# US 9,040,492 B2 · Double-stranded lipid-modified RNA having high RNA interference effect

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- Number: US 9,040,492 B2
- Document ID: US9040492B2
- Status: Granted
- Kind: B2
- Application number: 12/935,546
- Filed: 2009-03-31
- Published: 2011-02-10
- Granted: 2015-05-26
- Earliest claimed: 2008-03-31
- Assignees: National Institute of Advanced Industrial Science and Technology; Otsuka Pharmaceutical Co., Ltd.
- Applicants: Takanori Kubo; Hideki Ohba; Hidekazu Toyobuku; Hirotake Hayashi
- Inventors: Takanori Kubo; Hideki Ohba; Hidekazu Toyobuku; Hirotake Hayashi
- Web page: https://patentgrep.com/patent/US9040492B2

## CPC

- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- A61P 31/18: for HIV
- A61P 35/00: Antineoplastic agents
- C12N 2310/3515: Lipophilic moiety, e.g. cholesterol
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- 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

## Abstract

An object of the present invention is to provide a novel double-stranded RNA that has high resistance to nuclease and cellular uptake efficiency and that can produce an excellent RNA interference effect. The present invention provides a double-stranded lipid-modified RNA comprising an antisense strand having a nucleotide sequence complementary to a target sequence in a target gene, and a sense strand having a nucleotide sequence complementary to the antisense strand, the double-stranded RNA being capable of suppressing the expression of the target gene, and the sense strand having a double-stranded lipid bound directly or via a linker to at least one of the first to sixth nucleotides from the 5′ end.

## Claims

### Claim 1

A double-stranded lipid-modified RNA comprising an antisense strand having a nucleotide sequence complementary to a target sequence in a target gene, and a sense strand having a nucleotide sequence complementary to the antisense strand, the double-stranded lipid modified RNA being capable of suppressing expression of the target gene, and the sense strand having a double-stranded lipid bound directly or via a linker to at least one of the first to sixth nucleotides from the 5′ end, wherein said RNA does not contain any other nucleotide modifications, and
wherein said double-stranded lipid is a glycerophospholipid, and wherein said glycerophospholipid comprises two hydrophobic groups which each comprise a fatty acid residue.

### Claim 2 (depends on 1)

The double-stranded lipid-modified RNA according to claim 1, which is blunt-ended on the 5′-end side of the sense strand, and is blunt-ended or has a dangling end on the 3′-end side of the sense strand.

### Claim 3 (depends on 1)

The double-stranded lipid-modified RNA according to claim 1, which has dangling ends on both the 5′- and 3′-end sides of the sense strand.

### Claim 4 (depends on 1)

The double-stranded lipid-modified RNA according to claim 1, wherein the sense strand consists of 21 to 27 nucleotides.

### Claim 5 (depends on 2)

The double-stranded lipid-modified RNA according to claim 2, which is blunt-ended on both the 5′- and 3′-end sides of the sense strand, each of the sense and antisense strands consisting of 27 nucleotides.

### Claim 6 (depends on 2)

The double-stranded lipid-modified RNA according to claim 2, which is blunt-ended on both the 5′- and 3′-end sides of the sense strand, each of the sense and antisense strands consisting of 23 nucleotides.

### Claim 7 (depends on 2)

The double-stranded lipid-modified RNA according to claim 2, which is blunt-ended on the 5′-end side of the sense strand, the sense strand consisting of 25 nucleotides, and the antisense strand consisting of 23 nucleotides.

### Claim 8 (depends on 3)

The double-stranded lipid-modified RNA according to claim 3, wherein each of the sense and antisense strands consists of 21 nucleotides.

### Claim 9 (depends on 1)

The double-stranded lipid-modified RNA according to claim 1, wherein two hydrophobic groups of the double-stranded lipid are the same or different, and each is a saturated or unsaturated fatty acid residue having 6 to 50 carbon atoms.

### Claim 10 (depends on 1)

The double-stranded lipid-modified RNA according to claim 1, wherein the double-stranded lipid is phosphatidylethanolamine.

### Claim 11 (depends on 10)

The double-stranded lipid-modified RNA according to claim 10, wherein the double-stranded lipid is at least one member selected from the group consisting of dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, 1-palmitoyl-2-oleyl-phosphatidylethanolamine, and dioleoylphosphatidylethanolamine.

### Claim 12 (depends on 1)

The double-stranded lipid-modified RNA according to claim 1, wherein the lipid is bound to at least one of the first to sixth nucleotides from the 5′ end of the sense strand via a linker represented by the formula (L-27)
—CO—(CH2)n3—CO—NH—(CH2)b4 (L-27)
wherein n3 and n4 are the same or different, and each represents an integer of 1 to 20.

### Claim 13 (depends on 1)

A pharmaceutical composition comprising the double-stranded lipid-modified RNA of claim 1, and a pharmaceutically acceptable carrier.

### Claim 14 (depends on 1)

A method for suppressing the expression of a target gene, comprising a step of introducing the double-stranded lipid-modified RNA of claim 1 into a cell.