# US 12,760,306 B2 · siRNAs with vinylphosphonate at the 5′ end of the antisense strand

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- Number: US 12,760,306 B2
- Document ID: US12760306B2
- Status: Granted
- Kind: B2
- Application number: 17/993,559
- Filed: 2022-11-23
- Published: 2023-08-31
- Granted: 2026-10-13
- Earliest claimed: 2018-04-05
- Assignees: Silence Therapeutics GmbH
- Applicants: Silence Therapeutics GmbH
- Inventors: Adrien Weingärtner; Lucas Bethge
- Web page: https://patentgrep.com/patent/US12760306B2

## CPC

- 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 47/549: {Sugars, nucleosides, nucleotides or nucleic acids}
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/312: Phosphonates
- C12N 2310/315: Phosphorothioates
- C12N 2310/321: 2'-O-R Modification
- C12N 2310/322: 2'-R Modification
- C12N 2310/345: having at least two different backbone modifications
- C12N 2310/346: having a combination of backbone and sugar modifications
- C12N 2310/3515: Lipophilic moiety, e.g. cholesterol
- C12N 2320/51: modulating the chemical stability, e.g. nuclease-resistance
- 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

The present invention relates to nucleic acids for inhibiting expression of a target gene in a cell, comprising at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from said target gene to be inhibited. The first strand of the nucleic acid has a terminal 5′ (E)-vinylphosphonate nucleotide that is linked to the second nucleotide in the first strand by a phosphodiester linkage.

## Claims

### Claim 1

A nucleic acid for inhibiting expression of a target gene in a cell, comprising at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from said target gene to be inhibited,
and wherein the first strand has a terminal 5′ (E)-vinylphosphonate nucleotide linked by a phosphodiester linkage to the second nucleotide of the first strand and no phosphorothioate internucleotide linkages at the 5′ end of the first strand,
wherein the second strand of the nucleic acid is conjugated to at least one ligand portion comprising a compound of formula 1:
[S—X1—P—X2]3-A-X3— (1)
wherein:
S represents a saccharide;
X1 represents C3-C6 alkylene or (—CH2—CH2—O)m(—CH2)2—wherein m is 1, 2, or 3;
P is a phosphate or modified phosphate;
X2 is alkylene or an alkylene ether of the formula (—CH2)n—O—CH2—where n=1-6;
A is a branching unit;
X3 represents a bridging unit;
and wherein the nucleic acid is conjugated to X3 via a phosphate or modified phosphate, or
wherein the second strand of the nucleic acid is conjugated to at least one ligand portion comprising a compound of formula 2:
[S—X1—P—X2]3-A-X3— (2)
wherein:
S represents a saccharide;
X1 represents C3-C6 alkylene or an ethylene glycol stem (—CH2—CH2—O)m(—CH2)2—
wherein m is 1, 2, or 3;
P is a phosphate or modified phosphate;
X2 is C1-C8 alkylene;
A is a branching unit;
X3 is a bridging unit;
and wherein the nucleic acid is conjugated to X3 via a phosphate or modified phosphate.

### Claim 2 (depends on 1)

The nucleic acid of claim 1, wherein the first strand comprises more than one phosphodiester linkage.

### Claim 3 (depends on 1)

The nucleic acid of claim 1, wherein the first strand comprises phosphodiester linkages between
i) at least the terminal three 5′ nucleotides; or
ii) at least the terminal four 5′ nucleotides.

### Claim 4 (depends on 1)

The nucleic acid of claim 1, wherein the first strand comprises at least one phosphorothioate (ps) linkage.

### Claim 5 (depends on 1)

The nucleic acid of claim 1, wherein the first strand comprises
i) a phosphorothioate linkage between the terminal two 3′ nucleotides; or
ii) phosphorothioate linkages between the terminal three 3′ nucleotides.

### Claim 6 (depends on 5)

The nucleic acid of claim 5, wherein the linkages between the other nucleotides in the first strand are phosphodiester linkages.

### Claim 7 (depends on 1)

The nucleic acid of claim 1, wherein the second strand comprises
i) a phosphorothioate linkage between the terminal two or three 3′ nucleotides; and/or
ii) a phosphorothioate linkage between the terminal two or three 5′ nucleotides.

### Claim 8 (depends on 1)

The nucleic acid of claim 1, wherein the terminal 5′ (E)-vinylphosphonate nucleotide is a DNA or RNA nucleotide.

### Claim 9 (depends on 1)

The nucleic acid of claim 1, wherein
i) the first strand of the nucleic acid has a length in the range of 15-30 nucleotides; and/or
ii) the second strand of the nucleic acid has a length in the range of 15-30 nucleotides.

### Claim 10 (depends on 9)

The nucleic acid of claim 9, wherein one or more nucleotides on the first strand and/or the second strand is/are modified, to form modified nucleotides.

### Claim 11 (depends on 10)

The nucleic acid of claim 10, wherein the modification is
(a) a modification at the 2′-OH group of the ribose sugar comprising a locked nucleotide, an abasic nucleotide, a non-natural base-comprising nucleotide, 2′-O—methyl modification or 2′-fluoro modifications, or
(b) a modification at the 2′ position of the deoxyribose sugar, comprising 2′-methoxyethyl, 2′-OCH3, 2′—O-allyl, 2′-C-allyl, or 2′-fluoro.

### Claim 12 (depends on 1)

The nucleic acid of claim 1, wherein the ligand portion comprises
i) one or more GalNAc ligand;
ii) one or more GalNAc ligand derivatives; or
iii) a GalNAc moiety conjugated at the 5′ end of the second strand of the nucleic acid, optionally through a linker moiety.

### Claim 13 (depends on 1)

A composition comprising a nucleic acid of claim 1 and a physiologically acceptable excipient.

### Claim 14 (depends on 1)

The nucleic acid of claim 1, wherein the modified phosphate is a thiophosphate.

### Claim 15 (depends on 1)

The nucleic acid of claim 1, wherein the terminal 5′ (E)-vinylphosphonate nucleotide is an RNA nucleotide.

### Claim 16 (depends on 9)

The nucleic acid of claim 9, wherein
i) the first strand of the nucleic acid has a length in the range of 19-25 nucleotides; and/or
ii) the second strand of the nucleic acid has a length in the range of 19-25 nucleotides.

### Claim 17 (depends on 1)

The nucleic acid of claim 1, having one of the following structures:
wherein Z is the nucleic acid.

### Claim 18 (depends on 1)

A method for treatment of a disease or disorder in a subject in need thereof, comprising administering a nucleic acid according to claim 1 to said subject.