Methods and compositions involving miRNA and miRNA inhibitor molecules

• US 9,068,219 B2
• Filed: 08/13/2014
• Issued: 06/30/2015
• Est. Priority Date: 11/12/2004
• Status: Expired due to Fees

• Current Assignee: Asuragen, Inc.
• 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:
  US14/459,074
• Publication Number:
  US 20140350086A1
• Time in Patent Office: 321 Days
• US Class Current: 1/1
• 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 comprising introducing into a cancer cell an effective amount of a synthetic miR-215 molecule comprising a miR-215 active strand and a separate passenger strand that is at least 60% complementary to the active strand.

2. The method of claim 1, wherein the synthetic miR-215 molecule is non-naturally occurring and markedly different in sequence from naturally occurring miR-215.

3. The method of claim 1, wherein the synthetic miR-215 molecule is non-naturally occurring and markedly different in chemical structure from naturally occurring miR-215.

4. The method of claim 1, wherein the sequence of the synthetic miR-215 molecule is not naturally occurring.

5. The method of claim 1, wherein at least one nucleobase of the synthetic miR-215 molecule is chemically modified.

6. The method of claim 1, wherein the complementary strand is not naturally occurring.

7. The method of claim 1, wherein the complementary strand comprises a chemical modification that improve uptake of the synthetic oligonucleotide.

8. The method of claim 1, wherein the complementary strand comprises a chemical modification that enhance activity of the synthetic oligonucleotide.

9. The method of claim 1, wherein the complementary strand comprises a chemical modification that enhance stability of the synthetic oligonucleotide.

10. The method of claim 1, wherein the complementary strand comprises a chemical modification that inhibit uptake of the complementary strand.

11. The method of claim 1, wherein the complementary strand comprises a chemical modification that inhibits activity of the complementary strand.

12. The method of claim 1, wherein the complementary strand comprise one or more nucleobases that are non-complementary with the synthetic oligonucleotide.

13. The method of claim 1, wherein the synthetic miR-215 molecule is in a pharmaceutical composition.

14. The method of claim 13, wherein the pharmaceutical composition is a sterile aqueous solution.

15. The method of claim 1, wherein the synthetic miR-215 molecule is in a liposomal pharmaceutical delivery system.

16. The method of claim 13, wherein the pharmaceutical composition comprises an antibacterial or antifungal agent.

17. The method of claim 13, wherein the pharmaceutical composition comprises about 2% to about 75% by weight (w/w) of the synthetic miR-215 molecule.

18. The method of claim 13, wherein the synthetic miR-215 molecule is at least about 95, 96, 97, 98, 99, or 100% pure.

19. The method of claim 13, wherein the pharmaceutical composition is aliquoted in a vial, test tube, flask, bottle, syringe, or container.

20. The method of claim 1, wherein introducing into the cell the effective amount of the synthetic miR-215 molecule comprises administering the synthetic miR-215 molecule to a subject comprising the cell.

21. The method of claim 20, wherein the effective amount is a dose of about 5 mg/kg body weight to about 100 mg/kg body weight of the subject.

22. The method of claim 20, wherein the pharmaceutical composition is administered to the subject topically, intravenously, by injection, transdermally, transmucosally, by inhalation, orally, or pulmonarily.

23. The method of claim 20, wherein the subject has a cancer.

24. The method of claim 20, wherein the synthetic miR-215 molecule induces apoptosis in the cell in the subject.

25. The method of claim 1, wherein the synthetic miR-215 molecule induces apoptosis in the cell.

26. The method of claim 1, wherein the synthetic miR-215 molecule comprises a sequence that is at least 80% identical to residues 27-47 of SEQ ID NO:

162.

27. The method of claim 1, wherein the synthetic miR-215 molecule comprises a sequence that is identical to residues 27-47 of SEQ ID NO:

162.

28. The method of claim 1, wherein the pharmaceutical composition comprises about 25% to about 60% by weight (w/w) of the synthetic miR-215 molecule.

29. The method of claim 1, wherein the synthetic miR-215 molecule is in a dose of about 5 mg/kg body weight to about 500 mg/kg body weight.

30. The method of claim 1, wherein the cancer cell is a breast cancer cell.

31. The method of claim 1, wherein the cancer cell is a cervical cancer cell.

32. The method of claim 1, wherein the cancer cell is a colon cancer cell.

33. The method of claim 1, wherein the cancer cell is a skin cancer cell.