Granted Patent B2

US 12,274,752 · App. 18/301,845 · Granted Apr 15, 2025

Inventors: Hongyan Zhang (Suzhou, CN); Shan Gao (Suzhou, CN); Daiwu Kang (Suzhou, CN)

Assignee: SUZHOU RIBO LIFE SCIENCE CO., LTD.

A61K47/549A61K31/713A61P3/06C07H1/00C12N15/113C12N2310/14C12N2310/315C12N2310/321C12N2310/322C12N2310/344C12N2310/346C12N2310/351

Abstract

Claims (143)

1. A modified siRNA, comprising a sense strand and an antisense strand, wherein each of the nucleotides in the sense strand and in the antisense strand is a modified nucleotide, wherein the sense strand and the antisense strand both comprise fluoro modified nucleotides and non-fluoro modified nucleotides, the sense strand comprises a nucleotide sequence I and the antisense strand comprises a nucleotide sequence II, said nucleotide sequence I and said nucleotide sequence II being at least partly reverse complementary to form a double-strand region, wherein the nucleotide sequence I comprises a nucleotide sequence represented by SEQ ID NO:60, and the nucleotide sequence II comprises a nucleotide sequence represented by SEQ ID NO:61:

5′-CAAUAAAGCUGGACAAGAZ A -3′ (SEQ ID NO: 60);

5′-Z′ B UCUUGUCCAGCUUUAUUG-3′ (SEQ ID NO: 61),

wherein, Z A is selected from A, U, G, or C, and Z′B is the first nucleotide at the 5′ terminal of the antisense strand and complementary to Z A ;

wherein the fluoro modified nucleotides are present in nucleotide sequence 60 and nucleotide sequence 61; in the direction from 5′ terminal to 3′ terminal, the nucleotides at positions 7, 8 and 9 of nucleotide sequence represented by SEQ ID NO:60 are fluoro modified nucleotides and the nucleotides at the other positions in the sense strand are non-fluoro modified nucleotides; and in the direction from 5′ terminal to 3′ terminal, the nucleotides at positions 2, 6, 14 and 16 of nucleotide sequence represented by SEQ ID NO:61 are fluoro modified nucleotides, and the nucleotides at the other positions in the antisense strand are non-fluoro modified nucleotides;

wherein “non-fluoro modified nucleotide” refers to a nucleotide formed by replacing 2′-hydroxy of the ribose group with a non-fluoro group, or a nucleotide analogue.

2. The siRNA according to claim 1 , wherein the sense strand and the antisense strand have the same or different length, wherein the sense strand has a length of 19 to 23 nucleotides, and the antisense strand has a length of 19 to 26 nucleotides.

3. The siRNA according to claim 1 , wherein the nucleotide sequence I further comprises nucleotide sequence III, the nucleotide sequence II further comprises nucleotide sequence IV, and the nucleotide sequence III and the nucleotide sequence IV each have a length of 1 to 4 nucleotides; the nucleotide sequence III is linked to 5′ terminal of the nucleotide sequence represented by SEQ ID NO 60, the nucleotide sequence IV is linked to 3′ terminal of the nucleotide sequence represented by SEQ ID NO 61, and the nucleotide sequence III and the nucleotide sequence IV have the same length and are reverse complementary.

4. The siRNA according to claim 1 , wherein the nucleotide sequence III and the nucleotide sequence IV both have a length of one nucleotide, and the base of the nucleotide sequence III is C;

or the nucleotide sequence III and the nucleotide sequence IV both have a length of 2 nucleotides, and in the direction from 5′ terminal to 3′ terminal, the bases of the nucleotide sequence III are sequentially C and C;

or, the nucleotide sequence III and the nucleotide sequence IV both have a length of 3 nucleotides, and in the direction from 5′ terminal to 3′ terminal, the bases of nucleotide sequence III are sequentially U, C and C;

or, the nucleotide sequence III and the nucleotide sequence IV both have a length of 4 nucleotides, and in the direction from 5′ terminal to 3′ terminal, the bases of nucleotide sequence III are sequentially C, U, C and C.

5. The siRNA according to claim 1 , wherein the nucleotide sequence II further comprises a nucleotide sequence V; the nucleotide sequence V has a length of 1 to 3 nucleotides, and is linked to the 3′terminal of the antisense strand, thereby forming a 3′ overhang terminal of the antisense strand.

6. The siRNA according to claim 1 , wherein, the nucleotide sequence V has a length 2 nucleotides; moreover the nucleotide sequence V is complementary to the nucleotides at the corresponding positions of the target mRNA, or the nucleotide sequence Vis 2 continuous thymine deoxyribonucleotides or 2 continuous uridine ribonucleotides.

7. The siRNA according to claim 1 , wherein the sense strand comprises the nucleotide sequence represented by SEQ ID NO:60, and the antisense strand comprises the nucleotide sequence represented by SEQ ID NO:3:

(SEQ ID No: 60)

5′-CAAUAAAGCUGGACAAGAZ A -3′

(SEQ ID No: 3)

5′-Z′ B UCUUGUCCAGCUUUAUUGGGG-3′

or, the sense strand comprises the nucleotide sequence represented by SEQ ID NO:4, and the antisense strand comprises the nucleotide sequence represented by SEQ ID NO:5:

(SEQ ID No: 4)

5′-CCCAAUAAAGCUGGACAAGAZ A -3′

(SEQ ID No: 5)

5′-Z′ B UCUUGUCCAGCUUUAUUGGGAG-3′

wherein, Z′ B is the first nucleotide at the 5′terminal of the antisense strand, Z A is selected from A, U, G or C, and Z′ B is a nucleotide complementary to Z A .

8. The siRNA according to claim 1 , wherein the siRNA is siAP1 or siAP2:

siAP1

sense strand:

(SEQ ID No: 6)

5′-CAAUAAAGCUGGACAAGAA-3′

antisense strand:

(SEQ ID No: 7)

5′-UUCUUGUCCAGCUUUAUUGGG-3′

siAP2

sense strand:

(SEQ ID No: 8)

5′-CCCAAUAAAGCUGGACAAGAA-3′

antisense strand:

(SEQ ID No: 9)

5′-UUCUUGUCCAGCUUUAUUGGGAG-3′.

9. The siRNA according to claim 1 , wherein each non-fluoro modified nucleotide is a methoxy modified nucleotide, and the methoxy modified nucleotide refers to a nucleotide formed by replacing 2′-hydroxy of the ribose group with a methoxy group.

10. The siRNA according to claim 9 , wherein the siRNA is siAP1-M2 or siAP2-M2:

siAP1-M2

sense strand:

(SEQ ID NO: 14)

5′-CmAmAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm-3′

antisense strand:

(SEQ ID NO: 15)

5′-UmUfCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGmGmGm-3′

siAP2-M2

sense strand:

(SEQ ID NO: 16)

5′-CmCmCmAmAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm-3′

antisense strand:

(SEQ ID NO: 17)

5′-mUfCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGmGmGmAmGm-

3′,

wherein C, G, U, and A represent the base composition of the nucleotides; m represents that the nucleotide adjacent to the left side of the letter m is a methoxy modified nucleotide; and f represents that the nucleotide adjacent to the left side of the letter f is a fluoro modified nucleotide.

11. The siRNA according to claim 1 , wherein in the siRNA, at least one phosphate group is a phosphorothioate group, and the phosphorothioate linkage is present at at least one of the following positions:

the position between the first and second nucleotides at 5′ terminal of the sense strand;

the position between the second and third nucleotides at 5′ terminal of the sense strand;

the position between the first and second nucleotides at 3′ terminal of the sense strand;

the position between the second and third nucleotides at 3′ terminal of the sense strand;

the position between the first and second nucleotides at 5′ terminal of the antisense strand;

the position between the second and third nucleotides at 5′ terminal of the antisense strand;

the position between the first and second nucleotides at 3′ terminal of the antisense strand; and

the position between the second and third nucleotides at 3′ terminal of the antisense strand.

12. The siRNA according to claim 11 , wherein the siRNA is siAP1-M2S or siAP2-M2S:

siAP1-M2S

sense strand:

(SEQ ID NO: 22)

5′-CmsAmsAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm-3′

antisense strand:

(SEQ ID NO: 23)

5′-UmsUfsCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGmsGmsGm-

3′

siAP2-M2S

sense strand:

(SEQ ID NO: 24)

5′-CmsCmsCmAmAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm-3′

antisense strand:

(SEQ ID NO: 25)

5′-UmsUfsCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGmGmGms

AmsGm-3′,

wherein C, G, U, and A represent the base composition of the nucleotides; m represents that the nucleotide adjacent to the left side of the letter m is a methoxy modified nucleotide; f represents that the nucleotide adjacent to the left side of the letter f is a fluoro modified nucleotide; and s represents that a phosphorothioate linkage is present between the two nucleotides adjacent to both sides of the letter s.

13. The siRNA according to claim 1 , wherein the nucleotide at 5′ terminal of the antisense strand is a 5′-phosphate nucleotide or a 5′-phosphate analogue modified nucleotide; or wherein the 5′-phosphate nucleotide is a nucleotide having the structure represented by Formula (102), and the 5′-phosphate analogue modified nucleotide is a nucleotide having the structure represented by any of formulae (103)-(106):

wherein R is selected from H, OH, methoxy, or F; “Base” represents a base selected from A, U, C, G, or T.

14. The siRNA according to claim 13 , wherein the siRNA is any one of siAP1-M2P1, siAP2-M2P1, siAP1-M2SP1, and siAP2-M2SP1:

siAP1-M2P1

sense strand:

(SEQ ID NO: 14)

5′-CmAmAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm-3′

antisense strand:

(SEQ ID NO: 28)

5′-P1-UmUfCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGmGmGm-3′ 

siAP2-M2P1

sense strand:

(SEQ ID NO: 16)

5′-CmCmCmAmAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm-3′

antisense strand:

(SEQ ID NO: 29)

5′-P1-UmUfCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGmGmGmAm

Gm-3′

siAP1-M2SP1

sense strand:

(SEQ ID NO: 22)

5′-CmsAmsAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm-3′

antisense strand:

(SEQ ID NO: 32)

5′-P1-UmsUfsCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGmsGms

Gm-3′

siAP2-M2SP1

sense strand:

(SEQ ID NO: 24)

5′-CmsCmsCmAmAmUmAmAmAfGfCfUmGmGmAmCmAmAmGmAmAm-3′

antisense strand:

(SEQ ID NO: 33)

5′-P1-UmsUfsCmUmUmGfUmCmCmAmGmCmUmUfUmAfUmUmGmGmGms

AmsGm-3′,

wherein C, G, U, and A represent the base composition of the nucleotides; m represents that the nucleotide adjacent to the left side of the letter m is a methoxy modified nucleotide; f represents that the nucleotide adjacent to the left side of the letter f is a fluoro modified nucleotide; s represents that a phosphorothioate linkage is present between the two nucleotides adjacent to both sides of the letter s; and P1 represents that the nucleotide adjacent to the right side of the letter P1 is a 5′-phosphate nucleotide or a 5′-phosphate analogue modified nucleotide.

15. A pharmaceutical composition, wherein the pharmaceutical composition comprises the siRNA according to claim 1 and a pharmaceutically acceptable carrier; wherein the weight ratio of the siRNA and the pharmaceutically acceptable carrier is 1: (1-500).

16. A siRNA conjugate, comprising the siRNA according to claim 1 and a conjugating group conjugated to the siRNA.

17. The siRNA conjugate according to claim 16 , wherein the conjugating group comprises a pharmaceutically acceptable targeting group and a linker, and the siRNA, the linker and the targeting group are sequentially linked covalently or non-covalently.

18. The siRNA conjugate according to claim 17 , wherein the linker has the structure represented by Formula (301):

wherein,

k is an integer of 1-3;

L A is a chain moiety comprising amide bond having the structure represented by Formula (302), each LA is respectively linked to a targeting group and the LC moiety through an ether bond at its two terminals;

L B is a chain moiety comprising N-acylpyrrolidine having the structure represented by Formula (303), wherein the chain moiety has a carbonyl at its one terminal and is linked to the LC moiety through an amide bond, and has an oxygen atom at the other terminal and is linked to the siRNA through a phosphoester bond;

L C is a bivalent to tetravalent linking group based on hydroxymethyl aminomethane, dihydroxymethyl aminomethane or trihydroxymethyl aminomethane, L C being linked to each L A moiety through ether bond via oxygen atom, and being linked to the L B moiety through amide bond via nitrogen atom.

19. The siRNA conjugate according to claim 16 , wherein the siRNA conjugate has the structure represented by Formula (305):

wherein, the double helix structure represents the siRNA.

20. The siRNA conjugate according to claim 17 , wherein the linker has the structure represented by Formula (306):

wherein,

l is an integer of 0-3;

* represents the site on the linker where the targeting group is linked through ether bond; and

# represents the site on the linker where the siRNA is linked through phosphoester bond.

21. The siRNA conjugate according to claim 16 , wherein the siRNA conjugate has the structure represented by Formula (307):

wherein the double helix structure represents the siRNA.

22. The siRNA conjugate according to claim 17 , wherein the linker is linked to the 3′ terminal of the sense strand of the siRNA, and each of the targeting groups is independently a ligand having affinity to asialoglycoprotein receptors on the surface of mammalian hepatocytes.

23. The siRNA conjugate according to claim 22 , wherein at least one or each of the targeting groups is galactose or N-acetylgalactosamine.

24. A method for treating dyslipidemia, wherein the method comprises administering an effective amount of the siRNA according to claim 1 to a subject suffering from dyslipidemia.

25. The method of claim 24 , wherein the dyslipidemia is hypercholesteremia, hypertriglyceridemia or atherosclerosis.

26. A method for inhibiting the expression of APOC3 gene in hepatocytes, wherein the method comprises contacting an effective amount of the siRNA according to claim 1 with the hepatocytes.

Assignments (1)

Priority Claims (2)

Continuity (2)

References Cited (400)

Cited By (1)

US 12,624,355