Enhanced antisense oligonucleotides

• US 20060063730A1
• Filed: 09/19/2005
• Published: 03/23/2006
• Est. Priority Date: 09/17/2004
• Status: Active Grant

• Current Assignee: Ionis Pharmaceuticals, Inc.
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Monia, Brett P., Bhanot, Sanjay, Siwkowski, Andrew M.
• Granted Patent: US 7,919,472 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: A61P 9/12 Antihypertensives
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/341 Gapmers, i.e. of the type ===---===
• CPC Class Codes: C12N 2310/346 having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3525 MOE, methoxyethoxy
• CPC Class Codes: C12N 2320/50 Methods for regulating/modulating their activity

Abstract

Described herein are gap-widened antisense oligonucleotides having improved therapeutic index as compared to 5-10-5 MOE gapmer antisense oligonucleotides of the same sequence. Also described are methods of reducing a target RNA in an animal using the gap-widened antisense oligonucleotides of the present invention. Further, are methods for selecting a gap-widened antisense oligonucleotides.

Claims

1. A method of reducing expression of a target RNA in an animal, in need of reducing expression of said target RNA, comprising administering to said animal a gap-widened antisense oligonucleotide 18-24 nucleotides in length comprising:

a gap region having greater than 11 contiguous 2′

-deoxyribonucleotides; and

a first wing region and a second wing region flanking the gap region, wherein each of said first and second wing regions independently have 1 to 8 2′

-O-(2-methoxyethyl) ribonucleotides, having an improved therapeutic index, improved potency, reduced tissue exposure, or reduced toxicity, or a combination thereof as compared to a corresponding 5-10-5 antisense oligonucleotide having a gap region of 10 contiguous 2′

-deoxyribonucleotides and a first wing region and a second wing region flanking the gap region of 5 2′

-O-(2-methoxyethyl) ribonucleotides.

2. The method of claim 1, wherein the improved therapeutic index is characterized by equal or increased potency and a reduction in tissue concentration, increased potency and equal tissue concentration, increased potency and decreased toxicity, or comparable potency and decreased toxicity as compared to a corresponding 5-10-5 antisense oligonucleotide.

3. The method of claim 1, wherein the gap-widened antisense oligonucleotide is characterized by an increased liver to kidney concentration as compared to corresponding 5-10-5 antisense oligonucleotide.

4. The method of claim 1, wherein the target RNA is associated with a metabolic or a cardiovascular disease or condition.

5. The method of claim 1, wherein the metabolic disease or condition is selected from metabolic syndrome, diabetes, obesity, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia, Type 2 diabetes, diet-induced obesity, hyperglycemia, insulin resistance, hepatic steatosis, fatty liver disease, or non-alcoholic steatohepatitis.

6. The method of claim 1, wherein the cardiovascular disease or condition is selected from familial hypercholesterolemia, nonfamilial hypercholesterolemia, mixed dyslipidemia, dysbetalipoproteinemia, atherosclerosis, coronary artery disease, myocardial infarction, hypertension, carotid artery diseases, stroke, cerebrovascular disease, carotid artery disease, stroke, cerebrovascular disease, peripheral vascular disease, thrombosis, or arterial aneurism.

7. The method of claim 1, wherein the gap-widened antisense oligonucleotide has a wing-gap-wing motif selected from 2-16-2, 3-14-3, or 4-12-4.

8. The method of claim 7, wherein the gap-widened antisense oligonucleotide has at least one phosphorothioate internucleotide linkage.

9. The method of claim 8, wherein the gap-widened antisense oligonucleotide has all phosphorothioate internucleotide linkages.

10. The method of claim 7, wherein gap-widened antisense oligonucleotide has at least one 5-methylcytosine.

11. A method of selecting a gap-widened antisense oligonucleotide with an improved therapeutic index, the method comprising:

screening in vitro a plurality of antisense oligonucleotides targeting a human RNA and having a single wing-gap-wing motif;

identifying a parent antisense oligonucleotide from the plurality of antisense oligonucleotides having a potent in vitro activity;

synthesizing a purality of gap-widened antisense oligonucleotides having the same sequence as the parent antisense oligonucleotide, wherein said gap-widened antisense oligonucleotide is 18 to 24 nucleotides in length comprising a gap region having greater than 11 contiguous 2′

-deoxyribonucleotides; and

a first wing region and a second wing region flanking the gap region, wherein each of said first and second wing regions independently has 1 to 8 2′

-O-(2-methoxyethyl) ribonucleotides;

testing said plurality of gap-widened antisense oligonucleotides in a plurality of animals;

obtaining tissue concentration data from said testing step; and

determining an optimized gap-widened oligonucleotide wing-gap-wing motif with an improved therapeutic index as compared to the parent antisense oligonucleotide.

12. The method of claim 11, further comprising the step of designing a rodent sequence analogous to said parent antisense oligonucleotide.

13. The method of claim 11, further comprising the step of designing a non-human primate sequence analogous to said parent antisense oligonucleotide.

14. The method of claim 13, wherein potency and concentration data are obtained from said testing step.

15. The method of claim 11, wherein determining the optimized gap-widened oligonucleotide wing-gap-wing motif with an improved therapeutic index is indicated by equal or increased potency as compared to the parent antisense oligonucleotide.

16. The method of claim 11, wherein for the step of screening, the plurality of antisense oligonucleotides has a wing-gap-wing motif selected from 2-16-2, 3-14-3, or 5-10-5.

17. The method of claim 11, wherein the screening is performed in primary hepatocytes, b.END, HepG2 or HeLa cells.

18. The method of claim 11, wherein for the step of selecting the potent in vitro activity is greater than 50% reduction in the target mRNA expression as compared to a saline control.

19. The method of claim 11, wherein the plurality of gap-widened antisense oligonucleotides each have a different wing-gap-wing motif.

20. The method of claim 11, wherein each gap-widened antisense oligonucleotides each have a 2′

-deoxyribonucleotide gap length selected from 12, 13, 14, 15, 16, 17, or 18.

21. The method of claim 11, wherein for the step of obtaining, the tissue concentration data are concentrations of full-length gap-widened antisense oligonucleotides.

22. The method of claim 11, wherein for the step of obtaining the tissue concentration data are obtained for liver, kidney, or adipose tissue.

23. Use of a gap-widened antisense oligonucleotide 18-24 nucleotides in length in the manufacture of a medicament for the treatment of disorders and diseases related to target RNA levels wherein said antisense oligonucleotide comprises a gap region having greater than 11 contiguous 2′

-deoxyribonucleotides; and

a first wing region and a second wing region flanking the gap region, wherein each of said first and second wing regions independently have 1 to 8 2′

-O-(2-methoxyethyl) ribonucleotides, and wherein said antisense oligonucleotide has an improved therapeutic index as compared to a corresponding 5-10-5 antisense oligonucleotide having a gap region of 10 contiguous 2′

-deoxyribonucleotides and a first wing region and a second wing region flanking the gap region of 5 2′

-O-(2-methoxyethyl) ribonucleotides.

24. A pharmaceutical composition comprising a gap-widened antisense oligonucleotide 18-24 nucleotides in length comprising:

a gap region having greater than 11 contiguous 2′

-deoxyribonucleotides; and

a first wing region and a second wing region flanking the gap region, wherein each of said first and second wing regions independently have 1 to 8 2′

-O-(2-methoxyethyl) ribonucleotides, having an improved therapeutic index as compared to a corresponding 5-10-5 antisense oligonucleotide having a gap region of 10 contiguous 2′

-deoxyribonucleotides and a first wing region and a second wing region flanking the gap region of 5 2′

-O-(2-methoxyethyl) ribonucleotides and optionally a pharmaceutically acceptable carrier, diluent, enhancer or excipient.

26. A method of modulating gene expression in an animal comprising the step of contacting said animal with the pharmaceutical composition of claim 24.

27. The method of claim 26 wherein the accumulation of the gap-widened antisense oligonucleotide in the kidney is less compared to a corresponding 5-10-5 antisense oligonucleotide having a gap region of 10 contiguous 2′

-deoxyribonucleotides and a first wing region and a second wing region flanking the gap region of 5 2′

-O-(2-methoxyethyl) ribonucleotides

28. The method of claim 27 wherein the kidney accumulation is measured by plasma protein binding capacity of said gap-widened antisense oligonucleotide.

25. A gap-widened antisense oligonucleotide 18-24 nucleotides in length comprising:

a gap region having greater than 11 contiguous 2′

-deoxyribonucleotides; and

a first wing region and a second wing region flanking the gap region, wherein each of said first and second wing regions independently have 1 to 8 2′

-O-(2-methoxyethyl) ribonucleotides, having lower kidney accumulation as compared to a corresponding 5-10-5 antisense oligonucleotide having a gap region of 10 contiguous 2′

-deoxyribonucleotides and a first wing region and a second wing region flanking the gap region of 5 2′

-O-(2-methoxyethyl) ribonucleotides as measured by plasma protein binding capacity of said gap-widened antisense oligonucleotide.