US 12,084,661 B2
Nucleic acid, composition and conjugate comprising the same, and preparation method and use thereof
Hongyan Zhang, Suzhou (CN); Shan Gao, Suzhou (CN); and Daiwu Kang, Suzhou (CN)
Assigned to SUZHOU RIBO LIFE SCIENCE CO., LTD., Jiangsu (CN)
Filed by SUZHOU RIBO LIFE SCIENCE CO., LTD., Suzhou (CN)
Filed on Jun. 29, 2022, as Appl. No. 17/852,888.
Application 17/852,888 is a continuation of application No. 16/763,058, granted, now 11,414,665, previously published as PCT/CN2018/118300, filed on Nov. 29, 2018.
Claims priority of application No. 201711249378.5 (CN), filed on Dec. 1, 2017; and application No. 201711478933.1 (CN), filed on Dec. 29, 2017.
Prior Publication US 2022/0389428 A1, Dec. 8, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/11 (2006.01); A61P 31/20 (2006.01); C12N 15/113 (2010.01); A61K 31/713 (2006.01)
CPC C12N 15/1131 (2013.01) [A61P 31/20 (2018.01); A61K 31/713 (2013.01); C12N 2310/14 (2013.01); C12N 2310/315 (2013.01); C12N 2310/321 (2013.01); C12N 2310/322 (2013.01); C12N 2310/344 (2013.01); C12N 2310/351 (2013.01); C12N 2320/32 (2013.01)] 18 Claims
 
1. A siRNA conjugate comprising siRNA and a conjugating group conjugated to the siRNA, and having a structure as shown by Formula (308):

OG Complex Work Unit Chemistry
wherein n1 is an integer of 1-2, and n3 is an integer of 0-[4]1, and n1+n3=2-3;
m1, m2, and m3 independently of one another are an integer of 2-10;
R10, R11, R12, R13, R14 and R15 independently of one another are H, or selected from the group consisting of C1-C10 alkyl, C1-C10 haloalkyl, and C1-C10 alkoxy;
R3 is a group having a structure as shown by Formula (A59):

OG Complex Work Unit Chemistry
wherein E1 is OH, SH or BH2; Nu is siRNA;
R2 is a linear alkylene of 1 to 20 carbon atoms in length, wherein one or more carbon atoms are optionally replaced with one or more groups 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 optionally has 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, —SC1-C10 haloalkyl, halo, —OH, —SH, —NH2, —C1-C10 alkyl-NH2, —N(C1-C10 alkyl)(C1-C10 alkyl), —NH(C1-C10 alkyl), cyano, nitro, —CO2H, —C(O)O(C1-C10 alkyl), —CON(C1-C10 alkyl)(C1-C10 alkyl), —CONH(C1-C10 alkyl), —CONH2, —NHC(O)(C1-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), —SO2NH2, —SO2NH(C1-C10 alkyl), —SO2NH(phenyl), —NHSO2(C1-C10 alkyl), —NHSO2(phenyl), and —NHSO2(C1-C10 haloalkyl);
each L1 is independently a linear alkylene of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally replaced with one or more groups 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 optionally has 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, —SC1-C10 haloalkyl, halo, —OH, —SH, —NH2, —C1-C10 alkyl-NH2, —N(C1-C10 alkyl)(C1-C10 alkyl), —NH(C1-C10 alkyl), cyano, nitro, —CO2H, —C(O)O(C1-C10 alkyl), —CON(C1-C10 alkyl)(C1-C10 alkyl), —CONH(C1-C10 alkyl), —CONH2, —NHC(O)(C1-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), —SO2NH2, —SO2NH(C1-C10 alkyl), —SO2NH(phenyl), —NHSO2(C1-C10 alkyl), —NHSO2(phenyl), and —NHSO2(C1-C10 haloalkyl);
custom character represents a site where a group is linked to the rest of the molecule;
M1 represents a targeting group, wherein each of the targeting groups is independently a ligand that has affinity to asialoglycoprotein receptors (ASGP-R) on a surface of mammalian hepatocytes;
wherein the siRNA comprises a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II;
the nucleotide sequence I and the nucleotide sequence II are at least partly reverse complementary to form a double stranded region; wherein the nucleotide sequence I comprises a nucleotide sequence A, which has the same length as the nucleotide sequence shown in SEQ ID NO: 1 with no more than 3 nucleotide differences; and the nucleotide sequence II comprises a nucleotide sequence B, which has the same length as the nucleotide sequence shown in SEQ ID NO: 2 with no more than 3 nucleotide differences:
 
 (SEQ ID NO: 1)
 
 5′-UGCUAUGCCUCAUCUUCUZ-3′;
 
 
 
 (SEQ ID NO: 2)
 
 5′-Z’AGAAGAUGAGGCAUAGCA-3′;
wherein,
Z is A; Z′ is U; and
the nucleotide sequence A comprises a nucleotide ZA at the position corresponding to Z; the nucleotide sequence B comprises a nucleotide Z′B at the position corresponding to Z′; the nucleotide Z′B is the first nucleotide at 5′ terminal of the antisense strand.