# US 8,772,469 B2 · Synthetic double-stranded oligonucleotides for specific inhibition of gene expression

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- Number: US 8,772,469 B2
- Document ID: US8772469B2
- Status: Granted
- Kind: B2
- Application number: 10/193,747
- Filed: 2002-07-11
- Published: 2003-07-24
- Granted: 2014-07-08
- Earliest claimed: 2001-07-12
- Assignees: Sanofi-Aventis Deutschland GmbH
- Applicants: Eugen Uhlmann; Jochen Huber; Niki Gunkel; Sandra Neumann
- Inventors: Eugen Uhlmann; Jochen Huber; Niki Gunkel; Sandra Neumann
- Web page: https://patentgrep.com/patent/US8772469B2

## CPC

- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 11/06: Antiasthmatics
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/04: Antibacterial agents
- A61P 31/12: Antivirals
- A61P 35/00: Antineoplastic agents
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/31: of the backbone
- C12N 2310/315: Phosphorothioates
- C12N 2310/319: linked by 2'-5' linkages, i.e. having a free 3'-position
- C12N 2310/53: partially self-complementary or closed
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- 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 oligonucleotide derivatives which are at least partly double-stranded and which have a 2′5′-linked oligonucleotide residue on at least one 3′ end and to the use thereof for specific inhibition of gene expression.

## Claims

### Claim 1

A double-stranded RNA oligonucleotide of Formula I
5′-(U)vX-(N)x-(W)-3′
3′-(Z)n-(N′)y-5′ Formula I
wherein
N and N′ are naturally occurring nucleotides bound by 3′,5′ internucleotide bonds, wherein a portion of the oligonucleotide is double-stranded between (N)x and (N′)y, and wherein the nucleotide strand (N)x, is sufficiently complementary to a target RNA to hybridize thereto and inhibit its expression through RNA interference, and wherein the nucleotide strand (N′)y is at least partially homologous to the target RNA, and wherein
N is selected from the group consisting of adenine (A), cytosine (C), guanine (G), and uracil (U);
x is independently 15-25;
y is independently 15-25;
n is independently 4 to 6;
v is independently 1 to 20, whereas: when v=1, X is selected from the group consisting of A, C, and G, and when v=2 to 20, X is absent;
W is selected from the group consisting of U2-20, UA, UC, and UG, or is absent; and Z is A; nucleotides of W being linked via a 3′,5′ internucleotide bond, and
wherein at least two nucleotides of Z are linked via a 2′,5′ internucleotide bond and are present in a single-stranded form that does not base pair with the target gene or with the RNA homologous thereto, thereby forming a 2′,5′ linked oligonucleotide residue on the 3′ end of the nucleotide strand (N′)y and wherein the internucleotide bonds in the double-stranded oligonucleotide are 3′,5′ or 2′,5′ internucleotide bonds.

### Claim 2 (depends on 1)

The double-stranded RNA of claim 1, wherein v is 2 to 10.

### Claim 3 (depends on 2)

The double-stranded RNA of claim 2, wherein v is 2 to 6.

### Claim 4 (depends on 1)

The double-stranded RNA of claim 1, wherein U2-20 is U2-10.

### Claim 5 (depends on 4)

The double-stranded RNA of claim 4, wherein U2-20 is U2-6.

### Claim 6 (depends on 1)

The double-stranded RNA of claim 1, wherein x is 16 to 23.

### Claim 7 (depends on 6)

The double-stranded RNA of claim 6, wherein x is 19 to 21.

### Claim 8 (depends on 1)

The double-stranded RNA of claim 1, wherein Z is adenosine or 3′-deoxyadenosine.

### Claim 9 (depends on 1)

The double-stranded RNA of claim 1, in which one or more natural phosphodiester bonds have been replaced by unnatural internucleotide bonds that stabilize against nuclease degradation.

### Claim 10 (depends on 9)

The double-stranded RNA of claim 9, in which one or more natural phosphodiester bonds have been replaced by phosphorothioate bonds.

### Claim 11 (depends on 10)

The double-stranded RNA of claim 10, in which a plurality of natural phosphodiester bonds have been replaced by phosphorothioate bonds, with said replacements being located on the 5′ or 3′ ends of the oligonucleotide, and on internal pyrimidine nucleotides of the oligonucleotide.

### Claim 12 (depends on 1)

The double-stranded RNA of claim 1, wherein one of nucleotide strands (N)x or (N′)y, is between 23 and 65 nucleotides in length and the other of the nucleotide strands is between 23 and 31 nucleotides in length.

### Claim 13 (depends on 1)

The double stranded RNA of claim 1, wherein the double stranded RNA comprises one or more 2′ O-methylribonucleotides.

### Claim 14 (depends on 1)

The double stranded RNA of claim 1, wherein the double stranded RNA comprises one or more 2′ fluoro-2′-deoxyribonucleotides.

### Claim 15 (depends on 1)

The double stranded RNA of claim 1, wherein the double stranded RNA comprises one or more locked nucleic acids (LNA).

### Claim 16 (depends on 1)

The double stranded RNA of claim 1, wherein the double stranded RNA further comprises a linker that links the two RNA strands.

### Claim 17 (depends on 16)

The double stranded RNA of claim 16, wherein the linker is a nucleotide linker.

### Claim 18 (depends on 16)

The double stranded RNA of claim 16, wherein the nucleotide linker comprises thymidine.

### Claim 19 (depends on 16)

The double stranded RNA of claim 16, wherein the nucleotide linker comprises 4-20 nucleotide residues.

### Claim 20 (depends on 19)

The double stranded RNA of claim 19, wherein the 4-20 nucleotide residues comprise one or more thymidines.

### Claim 21 (depends on 19)

The double stranded RNA of claim 19, wherein the 4-20 nucleotide residues are thymidines.

### Claim 22 (depends on 16)

The double stranded RNA of claim 16, wherein the nucleotide linker comprises 4-5 nucleotide residues.

### Claim 23 (depends on 22)

The double stranded RNA of claim 22, wherein the 4-5 nucleotide residues comprise one or more thymidines.

### Claim 24 (depends on 23)

The double stranded RNA of claim 23, wherein the 4-5 nucleotide residues are thymidines.

### Claim 25 (depends on 16)

The double stranded RNA of claim 16, wherein the linker is a non-nucleotide linker.