Ligand-Modified Double-Stranded Nucleic Acids

• US 20170305956A1
• Filed: 06/13/2017
• Published: 10/26/2017
• Est. Priority Date: 12/15/2014
• Status: Active Application

• Current Assignee: Dicerna Pharmaceuticals, Inc. (Novo Nordisk A/S)
• Original Assignee: Dicerna Pharmaceuticals, Inc. (Novo Nordisk A/S)
• Inventors: Brown, Bob D., Wang, Weimin
• Application Number:
  US15/621,395
• Publication Number:
  US 20170305956A1
• Time in Patent Office: Days
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 1/00 Processes for the preparation of sugar derivatives
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07H 99/00 Subject matter not provided for in other groups of this subclass
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 2310/351 Conjugate
• CPC Class Codes: C12N 2310/3515 Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2310/533 having a mismatch or nick in at least one of the strands
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance
• CPC Class Codes: C12N 2330/30 chemically synthesised

Abstract

The invention provides for double stranded nucleic acid molecules comprising a 5′ extension of the sense or antisense strand and further comprising a plurality of nucleotides that are conjugated to a ligand and methods of using the double-stranded nucleic acid molecules. Ligand-modified oligomers where the sense stands form a tetraloop provide new potent and stable RNA interference agents. These dsNA molecules are synthesized using a plurality of nucleotides that include ligand-modified monomers, nucleotide analog monomers, modified nucleotide monomers and the like, using standard nucleotide synthetic methods and systems.

Claims

1. A double stranded nucleic acid (dsNA) comprising:

a sense strand comprising 21 to 83 nucleotides;

an antisense strand comprising 15 to 39 nucleotides;

a duplex formed by said sense and antisense strand, having a length of 15 to 35 base pairs;

wherein the dsNA comprises a discontinuity between the 5′

terminus of the sense strand and the 3′

terminus of the antisense strand or between the 3′

terminus of the sense strand and the 5′

terminus of the antisense strand;

wherein the sense strand comprises a tetraloop and the tetraloop comprises at least one ligand conjugated nucleotide; and

wherein said antisense strand is sufficiently complementary to a target mRNA along at least 15 nucleotides of said second strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammal or a mammalian cell.

2. The dsNA of claim 1, wherein the tetraloop comprises one ligand conjugated nucleotide.

3. The dsNA of claim 1, wherein the tetraloop comprises two ligand conjugated nucleotides.

4. The dsNA of claim 1, wherein the tetraloop comprises three ligand conjugated nucleotides.

5. The dsNA of claim 1, where in the tetraloop comprises four ligand conjugated nucleotides.

6. The dsNA of claim 1, wherein at least one of the ligands is conjugated to a nucleotide of the tetraloop through the 2′

hydroxyl on the ribose of the nucleotide.

7. The dsNA of claim 6, wherein each ligand is conjugated to a nucleotide of the tetraloop through the 2′

hydroxyl on the ribose of the nucleotide.

8. The dsNA of claim 1, wherein the ligand is GalNAc.

9. The dsNA of claim 1, wherein the ligand is mannose-6-phosphate.

10. The dsNA of claim 1, wherein the tetraloop comprises three ligand conjugated nucleotides and each of the ligands is GalNAc.

11. The dsNA of claim 1, wherein the tetraloop comprises four ligand conjugated nucleotides and each of the ligands is GalNAc.

12. The dsNA of claim 2, 3, 4 or 5, wherein each of the ligands is GalNAc.

13. The dsNA of claim 1, wherein said antisense strand has a length range of:

15-30 nucleotides, 18-25 nucleotides or 19-24 nucleotides.

14. The dsNA of claim 1, wherein said duplex has a length range of:

15-22 nucleotides or 15-30 nucleotides.

15. The dsNA of claim 1, wherein said ligand is selected from the group consisting of:

a lipophile, a steroid, a protein, a vitamin, a carbohydrate, and a terpene.

16. The dsNA of claim 1, wherein said ligand is selected from the group consisting of:

N-acetyl galactosamine, cholesterol, cholic acid, adamantine acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), bile acid, PEG, folate, vitamin A, vitamin E, biotin, pyridoxal, a peptide, peptide mimic, mannose-6-phosphate, galactose, fructose, ribose, xylose, arabinose, lyxose, allose, altrose, gulose, iodose, glucose, talose, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, polysaccharide, an endosomolytic component, uvaol, hecigenin, diosgenin, triterpenesarsasapogenin, friedelin, epifriedelanol-derivatized lithocholic acid, a cationic lipid, and an antibody.

17. The dsNA of claim 1, wherein said dsNA comprises at least one modified nucleotide.

18. The dsNA of claim 17, wherein the modified nucleotide comprises a sugar modification selected from the group consisting of:

2′

-O-methyl, 2′

-methoxyethoxy, 2′

-fluoro, 2′

-allyl, 2′

-O-[2-(methylamino)-2-oxoethyl], 4′

-thio, 4′

-CH₂—

O-2′

-bridge, 4′

-(CH₂)₂—

O-2′

-bridge, 2′

-LNA, 2′

-amino, and 2′

-O—

(N-methylcarbamate) modification.

19. The dsNA of claim 1, comprising a backbone modification selected from the group consisting of:

phosphonate, phosphorothioate, phosphotriester, methylphosphonate, unlocked nucleic acid (UNA), locked nucleic acid (LNA), morpholino, SATE (S-acyl-2-thioethyl) modified phosphate, BMEG (Isobutyryl Mercapto Ethyl Glycol) modified phosphate and bicyclic furanose analog modification.

20. The dsNA of claim 1, comprising a region containing at least one phosphorothioate linkage.

21. The dsNA of claim 1 wherein both sense and antisense strands consist of up to 100% chemically modified nucleotides.