Double-stranded oligonucleotides

• US 20130045520A1
• Filed: 10/20/2011
• Published: 02/21/2013
• Est. Priority Date: 02/01/2002
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Woolf, Tod M.
• Granted Patent: US 8,815,821 B2
• Time in Patent Office: Days
• US Class Current: 435/173.6
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 31/20 for DNA viruses
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• 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 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 : 2'-O-R Modification
• CPC Class Codes: C12N 2310/33 : of the base
• CPC Class Codes: C12N 2310/351 : Conjugate
• CPC Class Codes: C12N 2310/53 : partially self-complementary or closed
• CPC Class Codes: C12N 2320/31 : Combination therapy
• CPC Class Codes: C12N 2320/50 : Methods for regulating/modulating their activity
• CPC Class Codes: C12N 2320/51 : modulating the chemical stability, e.g. nuclease-resistance
• CPC Class Codes: C12Y 207/11022 : Cyclin-dependent kinase (2.7.11.22)
• CPC Class Codes: C12Y 301/03048 : Protein-tyrosine-phosphatase (3.1.3.48)

Abstract

Antisense sequences, including duplex RNAi compositions, which possess improved properties over those taught in the prior art are disclosed. The invention provides optimized antisense oligomer compositions and method for making and using the both in in vitro systems and therapeutically. The invention also provides methods of making and using the improved antisense oligomer compositions.

Claims

1. A method for introducing a double-stranded nucleic acid molecule comprising a first strand and a second strand into a eukaryotic cell in vitro, the method comprising contacting the eukaryotic cell with the double-stranded nucleic acid molecule,

wherein from one to six of the nucleotides at the 5′

terminus of the first strand of the double-stranded nucleic acid molecule are chemically modified at the 2′

positions, wherein said modification is a 2′

-O-methyl modification;

wherein from one to six of the nucleotides at the 5′

terminus of the second strand of the double-stranded nucleic acid molecule are chemically modified at the 2′

positions, wherein said modification is a 2′

-O-methyl modification;

wherein the double-stranded nucleic acid molecule is between 18 and 30 nucleosides in length; and

wherein the double-stranded nucleic acid molecule is introduced into the eukaryotic cell and participates in RNA interference mediated degradation of RNA which shares sequence complementarity with at least one strand of the double-stranded nucleic acid molecule.

2. The method of claim 1, wherein the double-stranded nucleic acid molecule is between 20 and 30 nucleosides in length.

3. The method of claim 1, wherein the double-stranded nucleic acid molecule is 25 nucleosides in length.

4. The method of claim 1, wherein the double-stranded nucleic acid molecule contains an overhang of at least one nucleoside on at least one end.

5. The method of claim 4, wherein the overhang is a 3′

end overhang.

6. The method of claim 4, wherein the nucleosides of the 3′

end overhang are deoxy T-deoxy T.

7. The method of claim 1, wherein the 2′

chemical modification is on a ribose.

8. The method of claim 1, wherein the double-stranded nucleic acid molecule is RNA.

9. The method of claim 1, wherein the eukaryotic cell is contacted with the double-stranded nucleic molecule in the presence of a transfection reagent.

10. The method of claim 9, wherein the transfection reagent is a cationic lipid.

11. The method of claim 1, wherein the double-stranded nucleic acid molecule is introduced into the eukaryotic cell by electroporation.

12. The method of claim 1, wherein a strand of said double-stranded nucleic acid molecule is complementary to a sequence of an mRNA expressed in said eukaryotic cell.

13. The method of claim 12, wherein said double-stranded nucleic molecule is a double-stranded RNA molecule.