# US 8,541,385 B2 · Chemically modified oligonucleotides for use in modulation micro RNA and uses thereof

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- Number: US 8,541,385 B2
- Document ID: US8541385B2
- Status: Granted
- Kind: B2
- Application number: 12/714,863
- Filed: 2010-03-01
- Published: 2010-09-02
- Granted: 2013-09-24
- Earliest claimed: 2005-08-10
- Assignees: The Rockefeller University; Alnylam Pharmaceuticals, Inc.
- Applicants: Markus Stoffel; Muthiah Manoharan; Kallanthottathil G Rajeev
- Inventors: Markus Stoffel; Muthiah Manoharan; Kallanthottathil G Rajeev
- Web page: https://patentgrep.com/patent/US8541385B2

## 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)}
- C12N 2310/11: Antisense
- C12N 2310/315: Phosphorothioates
- C12N 2310/321: 2'-O-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 2310/3521: Methyl
- C12N 2310/3527: Other alkyl chain
- C12N 2310/3531: Hydrogen
- C12N 2310/3533: Halogen
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

This invention relates generally to chemically modified oligonuceotides useful for modulating expression of microRNAs and pre-microRNAs. More particularly, the invention relates to single stranded chemically modified oligonuceotides for inhibiting microRNA and pre-microRNA expression and to methods of making and using the modified oligonucleotides. Also included in the invention are compositions and methods for silencing microRNAs in the central nervous system.

## Claims

### Claim 1

A method of reducing plasma cholesterol levels in a mammal, the method comprising administering an antagomir to the mammal, wherein the antagomir is 15 to 25 nucleotides in length and has no more than two mismatches to the sequence ofmiR-122 (SEQ ID NO: 1), further wherein said antagomir comprises a non-nucleotide moiety at the 3′ -end, a 2′ -modification at each nucleotide, a phosphorothioate at the first and second internucleotide linkages at the 5′ end of the nucleotide sequence, a phosphorothioate at each of the first three internucleotide linkages at the 3′ end of the nucleotide sequence, and a phosphorothioate linkage between the 3′ terminal nucleotide and the non-nucleotide moiety, and wherein the remaining internucleotide linkages are phosphodiester.

### Claim 2 (depends on 1)

The method of claim 1, wherein the non-nucleotide moiety is a cholesterol moiety.

### Claim 3 (depends on 1)

The method of claim 1, wherein the 2′-modified nucleotide comprises a modification selected from the group consisting of: 2′-deoxy, 2′-deoxy-2′-fluoro,
2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and 2′-O——N-methylacetamido (2′-O-NMA).

### Claim 4 (depends on 3)

The method of claim 3, wherein the 2′-modified nucleotide comprises a 2′-O-methyl.

### Claim 5 (depends on 1)

The method of claim 1, wherein the antagomir has no more than 1 mismatches to the target sequence.

### Claim 6 (depends on 1)

The method of claim 1, wherein the antagomir has no mismatches to the target sequence.

### Claim 7 (depends on 1)

The method of claim 1, wherein the antagomir is 18 to 25 nucleotides in length.

### Claim 8 (depends on 1)

The method of claim 1, wherein the antagomir is present in a pharmaceutical composition.

### Claim 9 (depends on 1)

The method of claim 1, wherein the administering comprises parenteral administration.

### Claim 10 (depends on 1)

The method of claim 1, wherein the administering comprises intravenous administration.

### Claim 11 (depends on 1)

The method of claim 1, further comprising reducing HMG-CoA reductase activity.

### Claim 12 (depends on 1)

The method of claim 1, wherein the antagomir has the sequence of SEQ ID NO: 5, and the non-nucleotide moiety is a cholesterol moiety.