Oligoribonucleotides and ribonucleases for cleaving RNA

• US 7,629,321 B2
• Filed: 10/25/2002
• Issued: 12/08/2009
• Est. Priority Date: 06/06/1996
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Crooke, Stanley T.
• Primary Examiner(s): MCGARRY, SEAN
• Application Number:
  US10/281,349
• Publication Number:
  US 20030096784A1
• Time in Patent Office: 2,601 Days
• Field of Search: None
• US Class Current: 514/44.00R
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• 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: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. 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 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 2310/311 Phosphotriesters
• CPC Class Codes: C12N 2310/312 Phosphonates
• CPC Class Codes: C12N 2310/314 Phosphoramidates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/316 Phosphonothioates
• CPC Class Codes: C12N 2310/318 where the PO2 is completely replaced, e.g. MMI or formacetal
• CPC Class Codes: C12N 2310/3181 Peptide nucleic acid, PNA
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3341 5-Methylcytosine
• CPC Class Codes: C12N 2310/335 Modified T or U
• 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 2310/3521 : Methyl
• CPC Class Codes: C12N 2310/3525 : MOE, methoxyethoxy
• CPC Class Codes: C12N 2310/3527 : Other alkyl chain
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
• CPC Class Codes: Y02P 20/582 : Recycling of unreacted starting or intermediate materials

Abstract

Oligomeric compounds including oligoribonucleotides and oligoribonucleosides are provided that have subsequences of 2′-pentoribofuranosyl nucleosides that activate dsRNase. The oligoribonucleotides and oligoribonucleosides can include substituent groups for increasing binding affinity to complementary nucleic acid strand as well as substituent groups for increasing nuclease resistance. The oligomeric compounds are useful for diagnostics and other research purposes, for modulating the expression of a protein in organisms, and for the diagnosis, detection and treatment of other conditions susceptible to oligonucleotide therapeutics. Also included in the invention are mammalian ribonucleases, i.e., enzymes that degrade RNA, and substrates for such ribonucleases. Such a ribonuclease is referred to herein as a dsRNase, wherein “ds” indicates the RNase'"'"'s specificity for certain double-stranded RNA substrates. The artificial substrates for the dsRNases described herein are useful in preparing affinity matrices for purifying mammalian ribonuclease as well as non-degradative RNA-binding proteins.

Claims

1. A method for specifically cleaving a preselected target messenger RNA, comprising:

contacting the preselected target messenger RNA with a single-stranded oligomeric compound and a double-strand ribonuclease, wherein the single stranded oligomeric compound comprises an oligonucleotide consisting of 17 to 25 linked nucleosides wherein;

the oligonucleotide is specifically hybridizable with the preselected target messenger RNA;

the oligonucleotide comprises at least one sugar modified nucleoside, wherein the modification improves the affinity or specificity of the oligonucleotide for the preselected target messenger RNA or increases the resistance of the oligonucleotide to single-stranded nucleases, or both; and

the oligonucleotide comprises a plurality of nucleosides comprising a 2′

-hydroxyl pentofuranosyl sugar moiety.

2. The method of claim 1 wherein the oligonucleotide comprises at least four consecutive nucleosides comprising 2′

-hydroxyl pentofuranosyl sugar moieties.

3. The method of claim 1, wherein the oligonucleotide comprises at least two nucleosides comprising 2′

sugar modifications.

4. The method of claim 3 wherein the at least two 2′

sugar modifications are on consecutive nucleosides.

5. The method of claim 3 wherein the at least two 2′

sugar modifications are each independently selected from fluoro, C₁-C₂₀ alkoxy, C₁-C₉ aminoalkoxy, allyloxy, imidazolylalkoxy and polyethylene glycol.

6. The method of claim 5 wherein the C₁-C₂₀ alkoxy modification is 2′

-O-methoxyethyl or 2′

-O-methyl.

7. The method of claim 3 wherein the at least two 2′

modifications are at the 5′

or 3′

terminal regions of the oligonucleotide.

8. The method of claim 3 wherein the 3′

terminal four nucleosides of the oligonucleotide each comprise a 2′

sugar modification.

9. The method of claim 3 wherein the 3′

terminal five nucleosides of the oligonucleotide each comprise a 2′

sugar modification.

10. The method of claim 3 wherein the 3′

terminal six nucleosides of the oligonucleotide each comprise a 2′

sugar modification.

11. The method of claim 3 wherein the 3′

terminal seven nucleosides of the oligonucleotide each comprise a 2′

sugar modification.

12. The method of claim 3 wherein the 3′

terminal eight nucleosides of the-oligonucleotide each comprise a 2′

sugar modification.

13. The method of claim 3 wherein the four 5′

-most nucleosides and the four 3′

-most nucleosides of the oligonucleotide each independently comprise a 2′

sugar modification.

14. The method of claim 3 wherein the nucleosides comprising 2′

-hydroxyl pentofuranosyl sugar moieties are positioned between two segments comprising nucleosides comprising 2′

sugar modifications.

15. The method of claim 1 wherein the oligonucleotide comprises at least one internucleoside bond that is more stable to degradation as compared to a phosphodiester bond.

16. The method of claim 15 wherein the internucleoside bond is phosphorothioate.

17. The method of claim 16 wherein each internucleoside bond is phosphorothioate.

18. The method of claim 1 wherein the oligonucleotide comprises at least one nucleoside comprising a 2′

-fluoro modification.

19. The method of claim 1 wherein the oligonucleotide comprises four to twelve consecutive nucleosides each comprising a 2′

-hydroxyl pentofuranosyl sugar moiety.

20. The method of claim 2 wherein the oligonucleotide comprises five to nine consecutive nucleosides each comprising a 2′

-hydroxyl pentofuranosyl sugar moiety.

21. The method of claim 1 wherein the oligonucleotide consists of 17 to 20 linked nucleosides.

22. The method of claim 11 wherein the seven 3′

terminal nucleosides comprising a 2′

sugar modification each comprise a 2′

fluoro.

23. The method of claim 22 wherein each internucleoside linkage of the oligonucleotide is a phosphorothioate internucleoside linkage.

24. The method of claim 1 wherein contacting the pre-selected target messenger RNA with a single-stranded oligomeric compound occurs inside a cell.

25. The method of claim 24 wherein the double-strand ribonuclease is endogenous to the cell.