# US 10,478,500 B2 · Compositions and methods for inhibition of HAO1 (Hydroxyacid Oxidase 1 (Glycolate Oxidase)) gene expression

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- Number: US 10,478,500 B2
- Document ID: US10478500B2
- Status: Granted
- Kind: B2
- Application number: 15/517,471
- Filed: 2015-10-09
- Published: 2017-10-26
- Granted: 2019-11-19
- Earliest claimed: 2014-10-10
- Assignees: Alnylam Pharmaceuticals, Inc.
- Applicants: Alnylam Pharmaceuticals, Inc.
- Inventors: William Querbes; Kevin Fitzgerald; Brian Bettencourt; Abigail Liebow; David V. Erbe
- Web page: https://patentgrep.com/patent/US10478500B2

## CPC

- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61K 47/549: {Sugars, nucleosides, nucleotides or nucleic acids}
- A61P 13/00: Drugs for disorders of the urinary system
- A61P 13/12: of the kidneys
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- 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 15/1137: {against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138)}
- A61K 31/00: Medicinal preparations containing organic active ingredients
- A61K 45/00: Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/315: Phosphorothioates
- C12N 2310/344: Position-specific modifications, e.g. on every purine, at the 3'-end
- C12N 2310/346: having a combination of backbone and sugar modifications
- C12N 2310/351: Conjugate
- C12N 2310/3515: Lipophilic moiety, e.g. cholesterol
- C12Y 101/03015: (S)-2-Hydroxy-acid oxidase (1.1.3.15)
- 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)
- C12Y: ENZYMES

## Abstract

The invention relates to RNAi agents, e.g. double-stranded RNAi agents, targeting the HAO1 gene, and method of using such RNAi agents to inhibit expression of HAO1 and methods of treating subjects having, e.g., PH1. Described herein are double-stranded RNAi agents which inhibit the expression of a HA01 gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human having a HAO1 associated disorder, and uses of such double-stranded RNAi agents. In certain aspects of the invention, substantially all of the nucleotides of an iRNA of the invention are modified.

## Claims

### Claim 1

A double stranded RNAi agent capable of inhibiting expression of HAO1 in a cell, wherein said double stranded RNAi agent comprises a sense strand and an antisense strand forming a double-stranded region, wherein the sense strand nucleotide sequence consists of SEQ ID NO:589 and the antisense strand nucleotide sequence consists of SEQ ID NO:706;
wherein substantially all of the nucleotides of said sense strand and substantially all of the nucleotides of said antisense strand are modified nucleotides, and
wherein said sense strand is conjugated to a ligand attached at the 3′-terminus.

### Claim 2 (depends on 1)

The double stranded RNAi agent of claim 1, wherein all of the nucleotides of said sense strand and all of the nucleotides of said antisense strand are modified nucleotides.

### Claim 3 (depends on 1)

The double stranded RNAi agent of claim 1, wherein at least one of said modified nucleotides is selected from the group consisting of a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a nucleotide comprising a 5′ phosphate or 5′ phosphate mimic, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

### Claim 4 (depends on 1)

The double stranded RNAi agent of claim 1, wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

### Claim 5 (depends on 1)

The double stranded RNAi agent claim 1, wherein the ligand is

### Claim 6 (depends on 1)

The double stranded RNAi agent of claim 1, wherein the ligand is attached to the 3′ end of the sense strand.

### Claim 7 (depends on 6)

The double stranded RNAi agent of claim 6, wherein the RNAi agent is conjugated to the ligand as shown in the following schematic
wherein X is O or S.

### Claim 8 (depends on 1)

The double stranded RNAi agent of claim 1, wherein said agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

### Claim 9 (depends on 8)

The double stranded RNAi agent of claim 8, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminus of one strand.

### Claim 10 (depends on 8)

The double stranded RNAi agent of claim 8, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 5′-terminus of one strand.

### Claim 11 (depends on 8)

The double stranded RNAi agent of claim 8, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the both the 5′- and 3′-terminus of one strand.

### Claim 12 (depends on 1)

The double stranded RNAi agent of claim 1, wherein the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.

### Claim 13 (depends on 1)

The double stranded RNAi agent of claim 1, wherein the sense strand nucleotide sequence comprises SEQ ID NO:589 and the antisense strand nucleotide sequence comprises SEQ ID NO:706.

### Claim 14 (depends on 1)

The double stranded RNAi agent of claim 1, wherein the sense strand nucleotide sequence consists of SEQ ID NO:589 and the antisense strand nucleotide sequence consists of SEQ ID NO:706.

### Claim 15 (depends on 1)

A vector containing the double stranded RNAi agent of claim 1.

### Claim 16 (depends on 1)

A cell containing the double stranded RNAi agent of claim 1.

### Claim 17 (depends on 1)

A pharmaceutical composition comprising the double stranded RNAi agent of claim 1.

### Claim 18 (depends on 1)

A method of inhibiting HAO1 expression in a cell, the method comprising:
(a) contacting the cell with the double stranded RNAi agent of claim 1; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a HAO1 gene, thereby inhibiting expression of the HAO1 gene in the cell.

### Claim 19 (depends on 1)

The double stranded RNAi agent of claim 1, wherein the sense strand consists of SEQ ID NO:213 (gsascuuuCfaUfCfCfuggaaauauaL96) and the antisense strand consists of SEQ ID NO:330 (usAfsuauUfuCfCfaggaUfgAfaagucscsa) and each abbreviation represents a nucleotide monomer as follows:
Abbreviation
Nucleotide(s)
Af
2′-fluoroadenosine-3′-phosphate
Afs
2′-fluoroadenosine-3′-phosphorothioate
Cf
2′-fluorocytidine-3′-phosphate
U
Uridine-3′-phosphate
Uf
2′-fluorouridine-3′-phosphate
a
2′-O-methyladenosine-3′-phosphate
as
2′-O-methyladenosine-3′-phosphorothioate
c
2′-O-methylcytidine-3′-phosphate
cs
2′-O-methylcytidine-3′-phosphorothioate
g
2′-O-methylguanosine-3′-phosphate
gs
2′-O-methylguanosine-3′-phosphorothioate
u
2′-O-methyluridine-3′-phosphate
us
2′-O-methyluridine-3′-phosphorothioate
s
phosphorothioate linkage
L96
N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-
hydroxyprolinol Hyp-(GalNAc-alkyl)3.