# US 10,870,856 B2 · RNAi constructs for inhibiting ASGR1 expression and methods of use thereof

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- Number: US 10,870,856 B2
- Document ID: US10870856B2
- Status: Granted
- Kind: B2
- Application number: 16/328,221
- Filed: 2017-08-25
- Published: 2019-10-10
- Granted: 2020-12-22
- Earliest claimed: 2016-08-26
- Assignees: Amgen Inc.
- Applicants: Amgen Inc.
- Inventors: Michael Ollmann; Yang Li; Jun Zhang; Oliver Homann; Leslie P. Miranda; Justin K. Murray; Bin Wu; Oh Kyu Yoon; John Gordon Allen; Chawita Netirojjanakul
- Web page: https://patentgrep.com/patent/US10870856B2

## CPC

- C12N 15/1138: {against receptors or cell surface proteins}
- A61P 3/06: Antihyperlipidemics
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- C07K 16/2851: {against the lectin superfamily, e.g. CD23, CD72}
- C07K 2317/526: CH3 domain
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/315: Phosphorothioates
- C12N 2310/317: with an inverted bond, e.g. a cap structure
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/332: Abasic residue
- C12N 2310/351: Conjugate
- C12N 2310/3513: Protein; Peptide
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The present invention relates to RNAi constructs for reducing expression of the ASGR1 gene. Methods of using such RNAi constructs to treat or prevent cardiovascular disease, such as coronary artery disease and myocardial infarction, and to reduce serum non-HDL cholesterol levels are also described.

## Claims

### Claim 1

An RNAi construct comprising a sense strand and an antisense strand, wherein the antisense strand comprises a region having a sequence that is complementary to an ASGR1 mRNA sequence, and wherein said region comprises: (i) the sequence of nucleotides 1-19 of SEQ ID NO: 2651, (ii) the sequence of nucleotides 2-19 of SEQ ID NO: 2651, or (iii) the sequence of SEQ ID NO: 2651.

### Claim 2 (depends on 1)

The RNAi construct of claim 1, wherein the sense strand comprises a sequence that is sufficiently complementary to the sequence of the antisense strand to form a duplex region of about 15 to about 30 base pairs in length.

### Claim 3 (depends on 1)

The RNAi construct of claim 1, wherein the sense strand and the antisense strand are each about 18 to about 28 nucleotides in length.

### Claim 4 (depends on 3)

The RNAi construct of claim 3, wherein the sense strand and the antisense strand are each about 19 to about 27 nucleotides in length.

### Claim 5 (depends on 3)

The RNAi construct of claim 3, wherein the sense strand and the antisense strand are each about 21 to about 25 nucleotides in length.

### Claim 6 (depends on 1)

The RNAi construct of claim 1, wherein the RNAi construct comprises at least one blunt end.

### Claim 7 (depends on 1)

The RNAi construct of claim 1, wherein the RNAi construct comprises at least one nucleotide overhang of 1 to 4 unpaired nucleotides.

### Claim 8 (depends on 7)

The RNAi construct of claim 7, wherein the RNAi construct comprises a nucleotide overhang at the 3′ end of the sense strand, the 3′ end of the antisense strand, or the 3′ end of both the sense strand and the antisense strand.

### Claim 9 (depends on 1)

The RNAi construct of claim 1, wherein the RNAi construct comprises at least one modified nucleotide.

### Claim 10 (depends on 9)

The RNAi construct of claim 9, wherein the modified nucleotide is a 2′-modified nucleotide.

### Claim 11 (depends on 9)

The RNAi construct of claim 9, wherein the modified nucleotide is a 2′-fluoro modified nucleotide, a 2′-O-methyl modified nucleotide, a 2′-O-methoxyethyl modified nucleotide, a 2′-O-allyl modified nucleotide, a bicyclic nucleic acid (BNA), or combinations thereof.

### Claim 12 (depends on 9)

The RNAi construct of claim 9, wherein all of the nucleotides in the sense and antisense strands are modified nucleotides.

### Claim 13 (depends on 12)

The RNAi construct of claim 12, wherein the modified nucleotides are 2′-O-methyl modified nucleotides, 2′-fluoro modified nucleotides, or combinations thereof.

### Claim 14 (depends on 1)

The RNAi construct of claim 1, wherein the RNAi construct comprises at least one phosphorothioate internucleotide linkage.

### Claim 15 (depends on 14)

The RNAi construct of claim 14, wherein the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at the 3′ end of the antisense strand.

### Claim 16 (depends on 14)

The RNAi construct of claim 14, wherein the RNAi construct comprises two consecutive phosphorothioate internucleotide linkages at both the 3′ and 5′ ends of the antisense strand and two consecutive phosphorothioate internucleotide linkages at the 5′ end of the sense strand.

### Claim 17 (depends on 1)

The RNAi construct of claim 1, wherein the antisense strand comprises the sequence of SEQ ID NO: 2650 or SEQ ID NO: 2651.

### Claim 18 (depends on 17)

The RNAi construct of claim 17, wherein:
(a) the sense strand comprises the sequence of SEQ ID NO: 1147 and the antisense strand comprises the sequence of SEQ ID NO: 2650; or
(b) the sense strand comprises the sequence of SEQ ID NO: 1148 and the antisense strand comprises the sequence of SEQ ID NO: 2651.

### Claim 19 (depends on 18)

The RNAi construct of claim 18, wherein:
(a) the sense strand comprises the sequence of modified nucleotides according to SEQ ID NO: 3050 and the antisense strand comprises the sequence of modified nucleotides according to SEQ ID NO: 3701;
(b) the sense strand comprises the sequence of modified nucleotides according to SEQ ID NO: 3051 and the antisense strand comprises the sequence of modified nucleotides according to SEQ ID NO: 3702;
(c) the sense strand comprises the sequence of modified nucleotides according to SEQ ID NO: 3065 and the antisense strand comprises the sequence of modified nucleotides according to SEQ ID NO: 3716;
(d) the sense strand comprises the sequence of modified nucleotides according to SEQ ID NO: 3067 and the antisense strand comprises the sequence of modified nucleotides according to SEQ ID NO: 3718;
(e) the sense strand comprises the sequence of modified nucleotides according to SEQ ID NO: 3071 and the antisense strand comprises the sequence of modified nucleotides according to SEQ ID NO: 3722;
(f) the sense strand comprises the sequence of modified nucleotides according to SEQ ID NO: 4420 and the antisense strand comprises the sequence of modified nucleotides according to SEQ ID NO: 4600; or
(g) the sense strand comprises the sequence of modified nucleotides according to SEQ ID NO: 4698 and the antisense strand comprises the sequence of modified nucleotides according to SEQ ID NO: 4699.

### Claim 20 (depends on 1)

The RNAi construct of claim 1, wherein the sense strand comprises (i) the sequence of nucleotides 1-19 of SEQ ID NO: 1148, (ii) the sequence of nucleotides 2-19 of SEQ ID NO: 1148, or (iii) the sequence of SEQ ID NO: 1148.

### Claim 21 (depends on 1)

The RNAi construct of claim 1, wherein the RNAi construct further comprises a ligand.

### Claim 22 (depends on 21)

The RNAi construct of claim 21, wherein the ligand comprises a cholesterol moiety, a vitamin, a steroid, a bile acid, a folate moiety, a fatty acid, a carbohydrate, a glycoside, or antibody or antigen-binding fragment thereof.

### Claim 23 (depends on 21)

The RNAi construct of claim 21, wherein the ligand targets delivery of the RNAi construct to hepatocytes.

### Claim 24 (depends on 23)

The RNAi construct of claim 23, wherein the ligand comprises a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human ASGR1.

### Claim 25 (depends on 24)

The RNAi construct of claim 24, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a substitution of at least one amino acid with a cysteine amino acid, and wherein the sense strand is covalently attached to the monoclonal antibody or antigen-binding fragment thereof through the side chain of the cysteine amino acid.

### Claim 26 (depends on 21)

The RNAi construct of claim 21, wherein the ligand comprises galactose, galactosamine, or N-acetyl-galactosamine.

### Claim 27 (depends on 26)

The RNAi construct of claim 26, wherein the ligand comprises a multivalent galactose moiety or multivalent N-acetyl-galactosamine moiety.

### Claim 28 (depends on 27)

The RNAi construct of claim 27, wherein the multivalent galactose moiety or multivalent N-acetyl-galactosamine moiety is trivalent or tetravalent.

### Claim 29 (depends on 21)

The RNAi construct of claim 21, wherein the ligand is covalently attached to the sense strand optionally through a linker.

### Claim 30 (depends on 29)

The RNAi construct of claim 29, wherein the ligand is covalently attached to the 3′ end or 5′ end of the sense strand.

### Claim 31 (depends on 1)

The RNAi construct of claim 1, wherein the sense strand comprises a sequence that is sufficiently complementary to the sequence of the antisense strand to form a duplex region of about 19 to about 25 base pairs in length.

### Claim 32 (depends on 1)

A pharmaceutical composition comprising the RNAi construct of claim 1 and a pharmaceutically acceptable carrier, excipient, or diluent.

### Claim 33 (depends on 1)

A method for reducing the expression of ASGR1 in a patient in need thereof comprising administering to the patient the RNAi construct of claim 1.

### Claim 34 (depends on 1)

A method for reducing non-HDL cholesterol in a patient in need thereof comprising administering to the patient the RNAi construct of claim 1.

### Claim 35 (depends on 1)

A method for treating cardiovascular disease in a patient in need thereof comprising administering to the patient the RNAi construct of claim 1.

### Claim 36 (depends on 1)

A method for reducing the risk of myocardial infarction in a patient in need thereof comprising administering to the patient the RNAi construct of claim 1.