Single-stranded nucleic acid molecule for regulating expression of gene having delivering function

• US 10,238,752 B2
• Filed: 04/25/2017
• Issued: 03/26/2019
• Est. Priority Date: 05/26/2012
• Status: Active Grant

• Current Assignee: Bonac Corporation
• Original Assignee: Bonac Corporation
• Inventors: Ohgi, Tadaaki, Aoki, Eriko, Emura, Chisato, Hamasaki, Tomohiro
• Primary Examiner(s): McDonald, Jennifer Pitrak
• Application Number:
  US15/496,143
• Publication Number:
  US 20170306325A1
• Time in Patent Office: 700 Days
• Field of Search: None
• CPC Class Codes: A61K 47/542 Carboxylic acids, e.g. a fatty acid or an amino acid
• CPC Class Codes: A61K 47/543 Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• CPC Class Codes: A61K 47/544 Phospholipids
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61K 47/551 one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
• CPC Class Codes: A61K 47/554 the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
• CPC Class Codes: A61K 47/64 Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent peptidic linkers A61K47/65
• CPC Class Codes: A61K 47/6803 Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• CPC Class Codes: A61K 47/6851 the antibody targeting a determinant of a tumour cell
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: 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
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12N 2310/533 having a mismatch or nick in at least one of the strands

Abstract

The invention provides a single-stranded nucleic acid capable of inhibiting expression of a target gene having a delivery function. The nucleic acid contains, from the 5′-side to the 3′-side, a 5′-side region (Xc), a linker region (Lx), an inner region (Z), a linker region (Ly) and a 3′-side region (Yc) in this order, wherein the inner region (Z) is constituted by linkage of the inner 5′-side region (X) and the inner 3′-side region (Y), 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) comprises an expression inhibitory sequence that inhibits expression of a target gene, and at least one of the 5′-terminus, the 3′-terminus, the linker region (Lx) and the linker region (Ly) is bound to a bio-related substance.

Claims

1. An isolated molecule for inhibiting expression of a target gene, wherein the molecule has a delivery function and comprises, in the following order, a 5′

-side region (Xc), a linker region (Lx), an inner region (Z), a linker region (Ly) and a 3′

-side region (Yc) which regions are linked together, wherein

the inner region (Z) is constituted by linkage of an inner 5′

-side region (X) and an inner 3′

-side region (Y),

the 5′

-side region (Xc), the inner 5′

-side region (X), the inner 3′

-side region (Y) and the 3′

side region (Yc) are each composed of a nucleotide residue or a nucleotide sequence,

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) comprises an expression inhibitory sequence that inhibits expression of a target gene,

the linker region (Lx) is linked to the 3′

-end of the 5′

-side region (Xc) and the 5′

-end of the inner 5′

-side region (X),

the linker region (Ly) is linked to the 3′

-end of the inner 3′

-side region (Y) and the 5′

-end of the 3′

-side region (Yc),

the linker region (Lx) and the linker region (Ly) are each composed of a nucleotide or non-nucleotide residue or a sequence of at least one nucleotide and non-nucleotide residues, wherein at least one selected from the group consisting of the linker region (Lx) and the linker region (Ly) comprises a lysine residue in such a manner that (i) the 5′

-side region (Xc) and the inner 5′

-side region (X) or (ii) the inner 3′

-side region (Y) and the 3′

-side region (Yc) are linked together via the lysine residue,

a bio-related substance is covalently bound to the molecule via the lysine residue, and

the bio-related substance is at least one selected from the group consisting of a peptide, polyethylene glycol (PEG), a polyamine, a fluorescence molecule, biotin, an intercalator molecule, a sugar, a water-soluble vitamin, a metal chelating agent, and a cross-linking agent.

2. The molecule according to claim 1, wherein the bio-related substance is at least one selected from the group consisting of an antibody protein, a peptide thereof and a membrane-permeable peptide.

3. The molecule according to claim 1, wherein the peptide is at least one selected from the group consisting of a protein bindable to a membrane protein of a target cell or a peptide thereof;

a protein selectively incorporated into the target site or a peptide thereof;

a biological molecule acting on a particular receptor or a peptide thereof; and

an antibody protein reacting with an antigen expressed on a cellular surface or a peptide thereof.

4. The molecule according to claim 1, wherein the peptide is at least one selected from the group consisting of an arginine peptide, and a Fab domain of an antibody protein reacting with an antigen expressed on a cellular surface.

5. The molecule according to claim 1, wherein a lipid is covalently bound to at least one selected from the group consisting of the 5′

-terminus of the 5′

-side region (Xc), the 3′

-terminus of the 3′

-side region (Yc), the linker region (Lx) and the linker region (Ly).

6. The molecule according to claim 5, wherein the lipid is at least one selected from the group consisting of a simple lipid, a complex lipid, a derived lipid, a single-chain lipid, a double-chain lipid, a glycolipid, a liposoluble vitamin and a steroid.

7. The molecule according to claim 5, wherein the lipid is at least one selected from the group consisting of palmitic acid, myristic acid, stearic acid and oleic acid.

8. The molecule according to claim 1, which satisfies the following conditions of the formulas (1) and (2):

Z=X+Y 

 

(1)

Z□

Xc+Yc 

 

(2)

wherein Z is a base number of the inner region (Z), X is a base number of the inner 5′

-side region (X), Y is a base number of the inner 3′

-side region (Y), Xc is a base number of the 5′

-side region (Xc) and Yc is a base number of the 3′

-side region (Yc).

9. The molecule according to claim 1, which comprises at least one modified nucleotide residue.

10. The molecule according to claim 1, which comprises a stable isotope.

11. The molecule according to claim 1, which comprises a non-modified nucleotide residue and/or a modified nucleotide residue.

12. The molecule according to claim 1, wherein the linker region (Lx) or the linker region (Ly), which does not comprise a lysine residue, is composed of any of the residues of the following (1)-(7):

(1) a non-modified nucleotide residue,

(2) a modified nucleotide residue,

(3) a non-modified nucleotide residue and a modified nucleotide residue,

(4) a non-nucleotide residue,

(5) a non-nucleotide residue and a non-modified nucleotide residue,

(6) a non-nucleotide residue and a modified nucleotide residue, and

(7) a non-nucleotide residue, a non-modified nucleotide residue and a modified nucleotide residue.

13. The molecule according to claim 1, wherein inhibiting expression of the target gene is effected by RNA interference.

14. The molecule according to claim 1, wherein the sequence of the residues in the molecule is the sequence of SEQ ID NO:

11, 13, or 15.

15. A composition for inhibiting an expression of a target gene, comprising a molecule according to claim 1.

16. A pharmaceutical composition comprising a molecule according to claim 1.

17. A method of inhibiting an expression of a target gene, comprising contacting a cell with a molecule according to claim 1.

18. The method according to claim 17, comprising a step of administering the molecule to a cell, tissue or organ.

19. The method according to claim 18, wherein the molecule is administered in vivo or in vitro.

20. The method according to claim 17, wherein the gene expression inhibition is an expression inhibition by RNA interference.

21. A method of inducing RNA interference to inhibit expression of a target gene, comprising contacting a cell with the molecule according to claim 1.

22. A method of treating disease, comprising a step of administering a molecule according to claim 1 to a patient to inhibit expression of a target gene causing disease.