High-efficiency nanoparticle-type double-helical oligo-RNA structure and method for preparing same

Patent metadata

Field	Value
Publication number	US-9326941-B2
Application number	US-201314370035-A
Country	US
Kind code	B2
Filing date	Jan 4, 2013
Priority date	Jan 5, 2012
Publication date	May 3, 2016
Grant date	May 3, 2016

Abstract

Provided are a double-stranded oligo RNA structure and a method of preparing the same, and more specifically, a double-stranded oligo RNA structure in which a polymer compound is covalently bound to a double-stranded oligo RNA in order to improve stability in vivo and a cell delivery efficiency of the double-stranded oligo RNA, and a method of preparing the same.
The double-stranded oligo RNA structure having the optimized structure according to the present invention may not inhibit functions of the double-stranded oligo RNA, but effectively improve stability and cell membrane permeability of the double-stranded oligo RNA, such that the double-stranded oligo RNA may be delivered into the cell even at a low concentration dosage thereof to be significantly used as a tool for treatment of cancer, infectious diseases, and the like, as well as a new delivery system of the double-stranded oligo RNA.

First claim

The invention claimed is:

1. A double-stranded oligo RNA structure to which a chemical material having a structure represented by the following formula 1 is bound:

wherein one of A and B is a hydrophilic material, the other one is a hydrophobic material, X and Y are each a simple covalent bond or a linker-mediated covalent bond independently of each other, S is a sense strand of the double-stranded oligo RNA, and AS is an antisense strand of the double-stranded oligo RNA.

2. The double-stranded oligo RNA structure according to claim 1 , wherein the hydrophobic material is bound to 5′ end of a sense strand of the double-stranded oligo RNA and the hydrophilic material is bound to 3′ end thereof as shown in the following formula 3:

wherein A is a hydrophobic material, B is a hydrophilic material, X and Y are each a simple covalent bond or a linker-mediated covalent bond independently of each other, S is a sense strand of the double-stranded oligo RNA, and AS is an antisense strand of the double-stranded oligo RNA.

3. The double-stranded oligo RNA structure according to claim 1 , wherein the sense and antisense strand of the double-stranded oligo RNA strand consists of 19 to 31 nucleotides.

4. The double-stranded oligo RNA structure according to claim 3 , wherein nucleotide comprises one or two or more combinations of modifications selected from modification in which —OH group at 2′ carbon in a sugar structure in one or more nucleotides is substituted with —CH 3 (methyl), —OCH 3 (methoxy), —NH 2 , —F(fluorine), —O-2-methoxyethyl-O-propyl, —O-2-methylthioethyl, —O-3-aminopropyl, —O-3-dimethylaminopropyl, —O—N-methylacetamido or —O-dimethylamidooxyethyl; modification in which oxygen in a sugar structure in nucleotides is substituted with sulfur; and modification to phosphorothioate or boranophosphophate, methyl phosphonate bindings from bindings among nucleotides, and modification to peptide nucleic acid (PNA), locked nucleic acid (LNA) or unlocked nucleic acid (UNA).

5. The double-stranded oligo RNA structure according to claim 1 , wherein phosphate group(s) is bound to 5′ end of the antisense strand.

6. The double-stranded oligo RNA structure according to claim 5 , wherein one to three phosphate group(s) is bound to 5′ end of the antisense strand.

7. The double-stranded oligo RNA structure according to claim 1 , wherein the hydrophobic material has a molecular weight of 250 to 1,000.

8. The double-stranded oligo RNA structure according to claim 7 , wherein the hydrophobic material is selected from a group consisting of steroid derivative, a glyceride derivative, glycerol ether, polypropylene glycol, C12 to C50 unsaturated or saturated hydrocarbons, diacylphosphatidylcholine, fatty acid, phospholipid, lipopolyamine.

9. The double-stranded oligo RNA structure according to claim 8 , wherein the steroid derivative is selected from a group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate, and cholestanyl amine.

10. The double-stranded oligo RNA structure according to claim 8 , wherein the glyceride derivative is selected from a group mono-, di- and tri-glyceride.

11. The double-stranded oligo RNA structure according to claim 1 , wherein the hydrophilic material has a molecular weight of 200 to 10,000.

12. The double-stranded oligo RNA structure according to claim 11 , wherein the hydrophilic material selected from a group consisting of polyethylene glycol, polyvinyl pyrrolidone, polyoxazoline.

13. The double-stranded oligo RNA structure according to claim 1 , wherein the covalent bond is non-degradable bond or a degradable bond.

14. The double-stranded oligo RNA structure according to claim 13 , wherein non-degradable bond is an amide bond or a phosphorylation bond.

15. The double-stranded oligo RNA structure according to claim 13 , wherein the degradable bond is a disulfide bond, an acid degradable bond, an ester bond, an anhydride bond, a biodegradable bond or an enzymatically degradable bond.

16. The double-stranded oligo RNA structure according to claim 1 , wherein the double-stranded oligo RNA specifically binds to a sequence of survivin mRNA.

17. The double-stranded oligo RNA structure according to claim 16 , wherein sense strand of the double-stranded oligo RNA has the sequence of 5′-AAG GAG AUC AAC AUU UUC A-3′ (SEQ ID NO:1).

18. A double-stranded oligo RNA structure containing a ligand bound thereto comprising a ligand bound to a hydrophilic material of the double-stranded oligo RNA structure according to claim 1 .

19. The double-stranded oligo RNA structure containing a ligand bound thereto according to claim 18 , wherein the ligand comprises a targeting moiety specifically bound to a receptor promoting internalization of a target cell, through receptor-mediated endocytosis (RME).

20. The double-stranded oligo RNA structure containing a ligand bound thereto according to claim 19 , wherein the ligand is selected from a group consisting of target specific antibody, aptamer, peptide, and receptor specific chemical material.

21. The double-stranded oligo RNA structure containing a ligand bound thereto according to claim 20 , wherein the receptor specific chemical material is selected from a group consisting of folate, N-acetyl galactosamine (NAG) and mannose.

22. A method of preparing a double-stranded oligo RNA structure comprising:
(1) synthesizing an RNA single strand based on a solid support containing a hydrophilic material bound thereto;
(2) preparing an RNA-polymer structure by covalently binding a hydrophobic material to 5′ end of the RNA containing the hydrophilic material bound thereto;
(3) separating the RNA-polymer structure from the solid support; and
(4) forming the double-stranded oligo RNA structure by annealing the RNA-polymer structure and an RNA single strand of a complementary sequence thereto.

23. The method of preparing according to claim 22 , wherein a complementary sequence of the RNA single strand in the step (1) comprises phosphate group(s) bound to 5′ end of the RNA single strand.

24. A method of preparing a double-stranded oligo RNA structure comprising:
(1) synthesizing an RNA single strand based on a solid support containing a functional group bound thereto;
(2) covalently binding a hydrophilic material to the material obtained by the step (1);
(3) separating the material obtained by the step (2) from the solid support;
(4) forming the RNA-polymer structure by covalently binding a hydrophobic material through a functional group bound to 3′ end of the material obtained by the step (3); and
(5) forming the double-stranded oligo RNA structure by annealing the RNA-polymer structure prepared by the step (4) and an RNA single strand of a complementary sequence thereto.

25. The method of preparing according to claim 24 , wherein a complementary sequence of the RNA single strand in the step (1) comprises phosphate group(s) bound to 5′ end of the RNA single strand.

26. A nanoparticle comprising a double-stranded oligo RNA structure according to claim 1 .

27. A nanoparticle comprising a double-stranded oligo RNA structure containing a ligand bound thereto according to claim 18 .

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Assignees

• Bioneer Corp

Inventors

• Chae Jeiwook
• Han Boram
• Kim Han-Na
• Park Han Oh

Classifications

• A61P31/00

  Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics · CPC title

• C12N2310/3515

  Lipophilic moiety, e.g. cholesterol · CPC title

• C12N15/113

  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 title

• C12N2310/14

  interfering nucleic acids [NA] · CPC title

• C12N2320/30

  Special therapeutic applications · CPC title