# US 11,027,023 B2 · Natural type miRNA for controlling gene expression, and use of same

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- Number: US 11,027,023 B2
- Document ID: US11027023B2
- Status: Granted
- Kind: B2
- Application number: 15/539,226
- Filed: 2015-12-25
- Published: 2018-11-15
- Granted: 2021-06-08
- Earliest claimed: 2014-12-27
- Assignees: Bonac Corporation
- Applicants: Bonac Corporation
- Inventors: Eriko Aoki; Yasuhiko Yoshida; Shiori Kato; Tadaaki Ohgi
- Web page: https://patentgrep.com/patent/US11027023B2

## CPC

- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- 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 47/18: Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K 47/34: Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers (A61K47/10 takes precedence)
- A61K 47/42: Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein (oligopeptides having up to five amino acids {A61K47/183}; polyamino acids A61K47/34)
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61P 35/00: Antineoplastic agents
- 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 2310/141: MicroRNAs, miRNAs
- C12N 2310/318: where the PO2 is completely replaced, e.g. MMI or formacetal
- C12N 2310/3181: Peptide nucleic acid, PNA
- C12N 2310/531: Stem-loop; Hairpin
- C12N 2330/30: chemically synthesised
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention provides a natural type miRNA, which is a single-stranded nucleic acid containing X region and Y region, wherein the 3′-terminus of said X region and the 5′-terminus of said Y region are linked via a linker region of a non-nucleotide structure, the X region contains (a) a guide strand sequence or (b) a passenger strand sequence of a mature miRNA, when the X region contains (a), the Y region contains a passenger strand sequence of the mature miRNA, when the X region contains (b), the Y region contains a guide strand sequence of the mature miRNA, and the guide strand sequence and the passenger strand sequence form a double-stranded structure.

## Claims

### Claim 1

A single-stranded nucleic acid comprising X region and Y region,
wherein
the 3′-terminus of said X region and the 5′-terminus of said Y region are linked via a linker region of a non-nucleotide structure,
said X region comprises (a) a guide strand sequence or (b) a passenger strand sequence of a mature miRNA,
when the X region comprises (a), said Y region comprises a passenger strand sequence of said mature miRNA,
when the X region comprises (b), said Y region comprises a guide strand sequence of said mature miRNA,
said X region further comprises an additional sequence of 3-5 base length, and
said guide strand sequence and said passenger strand sequence form a double-stranded structure.

### Claim 2 (depends on 1)

The single-stranded nucleic acid according to claim 1, wherein when said Y region and said X region are aligned, said Y region has an overhang on the 3′-terminus.

### Claim 3 (depends on 2)

The single-stranded nucleic acid according to claim 2, wherein said overhang has a 1-4 base length.

### Claim 4 (depends on 1)

The single-stranded nucleic acid according to claim 1, wherein said X region comprises said guide strand or passenger strand sequence and said additional sequence, and said additional sequence is linked to the 3′-terminus of said guide strand or passenger strand sequence.

### Claim 5 (depends on 1)

The single-stranded nucleic acid according to claim 1, wherein said X region has a length of 19-35 bases, and/or said Y region has a length of 21-37 bases, and/or the full length miRNA has a 40-68 base length.

### Claim 6 (depends on 1)

The single-stranded nucleic acid according to claim 1, wherein said linker region comprises at least one selected from the group consisting of an amino acid residue, a polyamine residue, and a polycarboxylic acid residue.

### Claim 7 (depends on 6)

The single-stranded nucleic acid according to claim 6, wherein said polycarboxylic acid residue is a terephthalic acid residue.

### Claim 8 (depends on 6)

The single-stranded nucleic acid according to claim 6, wherein said amino acid residue is a glycine residue, a terephthalic acid amide residue, a proline residue or a lysine residue.

### Claim 9 (depends on 6)

The single-stranded nucleic acid according to claim 6, wherein said amino acid residue comprises a plurality of amino acid residues linked to each other.

### Claim 10 (depends on 9)

The single-stranded nucleic acid according to claim 9, wherein said plurality of amino acid residues are linked to form a residue of a glycine dimer or trimer.

### Claim 11 (depends on 1)

The single-stranded nucleic acid according to claim 1, wherein said linker residue is represented by the following chemical formula (I-0):
in said chemical formula (I-0),
Q11 and Q12 are each independently a single bond, CH2 (a methylene group), NH (an imino group), C═O (a carbonyl group), C═S (a thiocarbonyl group), C═NH (an iminomethylene group), O, or S,
Q1 and Q2 are each independently a single bond, CH2 (a methylene group), NH (an imino group), C═O (a carbonyl group), C═S (a thiocarbonyl group), C═NH (an iminomethylene group), O, or S,
Y1 and Y2 are each independently a single bond, CH2, NH, O, or S;
L1 is an alkylene chain having n carbon atoms, and a hydrogen atom on an alkylene carbon atom may or may not be substituted with OH, ORa, NH2, NHRa, NRaRb SH, or SRa, or,
L1 is a polyether chain obtained by substituting at least one carbon atom on said alkylene chain with an oxygen atom,
provided that: when Y1 is NH, O, or S, an atom bound to Y1 in L1 is carbon, an atom bound to OR1 in L1 is carbon, and oxygen atoms are not adjacent to each other;
L2 is an alkylene chain having m carbon atoms, and a hydrogen atom on an alkylene carbon atom may or may not be substituted with OH, ORc, NH2, NHRc, NRcRd, SH, or SRc, or
L2 is a polyether chain obtained by substituting at least one carbon atom on said alkylene chain with an oxygen atom,
provided that: when Y2 is NH, O, or S, an atom bound to Y2 in L2 is carbon, an atom bound to OR2 in L2 is carbon, and oxygen atoms are not adjacent to each other;
Ra, Rb, Rc, and Rd are each independently a substituent or a protecting group;
m is an integer in the range from 0 to 30;
n is an integer in the range from 0 to 30;
said regions X and Y are each linked to said linker residue via —OR1— or —OR2—,
wherein R1 and R2 may or may not be present, and when they are present, R1 and R2 are each independently a nucleotide residue or said structure (I-0); and
A is any atomic group.

### Claim 12 (depends on 1)

The single-stranded nucleic acid according to claim 1, wherein said linker region comprises an amino acid residue, and said amino acid residue is represented by the following chemical formula (I):
in said chemical formula (I),
X1 and X2 are each independently H2, O, S or NH;
Y1 and Y2 are each independently a single bond, CH2, NH, O or S;
L1 is an alkylene chain having n carbon atoms, and a hydrogen atom on an alkylene carbon atom may or may not be substituted with OH, ORa, NH2, NHRa, NRaRb, SH, or SRa, or,
L1 is a polyether chain obtained by substituting at least one carbon atom on said alkylene chain with an oxygen atom,
provided that: when Y1 is NH, O, or S, an atom bound to Y1 in L1 is carbon, an atom bound to OR1 in L1 is carbon, and oxygen atoms are not adjacent to each other;
L2 is an alkylene chain having m carbon atoms, and a hydrogen atom on an alkylene carbon atom may or may not be substituted with OH, ORc, NH2, NHRc, NRcRd, SH, or SRc, or
L2 is a polyether chain obtained by substituting at least one carbon atom on said alkylene chain with an oxygen atom,
provided that: when Y2 is NH, O, or S, an atom bound to Y2 in L2 is carbon, an atom bound to OR2 in L2 is carbon, and oxygen atoms are not adjacent to each other;
Ra, Rb, Rc, and Rd are each independently a substituent or a protecting group;
m is an integer in the range from 0 to 30;
n is an integer in the range from 0 to 30;
said regions X and Y are each linked to said amino acid residue via —OR1— or —OR2—,
wherein R1 and R2 may or may not be present, and when they are present, R1 and R2 are each independently a nucleotide residue or said structure (I); and
A is any atomic group, provided that the following chemical formula (Ia) is an amino acid or peptide:

### Claim 13 (depends on 11)

The single-stranded nucleic acid according to claim 11, wherein said chemical formula (I-0) or (I) has a structure of the following chemical formulae (I-1)-(I-7) and, in the following chemical formulae (I-1)-(I-7), n is an integer of 0-30, and m is an integer of 0-30:

### Claim 14 (depends on 13)

The single-stranded nucleic acid according to claim 13, wherein,
(i) in said chemical formula (I-1), n=11 and m=12, or n=5 and m=4;
(ii) in said chemical formula (I-4), n=5 and m=4;
(iii) in said chemical formula (I-6), n=4 and m=4; or
(iv) in said chemical formula (I-7), n=5 and m=4.

### Claim 15 (depends on 1)

The single-stranded nucleic acid according to claim 1, wherein a non-nucleotide structure of said linker region comprises at least one of a pyrrolidine skeleton and a piperidine skeleton.

### Claim 16 (depends on 1)

The single-stranded nucleic acid according to claim 1, wherein said non-nucleotide structure is represented by the following formula (II):
wherein
X1 and X2 are each independently H2, O, S, or NH;
Y1 and Y2 are each independently a single bond, CH2, NH, O, or S;
R3 is a hydrogen atom or a substituent which is bonded to C-3, C-4, C-5 or C-6 on ring A,
L1 is an alkylene chain having n atoms, and a hydrogen atom on an alkylene carbon atom may or may not be substituted with OH, ORa, NH2, NHRa, NRaRb, SH, or SRa, or,
L1 is a polyether chain obtained by substituting at least one carbon atom on said alkylene chain with an oxygen atom,
provided that: when Y1 is NH, O, or S, an atom bound to Y1 in L1 is carbon, an atom bound to OR1 in L1 is carbon, and oxygen atoms are not adjacent to each other;
L2 is an alkylene chain having m atoms, and a hydrogen atom on an alkylene carbon atom may or may not be substituted with OH, ORc, NH2, NHRc, NRcRd, SH, or SRc, or
L2 is a polyether chain obtained by substituting at least one carbon atom on said alkylene chain with an oxygen atom,
provided that: when Y2 is NH, O, or S, an atom bound to Y2 in L2 is carbon, an atom bound to OR2 in L2 is carbon, and oxygen atoms are not adjacent to each other;
Ra, Rb, Rc, and Rd are each independently a substituent or a protecting group;
l is 1 or 2;
m is an integer in the range from 0 to 30;
n is an integer in the range from 0 to 30; and
in ring A, one carbon atom other than said C-2 on ring A may be substituted by nitrogen, oxygen or sulfur, and may contain, in said ring A, a carbon-carbon double bond or a carbon-nitrogen double bond,
said regions (X) and (Y) are each linked to said non-nucleotide structure via —OR1— or —OR2—,
wherein R1 and R2 may or may not be present, and when they are present, R1 and R2 are each independently a nucleotide residue or said structure (II).

### Claim 17 (depends on 1)

The single-stranded nucleic acid according to claim 1, wherein said X region comprises a guide strand sequence of a mature miRNA selected from the group consisting of hsa-miR-34 or hsa-let-7.

### Claim 18 (depends on 1)

The single-stranded nucleic acid according to claim 1, wherein said X region comprises a passenger strand sequence of a mature miRNA of hsa-miR-29b.

### Claim 19 (depends on 1)

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