# US 12,600,965 B2 · Modified double stranded oligonucleotide

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- Number: US 12,600,965 B2
- Document ID: US12600965B2
- Status: Granted
- Kind: B2
- Application number: 17/291,775
- Filed: 2019-11-05
- Published: 2021-12-16
- Granted: 2026-04-14
- Earliest claimed: 2018-11-09
- Assignees: Alnylam Pharmaceuticals, Inc.
- Applicants: Alnylam Pharmaceuticals, Inc.
- Inventors: Donald Foster; Mark K. Schlegel; Christopher Brown
- Web page: https://patentgrep.com/patent/US12600965B2

## CPC

- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- 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)}
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- C12N 2310/11: Antisense
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/315: Phosphorothioates
- C12N 2310/343: having patterns, e.g. ==--==--==--
- C12N 2310/344: Position-specific modifications, e.g. on every purine, at the 3'-end
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)

## Abstract

One aspect of the present invention relates to double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene. Other aspects of the invention relate to pharmaceutical compositions comprising these dsRNA molecules suitable for therapeutic use, and methods of inhibiting the expression of a target gene by administering these dsRNA molecules, e.g., for the treatment of various disease conditions.

## Claims

### Claim 1

A dsRNA agent comprising:
a sense strand having a length of 17-35 nucleotides;
an antisense strand having a length of 17-35 nucleotides;
at least two phosphorothioate internucleotide linkages between the first five nucleotides of the antisense strand, counting from the 5′ end of the antisense strand; and
only three, four, five or six 2′-deoxy modifications on the antisense strand;
wherein:
at least two 2′-deoxy modifications are in a central region of the sense strand, wherein the central region of the sense strand is positions 7, 8, 9, 10, 11, 12, and 13 counting from the 5′-end of the sense strand;
three of the 2′-deoxy modifications of the antisense strand are at positions 2, 12, and 14, counting from the 5′-end of the antisense strand;
the sense strand and the antisense strand form a duplex region containing between 17 to 25 base pairs;
the dsRNA agent comprises a ligand;
the sense strand does not comprise a glycol nucleic acid (GNA); and
the 2′-deoxy modifications are not 2′-deoxy-2′-fluoro (2′-F) nucleoside.

### Claim 2 (depends on 1)

The dsRNA agent of claim 1, wherein the dsRNA agents have all natural nucleotides, or less than 20% non-natural nucleotides.

### Claim 3 (depends on 1)

The dsRNA agent of claim 1, wherein the dsRNA comprises a sense strand having a length of 18-30 nucleotides.

### Claim 4 (depends on 1)

The dsRNA agent of claim 1, wherein the dsRNA comprises an antisense strand having a length of 18-30 nucleotides.

### Claim 5 (depends on 1)

The dsRNA agent of claim 1, wherein the dsRNA comprises an antisense strand having a length of 18-23 nucleotides, and five 2′-deoxy modifications in the antisense strand at positions 2, 5, 7, 12 and 14 counting from the 5′-end of the antisense strand.

### Claim 6 (depends on 1)

The dsRNA agent of claim 1, wherein at least one of the 2′-deoxy modifications is in the sense strand at position 11, counting from the 5′-end of the sense strand.

### Claim 7 (depends on 1)

The dsRNA agent of claim 1, wherein at least two of the 2′-deoxy modifications are in the sense strand at positions 9 and 11 counting from the 5′-end of the sense strand.

### Claim 8 (depends on 1)

The dsRNA agent of claim 1, wherein at least five of the 2′-deoxy modifications are in the antisense strand at positions 2, 5, 7, 12 and 14 counting from the 5′-end of the antisense strand, and at least two of the 2′-deoxy modifications are in the sense strand at positions 9 and 11 counting from the 5′-end of the sense strand.

### Claim 9 (depends on 2)

The dsRNA agent of claim 2, wherein the non-natural nucleotide is selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA) nucleotides, hexitol nucleic acid (HNA) nucleotides, cyclohexenyl nucleic acid (CeNA) nucleotides, 2′-methoxyethyl nucleotides, 2′-O-allyl nucleotides, 2′-C-allyl nucleotides, 2′-fluoro nucleotides, 2′-O—N-methylacetamido (2′-O-NMA) nucleotides, 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotides, 2′-O-aminopropyl (2′-O-AP) nucleotides, and 2′-ara-F nucleotides.

### Claim 10 (depends on 2)

The dsRNA agent of claim 2, wherein the natural nucleotide is selected from the group consisting of 2′-OH nucleotides, 2′-OMe nucleotides, and 2′-deoxy nucleotides.

### Claim 11 (depends on 1)

The dsRNA agent of claim 1, wherein the ligand is an ASGPR ligand.

### Claim 12 (depends on 1)

The dsRNA agent of claim 1, wherein:
the sense strand has:
a length of 19-25 nucleotides and only two 2′-deoxy modifications in a central region of the sense strand, wherein the central region of the sense strand is positions 7, 8, 9, 10, 11, 12 and 13, counting from 5′-end of the sense strand;
and
the antisense strand has:
a length of 19-25 nucleotides, and
three, four, or five 2′-deoxy modifications in the antisense strand, and wherein three of the 2′-deoxy modifications in the antisense strand are at positions 2, 12 and 14, counting from the 5′-end of the antisense strand,
and
wherein the duplex region contains between 17 to 25 base pairs.

### Claim 13 (depends on 1)

The dsRNA agent of claim 1, wherein the dsRNA does not comprise a 2′-fluoro nucleotide.

### Claim 14 (depends on 1)

The dsRNA agent of claim 1, wherein the sense strand contains only two 2′-deoxy modifications and the antisense strand contains only three, four, five or six 2′-deoxy modifications.

### Claim 15 (depends on 1)

The dsRNA agent of claim 1, wherein the ligand is coupled to the 3′-end, 5′-end, and/or at an internal position of the sense strand.

### Claim 16 (depends on 1)

The dsRNA agent of claim 1, wherein the ligand is a cell or tissue targeting agent.

### Claim 17 (depends on 16)

The dsRNA agent of claim 16, wherein the ligand is an antibody.

### Claim 18 (depends on 1)

The dsRNA agent of claim 1, wherein the ligand is an integrin receptor ligand.

### Claim 19 (depends on 1)

The dsRNA agent of claim 1, wherein the ligand is a lipid or cholesterol.

### Claim 20 (depends on 1)

The dsRNA agent of claim 1, comprising a two nucleotide overhang at the 3′-end of the antisense strand and a blunt end at the 5′-end of the antisense strand.

### Claim 21 (depends on 1)

The dsRNA agent of claim 1, comprising blunt ends at both of the 5′-end and 3′-end of the dsRNA agent.

### Claim 22 (depends on 12)

The dsRNA agent of claim 12, wherein the dsRNA agents have all natural nucleotides, or less than 20% non-natural nucleotides.

### Claim 23 (depends on 12)

The dsRNA agent of claim 12, comprising a sense strand having a length of 15-17 nucleotides and an antisense strand having a length of 19-23 nucleotides.

### Claim 24 (depends on 22)

The dsRNA agent of claim 22, comprising a two nucleotide overhang at the 3′-end of the antisense strand and a blunt end at the 5′-end of the antisense strand.

### Claim 25 (depends on 22)

The dsRNA agent of claim 22, comprising blunt ends at both of the 5′-end and 3′-end of the dsRNA agent.

### Claim 26 (depends on 12)

The dsRNA agent of claim 12, wherein five 2′-deoxy modifications in the antisense strand at positions 2, 5, 7, 12 and 14 counting from the 5′-end of the antisense strand.

### Claim 27 (depends on 12)

The dsRNA agent of claim 12, wherein at least one of the 2′-deoxy modifications is in the sense strand at position 11, counting from the 5′-end of the sense strand.

### Claim 28 (depends on 12)

The dsRNA agent of claim 12, wherein at least two of the 2′-deoxy modifications are in the sense strand at positions 9 and 11 counting from the 5′-end of the sense strand.

### Claim 29 (depends on 12)

The dsRNA agent of claim 12, wherein five of the 2′-deoxy modifications are in the antisense strand at positions 2, 5, 7, 12 and 14 counting from the 5′-end of the antisense strand, and at least two of the 2′-deoxy modifications are in the sense strand at positions 9 and 11 counting from the 5′-end of the sense strand.

### Claim 30 (depends on 22)

The dsRNA agent of claim 22, wherein
the non-natural nucleotide is selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA) nucleotides, hexitol nucleic acid (HNA) nucleotides, cyclohexenyl nucleic acid (CeNA) nucleotides, 2′-methoxyethyl nucleotides, 2′-O-allyl nucleotides, 2′-C-allyl nucleotides, 2′-fluoro nucleotides, 2′-O—N-methylacetamido (2′-O-NMA) nucleotides, 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotides, 2′-O-aminopropyl (2′-O-AP) nucleotides, and 2′-ara-F nucleotides; and
the natural nucleotide is selected from the group consisting of 2′-OH nucleotides, 2′-OMe nucleotides, and 2′-deoxy nucleotides.

### Claim 31 (depends on 12)

The dsRNA agent of claim 12, wherein the ligand is an ASGPR ligand.

### Claim 32 (depends on 12)

The dsRNA agent of claim 12, wherein the dsRNA does not comprise a 2′-fluoro nucleotide.

### Claim 33 (depends on 12)

The dsRNA agent of claim 12, wherein the sense strand contains only two 2′-deoxy modifications and the antisense strand only contains three, four, five or six 2′-deoxy modifications.

### Claim 34 (depends on 12)

The dsRNA agent of claim 12, wherein the ligand is coupled to the 3′-end, 5′-end, and/or at an internal position of the sense strand.

### Claim 35 (depends on 12)

The dsRNA agent of claim 12, wherein the ligand is a cell or tissue targeting agent.

### Claim 36 (depends on 35)

The dsRNA agent of claim 35, wherein the ligand is an antibody.

### Claim 37 (depends on 12)

The dsRNA agent of claim 12, wherein the ligand is an integrin receptor ligand.

### Claim 38 (depends on 12)

The dsRNA agent of claim 12, wherein the ligand is a lipid or cholesterol.