# US 8,785,121 B2 · Single-stranded nucleic acid molecule for controlling gene expression

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- Number: US 8,785,121 B2
- Document ID: US8785121B2
- Status: Granted
- Kind: B2
- Application number: 13/254,150
- Filed: 2011-07-08
- Published: 2012-01-12
- Granted: 2014-07-22
- Earliest claimed: 2010-07-08
- Assignees: Bonac Corporation
- Applicants: Tadaaki Ohgi; Hirotake Hayashi; Hisao Shirohzu; Tomohiro Hamasaki; Akihiro Itoh; Hiroshi Suzuki
- Inventors: Tadaaki Ohgi; Hirotake Hayashi; Hisao Shirohzu; Tomohiro Hamasaki; Akihiro Itoh; Hiroshi Suzuki
- Web page: https://patentgrep.com/patent/US8785121B2

## 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 31/7105: Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
- A61K 31/7115: Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
- A61K 31/712: Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
- A61K 31/7125: Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2310/318: where the PO2 is completely replaced, e.g. MMI or formacetal
- C12N 2310/53: partially self-complementary or closed
- C12N 2310/531: Stem-loop; Hairpin
- C12N 2310/533: having a mismatch or nick in at least one of the strands
- C12N 2320/30: Special therapeutic applications
- C12N 2330/51: Specially adapted vectors
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

Provided is a novel nucleic acid molecule that is a single-stranded nucleic acid molecule including an expression inhibitory sequence that inhibits expression of a target gene. The single-stranded nucleic acid molecule includes, in sequence from the 5′ side to the 3′ side: a 5′ side region (Xc); an inner region (Z); and a 3′ side region (Yc). The inner region (Z) is composed of an inner 5′ side region (X) and an inner 3′ side region (Y) that are linked to each other. The 5′ side region (Xc) is complementary to the inner 5′ side region (X). The 3′ side region (Yc) is complementary to the inner 3′ side region (Y). At least one of the inner region (Z), the 5′ side region (Xc), and the 3′ side region (Yc) includes the expression inhibitory sequence.

## Claims

### Claim 1

A single-stranded nucleic acid molecule comprising:
an expression inhibitory sequence that inhibits expression of a target gene, wherein
the single-stranded nucleic acid molecule comprises, in sequence from a 5′ side to a 3′ side: a 5′ side region (Xc); a linker region (Lx); an inner 5′ side region (X); an inner 3′ side region (Y); a linker region (Ly); and a 3′ side region (Yc), the 5′ end and the 3′ end of the single-stranded nucleic acid molecule are not linked to each other,
the 5′ side region (Xc) and the inner 5′ side region (X) are linked to each other by the linker region (Lx), wherein the 5′ side region (Xc) has 1 to 29 bases,
the 3′ side region (Yc) and the inner 3′ side region (Y) are linked to each other by the linker region (Ly), wherein the 3′ side region (Yc) has 1 to 29 bases,
a inner region (Z) consists of the inner 5′ side region (X) and the inner 3′ side region (Y) that are linked to each other, and has 19 to 30 bases,
the 5′ side region (Xc) is complementary to the inner 5′ side region (X),
the 3′ side region (Yc) is complementary to the inner 3′ side region (Y), and
the inner region (Z), comprises the expression inhibitory sequence, and the expression inhibitory sequence is a ribonucleic acid (RNA) molecule.

### Claim 2 (depends on 1)

The single-stranded nucleic acid molecule according to claim 1, wherein the number of bases (Z) in the inner region (Z), the number of bases (X) in the inner 5′ side region (X), the number of bases (Y) in the inner 3′ side region (Y), the number of bases (Xc) in the 5′ side region (Xc), and the number of bases (Yc) in the 3′ side region (Yc) satisfy conditions of Expressions (1) and (2):
Z=X+Y (1)
Z≧Xc+Yc (2).

### Claim 3 (depends on 1)

The single-stranded nucleic acid molecule according to claim 1, wherein the number of bases (X) in the inner 5′ side region (X), the number of bases (Xc) in the 5′ side region (Xc), the number of bases (Y) in the inner 3′ side region (Y), and the number of bases (Yc) in the 3′ side region (Yc) satisfy any of conditions (a) to (d):
(a) Conditions of Expressions (3) and (4) are satisfied;
X>Xc (3)
Y=Yc (4)
(b) Conditions of Expressions (5) and (6) are satisfied;
X=Xc (5)
Y>Yc (6)
(c) Conditions of Expressions (7) and (8) are satisfied;
X>Xc (7)
Y>Yc (8)
(d) Conditions of Expressions (9) and (10) are satisfied;
X=Xc (9)
Y=Yc (10).

### Claim 4 (depends on 3)

The single-stranded nucleic acid molecule according to claim 3, wherein, in the conditions (a) to (d), the difference between the number of bases (X) in the inner 5′ side region (X) and the number of bases (Xc) in the 5′ side region (Xc), and the difference between the number of bases (Y) in the inner 3′ side region (Y) and the number of bases (Yc) in the 3′ side region (Yc) satisfy the following conditions:
(a) Conditions of Expressions (11) and (12) are satisfied;
X−Xc=1, 2 or 3 (11)
Y−Yc=0 (12)
(b) Conditions of Expressions (13) and (14) are satisfied;
X−Xc=0 (13)
Y−Yc=1, 2 or 3 (14)
(c) Conditions of Expressions (15) and (16) are satisfied;
X−Xc=1, 2 or 3 (15)
Y−Yc=1, 2 or 3 (16)
(d) Conditions of Expressions (17) and (18) are satisfied;
X−Xc=0 (17)
Y−Yc=0 (18).

### Claim 5 (depends on 1)

The single-stranded nucleic acid molecule according to claim 1, wherein the number of bases (Xc) in the 5′ side region (Xc) is 1 to 11.

### Claim 6 (depends on 5)

The single-stranded nucleic acid molecule according to claim 5, wherein the number of bases (Xc) in the 5′ side region (Xc) is 1 to 7.

### Claim 7 (depends on 5)

The single-stranded nucleic acid molecule according to claim 5, wherein the number of bases (Xc) in the 5′ side region (Xc) is 1 to 3.

### Claim 8 (depends on 1)

The single-stranded nucleic acid molecule according to claim 1, wherein the number of bases (Yc) in the 3′ side region (Yc) 1 to 11.

### Claim 9 (depends on 8)

The single-stranded nucleic acid molecule according to claim 8, wherein the number of bases (Yc) in the 3′ side region (Yc) is 1 to 7.

### Claim 10 (depends on 8)

The single-stranded nucleic acid molecule according to claim 8, wherein the number of bases (Yc) in the 3′ side region (Yc) is 1 to 3.

### Claim 11 (depends on 1)

The single-stranded nucleic acid molecule according to claim 1, wherein the single-stranded nucleic acid molecule comprises at least one modified residue.

### Claim 12 (depends on 1)

The single-stranded nucleic acid molecule according to claim 1, further comprising a labeling substance.

### Claim 13 (depends on 1)

The single-stranded nucleic acid molecule according to claim 1, further comprising a stable isotope.

### Claim 14 (depends on 1)

The single-stranded nucleic acid molecule according to claim 1, which is an RNA molecule.

### Claim 15 (depends on 1)

The single-stranded nucleic acid molecule according to claim 1, wherein the linker region (Lx) and/or the linker region (Ly) is composed of at least one of a nucleotide residue and a non-nucleotide residue.

### Claim 16 (depends on 15)

The single-stranded nucleic acid molecule according to claim 15, wherein the nucleotide residue is an unmodified nucleotide residue and/or a modified nucleotide residue.

### Claim 17 (depends on 15)

The single-stranded nucleic acid molecule according to claim 15, wherein the linker region (Lx) and/or the linker region (Ly) is composed of any of residues (1) to (7):
(1) an unmodified nucleotide residue
(2) a modified nucleotide residue
(3) an unmodified nucleotide residue and a modified nucleotide residue
(4) a non-nucleotide residue
(5) a non-nucleotide residue and an unmodified nucleotide residue
(6) a non-nucleotide residue and a modified nucleotide residue
(7) a non-nucleotide residue, an unmodified nucleotide residue, and a modified nucleotide residue.

### Claim 18 (depends on 1)

The single-stranded nucleic acid molecule according to claim 1, wherein the total number of bases in the single-stranded nucleic acid molecule is 50 or more.

### Claim 19 (depends on 1)

The single-stranded nucleic acid molecule according to claim 1, wherein expression of the gene is inhibited by RNA interference.

### Claim 20 (depends on 1)

The single-stranded nucleic acid molecule according to claim 1, wherein a base sequence of the single-stranded nucleic acid molecule is any of SEQ ID NOs: 2, 7, 8, 13, 14, 29 to 35, 37, 43, 44, 47, 48, and 51 to 80.

### Claim 21 (depends on 1)

A composition for inhibiting expression of a target gene, the composition comprising: the single-stranded nucleic acid molecule according to claim 1.

### Claim 22 (depends on 1)

A pharmaceutical composition comprising: the single-stranded nucleic acid molecule according to claim 1.

### Claim 23 (depends on 1)

A method for inhibiting expression of a target gene, the method comprising the step of: using the single-stranded nucleic acid molecule according to claim 1.

### Claim 24 (depends on 23)

The method according to claim 23, comprising the step of: administering the single-stranded nucleic acid molecule to a cell, a tissue, or an organ.

### Claim 25 (depends on 24)

The method according to claim 24, wherein the single-stranded nucleic acid molecule is administered in vivo or in vitro.

### Claim 26 (depends on 23)

The method according to claim 23, wherein expression of the gene is inhibited by RNA interference.

### Claim 27 (depends on 1)

A method for inducing RNA interference that inhibits expression of a target gene, the method comprising the step of: using the single-stranded nucleic acid molecule according to claim 1.

### Claim 28 (depends on 1)

The single-stranded nucleic acid molecule according to claim 1, wherein the linker region (Lx) and/or the linker region (Ly) has 3 to 50 bases and at least one non-nucleotide residue.

### Claim 29 (depends on 1)

The single-stranded nucleic acid molecule according to claim 1, wherein the expression inhibitory sequence inhibits expression of a GAPDH gene, TGF-β1 gene, LAMA1 gene, or LMNA gene.

### Claim 30 (depends on 1)

A method of treating an acute lung injury comprising administering the single-stranded nucleic acid molecule according to claim 1 to a patient, wherein the single-stranded nucleic acid molecule comprises, as the expression inhibitory sequence, a sequence that inhibits expression of a TGF-β1 gene expressed during the acute lung injury.