Methods and compositions involving miRNA and miRNA inhibitor molecules

• US 8,173,611 B2
• Filed: 11/14/2005
• Issued: 05/08/2012
• Est. Priority Date: 11/12/2004
• Status: Expired due to Fees

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Asuragen, Inc.
• Inventors: Brown, David, Ford, Lance, Cheng, Angie, Jarvis, Rich, Byrom, Mike, Ovcharenko, Dmitriy, Devroe, Eric, Kelnar, Kevin
• Primary Examiner(s): WHITEMAN, BRIAN A
• Application Number:
  US11/273,640
• Publication Number:
  US 20090176723A1
• Time in Patent Office: 2,367 Days
• Field of Search: 514/44.A
• US Class Current: 514/44.A
• CPC Class Codes: A61K 31/7088 Compounds having three or more nucleosides or nucleotides
• CPC Class Codes: A61K 31/7105 Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61K 9/127 Liposomes
• CPC Class Codes: A61N 5/10 X-ray therapy; Gamma-ray therapy; Particle-irradiation therapy A61N5/01 takes precedence
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 15/08 for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/06 for tuberculosis
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• 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: 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 15/1136 : against growth factors, growth regulators, cytokines, lymphokines or hormones
• CPC Class Codes: C12N 2310/14 : interfering N.A.
• CPC Class Codes: C12N 2310/141 : MicroRNAs, miRNAs
• CPC Class Codes: C12N 2310/312 : Phosphonates
• CPC Class Codes: C12N 2310/321 : 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 : 2'-R Modification
• CPC Class Codes: C12N 2310/33 : of the base
• CPC Class Codes: C12N 2310/344 : Position-specific modifications, e.g. on every purine, at the 3'-end
• CPC Class Codes: C12N 2310/35 : Nature of the modification
• CPC Class Codes: C12N 2310/351 : Conjugate
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2310/3527 : Other alkyl chain
• CPC Class Codes: C12N 2310/3535 : Nitrogen
• CPC Class Codes: C12N 2310/533 : having a mismatch or nick in at least one of the strands
• CPC Class Codes: C12N 2320/12 : in functional genomics, i.e. for the determination of gene function
• CPC Class Codes: C12N 2320/30 : Special therapeutic applications
• CPC Class Codes: C12N 2320/50 : Methods for regulating/modulating their activity
• CPC Class Codes: C12N 2330/10 : naturally occurring
• CPC Class Codes: C12Q 1/68 : involving nucleic acids
• CPC Class Codes: C12Q 1/6876 : Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: C12Q 2600/178 : miRNA, siRNA or ncRNA

Abstract

The present invention concerns methods and compositions for introducing miRNA activity or function into cells using synthetic nucleic acid molecules. Moreover, the present invention concerns methods and compositions for identifying miRNAs with specific cellular functions that are relevant to therapeutic, diagnostic, and prognostic applications wherein synthetic miRNAs and/or miRNA inhibitors are used in library screening assays.

Claims

1. A method for reducing or inhibiting cell proliferation in a human cancer cell comprising introducing into the cell an effective amount of at least one synthetic double-stranded RNA molecule of between 17 and 25 residues in length comprising

a) an active strand comprising a sequence from 5′

to 3′

that is identical to a mature human miRNA sequence, and

b) a separate complementary strand whose sequence from 5′

to 3′

is 100% complementary to the active strand, wherein the mature miRNA sequence is selected from the group consisting of;

miR-16, miR-96, miR-101, miR-105, miR-124, miR-142, miR-147, miR-206, or miR-346, wherein the complementary strand comprises a chemical modification at the 5′

end that enhances activity of the active strand.

2. The method of claim 1, further comprising identifying a cell or a patient having the cell as in need of reducing or inhibiting cell proliferation, wherein the cell is a cancer cell.

3. The method of claim 1, wherein the chemical modification is an NH₂, biotin, an amine group, a lower alkylamine group, or an acetyl group.

4. The method of claim 1, wherein the synthetic double-stranded RNA molecule has a complementary strand that has a 5′

terminal nucleotide with a sugar modification.

5. The method of claim 4, wherein the sugar modification is 2′

OMe.

6. The method of claim 1, wherein the mature miRNA sequence is miR-16.

7. The method of claim 1, wherein the mature miRNA sequence is miR-96.

8. The method of claim 1, wherein the mature miRNA sequence is miR-101.

9. The method of claim 1, wherein the mature miRNA sequence is miR-105.

10. The method of claim 1, wherein the mature miRNA sequence is miR-124.

11. The method of claim 1, wherein the mature miRNA sequence is miR-142.

12. The method of claim 1, wherein the mature miRNA sequence is miR-147.

13. The method of claim 1, wherein the mature miRNA sequence is miR-206.

14. The method of claim 1, wherein the mature miRNA sequence is miR-346.

15. A method for treating lung cancer in a human subject comprising administering to the subject an effective amount of one or more synthetic double-stranded miRNA molecules corresponding to human miR-124, miR-126, or miR-147, wherein the synthetic miRNA molecule is 17 to 30 residues in length and comprises:

a) an active strand comprising a sequence of a mature human miRNA, and

b) a separate strand comprising a sequence that is fully complementary to mature miRNA sequence and having at least one chemical modification at the 5′

end that enhances the activity of the active strand.

16. The method of claim 15, wherein one of the synthetic double-stranded molecules corresponds to human miR-124.

17. The method of claim 15, wherein one of the synthetic double-stranded molecules corresponds to human miR-126.

18. The method of claim 15, wherein one of the synthetic double-stranded molecules corresponds to human miR-147.

19. A method for treating cancer in a human subject comprising administering to the subject an effective amount of one or more synthetic double-stranded miRNA molecules corresponding to human miR-16, miR-124, miR-126, miR-147, or miR-194, wherein the synthetic miRNA molecule is 17-30 nucleotides in length and comprises:

a) an active strand comprising a sequence of a mature human miRNA, and

b) a separate complementary strand that is fully complementary to the active strand and that comprises at least one chemical modification at the 5′

end that enhances the activity of the active strand.

20. The method of claim 19, wherein the 5′

terminus is modified with an NH₂, biotin, an amine group, a lower alkylamine group, or an acetyl group.

21. The method of claim 19, wherein the synthetic double-stranded RNA molecule has a complementary strand that has a 5′

terminal nucleotide with a sugar modification.

22. The method of claim 21, wherein the sugar modification is 2′

OMe.

23. The method of claim 19, wherein one of the synthetic double-stranded molecules corresponds to human miR-16.

24. The method of claim 19, wherein one of the synthetic double-stranded molecules corresponds to human miR-124.

25. The method of claim 19, wherein one of the synthetic double-stranded molecules corresponds to human miR-126.

26. The method of claim 19, wherein one of the synthetic double-stranded molecules corresponds to human miR-147.

27. The method of claim 19, wherein one of the synthetic double-stranded molecules corresponds to human miR-194.