# US 2021/0275564 A1 · Nucleic acid, pharmaceutical composition and conjugate containing nucleic acid, and use thereof

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- Number: US 2021/0275564 A1
- Document ID: US20210275564A1
- Status: Published application
- Kind: A1
- Application number: 17/269,039
- Filed: 2019-08-20
- Published: 2021-09-09
- Earliest claimed: 2018-08-21
- Assignees: Suzhou Ribo Life Science Co., Ltd.
- Applicants: Suzhou Ribo Life Science Co., Ltd.
- Inventors: Hongyan ZHANG; Shan GAO; Daiwu KANG; Gengrong CHEN
- Web page: https://patentgrep.com/patent/US20210275564A1

## CPC

- A61K 31/7088: Compounds having three or more nucleosides or nucleotides
- A61K 31/712: Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61K 47/18: Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K 47/543: {Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine}
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/1131: {against viruses}
- C12N 2310/11: Antisense
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/315: Phosphorothioates
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/322: 2'-R Modification
- C12N 2310/3515: Lipophilic moiety, e.g. cholesterol
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided is a siRNA for inhibiting HBV gene expression, including a sense strand and an antisense strand. The sense strand includes a nucleotide sequence 1, and the antisense strand includes a nucleotide sequence 2; the nucleotide sequence 1 and the nucleotide sequence 2 are at least partially reversely complementary to form a double-stranded region; the nucleotide sequence 1 and the nucleotide sequence shown in SEQ ID NO: 1 are equal in length, and no more than 3 nucleotide differences are generated; the nucleotide sequence 2 and the nucleotide sequence shown in SEQ ID NO: 2 are equal in length, and no more than 3 nucleotide differences are generated; nucleotides of the 7th, 8th, and 9th bits of the nucleotide sequence 1 and the 2nd, 6th, 14th, and 16th of the nucleotide sequence 2 are fluoro-modified nucleotides. Also provided are a pharmaceutical composition and a conjugate containing the siRNA.

## Claims

### Claim 1

A siRNA capable of inhibiting the expression of HBV gene, comprising a sense strand and an antisense strand, wherein each nucleotide in the sense strand and 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; a “fluoro modified nucleotide” refers to a nucleotide formed by substituting the 2′-hydroxy of the ribose group of the nucleotide with a fluoro group, and a “non-fluoro modified nucleotide” refers to a nucleotide formed by substituting the 2′-hydroxy of the ribose group of the nucleotide with a non-fluoro group, or a nucleotide analogue; the sense strand comprises a nucleotide sequence 1; the antisense strand comprises a nucleotide sequence 2; the nucleotide sequence 1 and the nucleotide sequence 2 are at least partly reverse complementary to form a double-stranded region; wherein the nucleotide sequence 1 and the nucleotide sequence shown in SEQ ID NO: 1 have an equal length and no more than 3 nucleotide differences; and the nucleotide sequence 2 and the nucleotide sequence shown in SEQ ID NO: 2 have an equal length and no more than 3 nucleotide differences:
(SEQ ID NO: 1)
5′-GAAAGUAUGUCAACGAAUZ-3′,
(SEQ ID NO: 2)
5′-Z'AUUCGUUGACAUACUUUC-3′,
wherein Z is U, Z′ is A;
the nucleotide sequence 1 comprises a nucleotide ZA at the position corresponding to Z; the nucleotide sequence 2 comprises a nucleotide Z′B at the position corresponding to Z′; the nucleotide Z′B is the first nucleotide from 5′ terminal of the antisense strand; and
the fluoro modified nucleotides are located within the nucleotide sequence 1 and the nucleotide sequence 2; the nucleotide sequence 1 comprises no more than 5 fluoro modified nucleotides, and in the direction from 5′ terminal to 3′ terminal, the nucleotides at positions 7, 8 and 9 of the nucleotide sequence 1 are fluoro modified nucleotides; the nucleotide sequence 2 comprises no more than 7 fluoro modified nucleotides and the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence 2 are fluoro modified nucleotides.

### Claim at position 2

2-5. (canceled)

### Claim 6 (depends on 1)

The siRNA according to claim 1, wherein the sense strand further comprises a nucleotide sequence 3, and the antisense strand further comprises a nucleotide sequence 4; the nucleotide sequence 3 and the nucleotide sequence 4 have a length of 1-4 nucleotides, respectively; the nucleotide sequence 3 is linked to 5′ terminal of the nucleotide sequence 1; and the nucleotide sequence 4 is linked to 3′ terminal of the nucleotide sequence 2; the nucleotide sequence 3 and the nucleotide sequence 4 have equal length and are substantially reverse complementary or completely reverse complementary to each other; the “substantially reverse complementary” means that there is no more than 1 base mispairing between two nucleotide sequences; and the “completely reverse complementary” means that there is no mispairing between two nucleotide sequences.

### Claim 7 (depends on 6)

The siRNA according to claim 6, wherein the nucleotide sequence 3 and the nucleotide sequence 4 both have a length of 1 nucleotide and the base of the nucleotide sequence 3 is G; or
the nucleotide sequence 3 and the nucleotide sequence 4 both have a length of 2 nucleotides;
in the direction from 5′ terminal to 3′ terminal, the bases of the nucleotide sequence 3 are U and G in succession; or
the nucleotide sequence 3 and the nucleotide sequence 4 both have a length of 3 nucleotides;
in the direction from 5′ terminal to 3′ terminal, the bases of the nucleotide sequence 3 are U, U and G in succession; or
the nucleotide sequence 3 and the nucleotide sequence 4 both have a length of 4 nucleotides;
in the direction from 5′ terminal to 3′ terminal, the bases of the nucleotide sequence 3 are A, U, U, and G in succession.

### Claim 8 (depends on 1)

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

### Claim 9 (depends on 8)

The siRNA according to claim 8, wherein the nucleotide sequence 5 has a length of 2 nucleotides; and in the direction from 5′ terminal to 3′ terminal, the nucleotide sequence 5 is 2 consecutive thymidine deoxyribonucleotides, 2 consecutive uridine ribonucleotides or 2 nucleotides complementary to the target mRNA.

### Claim 10 (depends on 9)

The siRNA according to claim 9, wherein the nucleotide sequence 1 comprises the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; and the nucleotide sequence 2 comprises any one of the nucleotide sequences selected from the group consisting of the nucleotide sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8:
(SEQ ID NO: 3)
5′-GAAAGUAUGUCAACGAAUU-3′,
(SEQ ID NO: 4)
5′-GAAAGUAUGUCAACGAAUA-3′,
(SEQ ID NO: 5)
5′-AAUUCGUUGACAUACUUUCUU-3′,
(SEQ ID NO: 6)
5′-AAUUCGUUGACAUACUUUCCA-3′,
(SEQ ID NO: 7)
5′-UAUUCGUUGACAUACUUUCUU-3′,
(SEQ ID NO: 8)
5′-UAUUCGUUGACAUACUUUCCA-3′.

### Claim 11 (depends on 10)

The siRNA according to claim 10, wherein the siRNA is any one of the following siP1 to siP4:
siP1
Sense strand:
(SEQ ID NO: 3)
5′-GAAAGUAUGUCAACGAAUU-3′,
Antisense strand:
(SEQ ID NO: 5)
5′- AAUUCGUUGACAUACUUUCUU-3′,
siP2
Sense strand:
(SEQ ID NO: 3)
5′- GAAAGUAUGUCAACGAAUU-3′,
Antisense strand:
(SEQ ID NO: 6)
5′- AAUUCGUUGACAUACUUUCCA-3′,
siP3
Sense strand:
(SEQ ID NO: 4)
5′- GAAAGUAUGUCAACGAAUA-3′,
Antisense strand:
(SEQ ID NO: 7)
5′- UAUUCGUUGACAUACUUUCUU-3′,
siP4
Sense strand:
(SEQ ID NO: 4)
5′- GAAAGUAUGUCAACGAAUA-3′,
Antisense strand:
(SEQ ID NO: 8)
5′- UAUUCGUUGACAUACUUUCCA-3′.

### Claim 12

(canceled)

### Claim 13 (depends on 1)

The siRNA according to claim 1, wherein each non-fluoro modified nucleotide is independently selected from a nucleotide formed by substituting the 2′-hydroxy of the ribose group of the nucleotide with a non-fluoro group, or a nucleotide analogue.

### Claim 14

(canceled)

### Claim 15 (depends on 13)

The siRNA according to claim 13, wherein each non-fluoro modified nucleotide is a methoxy modified nucleotide which refers to a nucleotide formed by substituting the 2′-hydroxy of the ribose group with a methoxy group.

### Claim 16 (depends on 15)

The siRNA according to claim 15, wherein the siRNA is any one of the siRNAs listed in Table 1A.

### Claim at position 14

17-19. (canceled)

### Claim 20 (depends on 1)

The siRNA according to claim 1, wherein at least one of the phosphate groups in the phosphate-ribose backbone of at least one single strand of the sense strand and the antisense strand is a phosphorothioate group, and at least one linkage selected from the group consisting of the following inter-nucleotide linkages is a phosphorothioate linkage:
the linkage between the first and second nucleotides at 5′ terminal of the sense strand;
the linkage between the second and third nucleotides at 5′ terminal of the sense strand;
the linkage between the first and second nucleotides at 3′ terminal of the sense strand;
the linkage between the second and third nucleotides at 3′ terminal of the sense strand;
the linkage between the first and second nucleotides at 5′ terminal of the antisense strand;
the linkage between the second and third nucleotides at 5′ terminal of the antisense strand;
the linkage between the first and second nucleotides at 3′ terminal of the antisense strand; and
the linkage between the second and third nucleotides at 3′ terminal of the antisense strand.

### Claim 21 (depends on 20)

The siRNA according to claim 20, wherein the siRNA is any one of the siRNAs listed in Table 1B.

### Claim 22 (depends on 1)

The siRNA according to claim 1, wherein the nucleotide at 5′-terminal of the antisense strand of the siRNA is a 5′-phosphate nucleotide or a 5′-phosphate analogue modified nucleotide.

### Claim 23 (depends on 22)

The siRNA according to claim 22, wherein the siRNA is any one of the siRNAs listed in Table 1C or 1D.

### Claim 24

(canceled)

### Claim 25 (depends on 1)

A pharmaceutical composition comprising 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).

### Claim at position 21

26-27. (canceled)

### Claim 28 (depends on 25)

The pharmaceutical composition according to claim 25, wherein the pharmaceutically acceptable carrier comprises an organic amine, a helper lipid and a PEGylated lipid; wherein the organic amine is a compound as shown by Formula (201) and/or a pharmaceutically acceptable salt thereof:
wherein:
X101 and X102 independently of one another are selected from O, S, N-A and C-A, wherein A is hydrogen or a C1-C20 hydrocarbon chain;
Y101 and Z101 independently of one another are selected from C═O, C═S, S═O, CH—OH and SO2;
R101, R102, R103, R104, R105, R106 and R107 independently of one another are selected from hydrogen; a cyclic or an acyclic, substituted or unsubstituted, branched or unbranched aliphatic group; a cyclic or an acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic group; a substituted or unsubstituted, branched or unbranched acyl group; a substituted or unsubstituted, branched or unbranched aryl group; and a substituted or unsubstituted, branched or unbranched heteroaryl group;
x is an integer of 1-10;
n is an integer of 1-3, m is an integer of 0-20, p is an integer of 0 or 1, wherein if m and p are 0, then R102 is hydrogen; and
if at least one of n and m is 2, then R103 and nitrogen in Formula (201) form a structure as shown by Formula (202) or (203):
wherein g, e and f independently of one another are an integer of 1-6; “HCC” represents a hydrocarbon chain; and each *N indicates a nitrogen atom shown in Formula (201).

### Claim 29 (depends on 28)

The pharmaceutical composition according to claim 28, wherein the organic amine is an organic amine as shown by Formula (214) and/or an organic amine as shown by Formula (215):
the helper lipid is cholesterol, cholesterol analogs and/or cholesterol derivatives; and
the PEGylated lipid is 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)]-2000.

### Claim 30 (depends on 28)

The pharmaceutical composition according to claim 28, wherein the molar ratio among the organic amine, the helper lipid, and the PEGylated lipid is (19.7-80):(19.7-80):(0.3-50).

### Claim 31

(canceled)

### Claim 32 (depends on 1)

A siRNA conjugate comprising the siRNA according to claim 1 and a ligand conjugated to the siRNA.

### Claim 33 (depends on 32)

The siRNA conjugate according to claim 32, wherein the ligand is a pharmaceutically acceptable conjugation group which comprises a pharmaceutically acceptable targeting group and a linker; and the siRNA, the linker and the targeting group are covalently linked in succession;
wherein the linker has a structure as shown by Formula (301):
wherein k is an integer of 1-3;
LA is an amide bond-comprising chain moiety that has a structure as shown by Formula (302), two terminals of which are respectively linked to the targeting group and the LC moiety each via an ether bond:
LB is an N-acylpyrrolidine-comprising chain moiety that has a structure as shown by Formula (303), which has a carbonyl group and is linked to the LC moiety via an amide bond at one end, and has an oxygen atom and is linked to the siRNA via a phosphoester bond at the other end thereof:
LC is a bivalent to tetravalent linking group based on hydroxymethyl aminomethane, dihydroxymethyl aminomethane or trihydroxymethyl aminomethane, LC being linked to each of the LA moieties through an ether bond via an oxygen atom, and being linked to the LB moiety through an amide bond via the nitrogen atom; or
wherein the linker has a structure as shown by Formula (306):
wherein,
l is an integer of 0-3;
* represents the site on the linker linked to the targeting group via ether bond; and
# represents the site on the linker linked to the siRNA via phosphoester bond.

### Claim at position 28

34-42. (canceled)

### Claim 43 (depends on 33)

The siRNA conjugate according to claim 33, wherein the siRNA conjugate has a structure as shown by Formula (305) or by Formula (307):
wherein, the double helix structure represents the siRNA; and
the linker is linked to 3′-terminal of the sense strand of the siRNA.

### Claim 44 (depends on 1)

A siRNA conjugate having a structure as shown by Formula (401):
wherein,
n1 is an integer of 1-3, and n3 is an integer of 0-4;
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 one of H, methyl or ethyl;
R3 is a group having a structure as shown by Formula (A59):
wherein,
E1 is OH, SH or BH2; Nu is the siRNA according to claim 1;
R2 is any group capable of linking to the N atom on the nitrogenous backbone and linking to A59;
each L1 is independently selected from the linkage combinations of one or more of Formulae A1-A26:
wherein j1 is an integer of 1-20; j2 is an integer of 1-20;
R′ is a C1-C10 alkyl;
Ra is selected from one of Formulae A27-A45:
Rb is a C1-C10 alkyl; and
represents the site where a group is covalently linked;
each M1 is independently selected from one of the ligands that have affinity to the asialoglycoprotein receptors (ASGP-R) on the surface of mammalian hepatocytes.

### Claim 45 (depends on 44)

The siRNA conjugate according to claim 44, wherein L1 is selected from the linkage combinations of one or more of Formulae A1, A4, A5, A6, A8, A10, A11, and A13; or
L1 is selected from the linkage combinations of at least two of Formulae A1, A4, A8, A10, and A11.

### Claim at position 32

46-47. (canceled)

### Claim 48 (depends on 44)

The siRNA conjugate according to claim 44, wherein L1 has a length of 3 to 25 atoms; or L1 has a length of 4 to 15 atoms.

### Claim at position 34

49-52. (canceled)

### Claim 53 (depends on 44)

The siRNA conjugate according to claim 44, wherein m1, m2 and m3 independently of one another are an integer of 2-5; or wherein m1=m2=m3.

### Claim 54

(canceled)

### Claim 55 (depends on 44)

The siRNA conjugate according to claim 44, wherein each M, is independently selected from the group consisting of 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-glucopyranose, β-D-glucopyranose, 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-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, methyl 2,3,4-tris-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside, 4-thio-β-D-galactopyranose, ethyl 3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-glucoheptopyranoside, 2,5-anhydro-D-allononitrile, ribose, D-ribose, D-4-thioribose, L-ribose, and L-4-thioribose.

### Claim at position 38

56-57. (canceled)

### Claim 58 (depends on 44)

The siRNA conjugate according to claim 44, wherein R2 comprises both a site linking to the N atom on the nitrogenous backbone and a site linking to the P atom in R3; and
in R2, the site linking to the N atom on the nitrogenous backbone forms an amide bond with the N atom, and the site linking to the P atom in R3 forms a phosphoester bond with the P atom; or
wherein R2 is selected from B5, B6, B5′, or B6′:
wherein represents the site where a group is covalently linked; and q2 is an integer of 1-10.

### Claim at position 40

59-61. (canceled)

### Claim 62 (depends on 44)

The siRNA conjugate according to claim 44, wherein the conjugate has a structure as shown by Formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), or (422):

### Claim at position 42

63-64. (canceled)

### Claim 65 (depends on 62)

The siRNA conjugate according to claim 62, wherein the P atom in Formula A59 is linked to 3′ terminal of the sense strand.

### Claim at position 44

66-68. (canceled)

### Claim 69 (depends on 1)

A method for treating and/or preventing a pathological condition or disease caused by HBV infection, comprising administering to a patient an effective amount of the siRNA according to claim 1.

### Claim 70 (depends on 1)

A method for inhibiting the expression of HBV genes, comprising contacting an effective amount of the siRNA according to claim 1.

### Claim 71

(canceled)

### Claim 72 (depends on 69)

The method of claim 69, wherein the pathological condition or disease caused by HBV infection is selected from chronic liver diseases, hepatitis, hepatic fibrosis, and liver proliferative diseases.

### Claim 73 (depends on 25)

A method for treating and/or preventing a pathological condition or disease caused by HBV infection, comprising the pharmaceutical composition according to claim 25.

### Claim 74 (depends on 32)

A method for treating and/or preventing a pathological condition or disease caused by HBV infection, comprising the siRNA conjugate according to claim 32.

### Claim 75 (depends on 25)

A method for inhibiting the expression of HBV genes, comprising the pharmaceutical composition according to claim 25.

### Claim 76 (depends on 32)

A method for inhibiting the expression of HBV genes, comprising the siRNA conjugate according to claim 32 with hepatitis cells infected with HBV.