Methods and compositions involving miRNA and miRNA inhibitor molecules

• US 9,051,571 B2
• Filed: 04/16/2014
• Issued: 06/09/2015
• 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:
  US14/254,532
• Publication Number:
  US 20140314833A1
• Time in Patent Office: 419 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 synthetic RNA molecule comprising:

a) a first 15-25 residue oligonucleotide having a sequence that is 80-100% identical to a mature miR-7 sequence;

b) a second 15-25 residue oligonucleoLide having a sequence that is 60-100% identical to a sequence complementary to the mature miR-7 sequence; and

c) at least one of

i) a replacement group for the phosphate or the hydroxyl of the nucleotide at the 5′

end of the second oligonucleotide, and

ii) one or more sugar modifications in the first or last 1-6 residues of the second oligonucleotide.

2. The synthetic RNA molecule of claim 1, comprising a replacement group for the phosphate or the hydroxyl of the nucleotide at the 5′

end of the second oligonucleotide.

3. The synthetic RNA molecule of claim 2, wherein the replacement group is biotin, an amine group, a lower alkylamine group, an acetyl group, 2′

O-Me, DMTO, fluoroscein, a thiol, or acridine.

4. The synthetic RNA molecule of claim 2, wherein the phosphate of the nucleotide at the 5′

end of the second oligonucleotide is replaced.

5. The synthetic RNA molecule of claim 4, wherein the replacement group is a lower alkylamine group.

6. The synthetic RNA molecule of claim 2, wherein the hydroxyl of the nucleotide at the 5′

end of the second oligonucleotide is replaced.

7. The synthetic RNA molecule of claim 1, comprising one or more sugar modifications in the first or last 1-6 residues of the second oligonucleotide.

8. The synthetic RNA molecule of claim 7, wherein the sugar modification is a 2′

O-Me modification.

9. The synthetic RNA molecule of claim 7, comprising one or more sugar modifications in the first 1-6 residues of the second oligonucleotide.

10. The synthetic RNA molecule of claim 7, comprising one or more sugar modifications in the last 1-6 residues of the second oligonucleotide.

11. The synthetic RNA molecule of claim 7, comprising one or more sugar modifications in the first or last 2 to 4 residues of the second oligonucleotide.

12. The synthetic RNA molecule of claim 1, further comprising a noncomplementarity between one or more nucleotide in the last 1-5 residues at the 3′

end of the second oligonucleotide and the corresponding nucleotides of the First oligonucleotide.

13. The synthetic RNA molecules of claim 1, further comprising a noncomplementarity between two or more nucleotides in the last 1-5 residues at the 3′

end of the second oligonucleotide and the corresponding nucleotides of the first oligonucleotide.

14. The synthetic RNA molecule of claim 1, comprising i) and ii).

15. The synthetic RNA molecule of claim 12, comprising i) and ii).

16. The synthetic RNA molecule of claim 1, wherein time first oligonucleotide is 90-100% identical to the mature miR-7 sequence.

17. The synthetic RNA molecule of claim 1, wherein the first oligonucleotide is 100% identical to the mature miR-7 sequence.

18. A pharmaceutical composition comprising the synthetic RNA of claim 1 and a liposomal delivery vehicle.

19. A method of reducing lung cancer cell viability or inducing apoptosis in a lung cancer cell comprising introducing into the lung cancer cell an effective amount of a synthetic RNA molecule comprising:

a) a first 15-25 residue oligonucleotide having a sequence that is 80-100% identical to a mature miR-7 sequence; and

b) a second 15-25 residue oligonucleotide having a sequence that is 60-100% identical to a sequence complementary to the mature miR-7 sequence; and

c) at least one of

i) a replacement group for the phosphate or the hydroxyl of the nucleotide at the 5′

end of the second oligonucleotide, and

ii) one or more sugar modifications in the first or last 1-6 residues of the second oligonucleotide.

20. The method of claim 19, wherein:

i) the phosphate or the nucleotide at the 5′

end of the second oligonucleotide is replaced with a lower alkylamine group, or

ii) one or more sugar in the first or last 1-6 residues of the second oligonucleotide has a 2′

O-Me modification.

21. The method of claim 19, wherein the cancer cell is a human cell.

22. The method of claim 19, wherein the cancer cell is in a human.

27. The method or claim 19, wherein the first oligonucleotide is 100% identical to the mature miR-7 sequence.

23. A method of treating lung cancer comprising introducing into a lung cancer cell an effective amount of a synthetic RNA molecule comprising:

a) a first 15-25 residue oligonucleotide having a sequence that is 80-100% identical to a mature miR-7 sequence; and

a second 15-25 residue oligonucleotide having a sequence that is 60-100% identical to a sequence complementary to the mature miR-7 sequence; and

c) at least one of

i) a replacement group for the phosphate or the hydroxyl of the nucleotide at the 5′

end of the second oligonucleotide, and

ii) one or more sugar modifications in the first or last 1-6 residues of the second oligonucleotide.

24. The method of claim 23, wherein:

i) the phosphate of the nucleotide at the 5′

end of the second oligonucleotide is replaced with a lower alkylamine group, or

ii) one or more sugar in the first or last 1-6 residues of the second oligonucleotide has a 2′

O-Me modification.

25. The method of claim 23, wherein the cancer cell is a human cell.

26. The method of claim 23, wherein the cancer cell is in a human.

28. The method or claim 23, wherein the first oligonucleotide is 100% identical to the mature miR-7 sequence.