂盒(Promega公司，货号A3500)按其说明书的操作方法反转录得到cDNA。使用SYBR SelectMaster Mix(Applied biosystem，货号4472897)试剂盒，以cDNA为模板按照说明书的步骤进行FXII mRNA表达量的检测，并计算siRNA缀合物对FXII mRNA表达量的抑制率。其中，用于扩增FXII和作为内参基因的GAPDH的PCR引物如表7所示。Real-time fluorescence quantitative PCR was used to detect the expression of FXII mRNA in liver tissue. Specifically, a reverse transcription kit (Promega, Cat. No. A3500) was used to reverse transcribe cDNA according to the instructions. The SYBR Select Master Mix (Applied biosystem, Cat. No. 4472897) kit was used to detect the expression of FXII mRNA using cDNA as a template according to the instructions, and the inhibition rate of siRNA conjugate on the expression of FXII mRNA was calculated. Among them, the PCR primers used to amplify FXII and GAPDH as an internal reference gene are shown in Table 7.
FXII mRNA表达量，即剩余表达量，按如下等式计算：FXII mRNA表达量＝(测试组FXII mRNA的表达量/测试组GAPDH mRNA的表达量)/(对照组FXII mRNA的表达量/对照组GAPDH mRNA的表达量)×100％。The FXII mRNA expression level, i.e., the residual expression level, was calculated according to the following equation: FXII mRNA expression level = (FXII mRNA expression level in the test group/GAPDH mRNA expression level in the test group)/(FXII mRNA expression level in the control group/GAPDH mRNA expression level in the control group) × 100%.
siRNA缀合物对FXII mRNA表达量的抑制率为(1-FXII mRNA表达量)×100％。其中，对照组为本实验中施以PBS的对照组小鼠，各测试组为分别施以不同siRNA缀合物的给药组小鼠。The inhibition rate of siRNA conjugate on FXII mRNA expression is (1-FXII mRNA expression)×100%. The control group is the control group mice administered with PBS in this experiment, and each test group is the administration group mice administered with different siRNA conjugates.
图1是给予C57小鼠PBS或不同剂量的缀合物1、2或3后，C57小鼠肝脏组织FXIImRNA表达量(以GAPDH为内参的相对值)的散点图。由图1可以看出，给药后第7天，对比序列NC未显示出任何抑制作用；与此同时，给药量为5mg/kg剂量下的siRNA缀合物2对FXII mRNA表达量的抑制率高达94.9％，1mg/kg剂量下的siRNA缀合物2对FXII mRNA的表达量也显示出74.8％的高抑制率；对于siRNA缀合物3，在1mg/kg剂量下就显示出96.7％的高抑制率，在5mg/kg剂量下对FXII mRNA表达量的抑制率更是高达98.7％；另外，尽管siRNA缀合物1未在小鼠中显示出高的FXIImRNA表达量抑制率，然而考虑到该缀合物所包含的siRNA是对应于人FXIImRNA的siRNA，且与其具有相同靶mRNA位点的siRNA缀合物3显示出出人意料的高抑制活性，可以预期其在人体内也极有可能显示出高的FXIImRNA表达量的抑制率。FIG1 is a scatter plot of FXII mRNA expression in liver tissue of C57 mice (relative value with GAPDH as internal reference) after administration of PBS or different doses of conjugate 1, 2 or 3 to C57 mice. As can be seen from Figure 1, on the 7th day after administration, the control sequence NC did not show any inhibitory effect; at the same time, the inhibition rate of siRNA conjugate 2 at a dose of 5 mg/kg on the expression of FXII mRNA was as high as 94.9%, and the siRNA conjugate 2 at a dose of 1 mg/kg also showed a high inhibition rate of 74.8% on the expression of FXII mRNA; for siRNA conjugate 3, a high inhibition rate of 96.7% was shown at a dose of 1 mg/kg, and an inhibition rate of FXII mRNA expression was as high as 98.7% at a dose of 5 mg/kg; in addition, although siRNA conjugate 1 did not show a high inhibition rate of FXII mRNA expression in mice, considering that the siRNA contained in the conjugate is an siRNA corresponding to human FXII mRNA, and siRNA conjugate 3 having the same target mRNA site as it shows unexpectedly high inhibitory activity, it can be expected that it is very likely to show a high inhibition rate of FXII mRNA expression in humans.
(6-B)缀合物1、3、4、5和6在C57小鼠体内对肝脏组织中FXII mRNA表达量的抑制率(6-B) Inhibition rate of FXII mRNA expression in liver tissue of C57 mice by conjugates 1, 3, 4, 5 and 6
在本实验中，使用与(6-A)相同的实验条件和步骤，对缀合物1、3、4、5和6在C57小鼠体内对肝脏组织中FXII mRNA表达量的抑制率进行了检测，不同之处在于，使用的siRNA缀合物为缀合物1、3、4、5和6，PBS组使用了10只小鼠。In this experiment, the same experimental conditions and procedures as (6-A) were used to detect the inhibitory effect of conjugates 1, 3, 4, 5 and 6 on FXII mRNA expression in liver tissue of C57 mice. The difference was that the siRNA conjugates used were conjugates 1, 3, 4, 5 and 6, and 10 mice were used in the PBS group.
图2是给予C57小鼠PBS或不同剂量的缀合物1、3、4、5和6后，C57小鼠肝脏组织FXIImRNA表达量(以GAPDH为内参的相对值)的散点图。由图2结果可见，缀合物1和缀合物3显示出与(6-A)类似的抑制效果，特别是缀合物3在1mg/kg和5mg/kg剂量下分别显示出95.4％和97.4％的高FXIImRNA表达量抑制率；缀合物4、5和6分别在1mg/kg剂量下显示出54.9％、61.8％和41.2％的FXIImRNA表达量抑制率，在3mg/kg剂量下更是分别显示出83.9％、87.0％和83.8％的高FXIImRNA表达量抑制率。Figure 2 is a scatter plot of FXII mRNA expression in liver tissue of C57 mice (relative value with GAPDH as internal reference) after administration of PBS or different doses of conjugates 1, 3, 4, 5 and 6. As can be seen from the results in Figure 2, conjugates 1 and 3 showed similar inhibitory effects as (6-A), especially conjugate 3 showed high FXII mRNA expression inhibition rates of 95.4% and 97.4% at 1 mg/kg and 5 mg/kg, respectively; conjugates 4, 5 and 6 showed FXII mRNA expression inhibition rates of 54.9%, 61.8% and 41.2% at 1 mg/kg, respectively, and showed high FXII mRNA expression inhibition rates of 83.9%, 87.0% and 83.8% at 3 mg/kg, respectively.
由上述实验例4、5、(6-A)和(6-B)的结果可知，本公开的siRNA和siRNA缀合物在体外人/小鼠肝原代细胞实验中以及小鼠体内实验中均显示出优异的FXIImRNA抑制活性。From the results of Experimental Examples 4, 5, (6-A) and (6-B) above, it can be seen that the siRNA and siRNA conjugates disclosed in the present invention showed excellent FXII mRNA inhibitory activity in both in vitro human/mouse liver primary cell experiments and in vivo mouse experiments.
以上详细描述了本公开的一些实施方式，但是，本公开并不限于上述实施方式中的具体细节，在本公开的技术构思范围内，可以对本公开的技术方案进行多种简单变型，这些简单变型均属于本公开的保护范围。Some embodiments of the present disclosure are described in detail above; however, the present disclosure is not limited to the specific details in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.
另外需要说明的是，在上述一些实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本公开对各种可能的组合方式不再另行说明。It should also be noted that the various specific technical features described in some of the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further explain various possible combinations.
此外，本公开的各种不同的实施方式之间也可以进行任意组合，只要其不违背本公开的思想，其同样应当视为本公开所公开的内容。In addition, various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.
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