Methods and compositions involving miRNA and miRNA inhibitor molecules

• US 20090176723A1
• Filed: 11/14/2005
• Published: 07/09/2009
• Est. Priority Date: 11/12/2004
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Byrom, Mike, Ford, Lance, Devroe, Eric, Brown, David, Kelnar, Kevin, Ovcharenko, Dmitriy, Jarvis, Rich, Cheng, Angie
• Granted Patent: US 8,173,611 B2
• Time in Patent Office: Days
• US Class Current: 514/44.R
• 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 of between 17 and 125 residues in length comprising

a) an miRNA region whose sequence from 5′

to 3′

is identical to a mature miRNA sequence, and

b) a complementary region whose sequence from 5′

to 3′

is between 60% and 100% complementary to the miRNA sequence.

2. The RNA molecule of claim 1, further comprising one or more of the following:

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

terminus of the RNA molecule;

ii) one or more sugar modifications in the first or last 1 to 6 residues of the complementary region;

or,

iii) noncomplementarity between one or more nucleotides in the last 1 to 5 residues at the 3′

end of the complementary region and the corresponding nucleotides of the miRNA region.

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

terminus of the RNA molecule.

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

oxygen-methyl (2′

O-Me), 4,4′

-dimethoxytrityl with oxygen (DMTO), fluoroscein, a thiol, or acridine.

5. The RNA molecule of claim 3, wherein the phosphate of the nucleotide at the 5′

terminus of the RNA molecule has been replaced.

6. The RNA molecule of claim 3, wherein the hydroxyl of the nucleotide at the 5′

terminus of the RNA molecule has been replaced.

7. The RNA molecule of claim 2, comprising one or more sugar modifications in the first or last 1 to 6 residues of the complementary region.

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

O-Me modification.

12. The RNA molecule of claim 2, comprising noncomplementarity between one or more nucleotides in the last 1 to 5 residues at the 3′

end of the complementary region and the corresponding nucleotides of the miRNA region.

14. The RNA molecule of claim 2, comprising at least two of i), ii), or iii).

16. The RNA molecule of claim 1, wherein the RNA molecule is a single polynucleotide.

17. The RNA molecule of claim 16, further comprising a linker region between the miRNA region and the complementary region.

18. The RNA molecule of claim 17, wherein the linker region is between 3 and 30 residues in length.

19. The RNA molecule of claim 1, wherein the RNA molecule is double stranded.

20. A library of 10 or more different RNA molecules of claim 1.

27. A method of characterizing an miRNA activity or function in a cell comprising:

a) introducing into one or more cells the RNA molecule of claim 1;

b) comparing one or more characteristics of cell(s) having the RNA molecule with cells not having the RNA molecule.

28. The method of claim 27, wherein at least five RNA molecules are introduced into different cells.

29. The method of claim 27, further comprising assaying the cell for the presence of the RNA molecule.

30. The method of claim 27, further comprising synthesizing the RNA molecule.

31. The method of claim 30, wherein the RNA molecule further comprises one or more of the following:

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

terminus of the RNA molecule;

ii) one or more sugar modifications in the first or last 1 to 6 residues of the complementary region;

or,

iii) noncomplementarity between one or more nucleotides in the last 1 to 5 residues at the 3′

end of the complementary region and the corresponding nucleotides of the miRNA region.

32. The method of claim 31, wherein the RNA molecule comprises a replacement group for phosphate or hydroxyl of the nucleotide at the 5′

terminus of the RNA molecule.

99. A kit for evaluating miRNA molecules comprising, in suitable containers, at least five RNA molecules of claim 1.

9-11. -11. (canceled)

13. (canceled)

15. (canceled)

21. A library of 10 or more different miRNA inhibitor molecules, wherein each miRNA inhibitor molecule is between about 17 to 25 nucleotides in length and comprises a 5′

to 3′

sequence that is at least 90% complementary to the 5′

to 3′

sequence of a mature miRNA.

22. The library of claim 21, wherein the miRNA inhibitor molecules have a replacement group for the phosphate or hydroxyl of the nucleotide at the 5′

terminus of the molecule.

23. The library of claim 22, wherein the replacement group is an amine group, a lower alkylamine group, an acetyl group, or 2′

O-Me.

24-26. -26. (canceled)

33-49. -49. (canceled)

50. A method of reducing or eliminating activity of one or more miRNAs from a cell comprising:

a) introducing into a cell an miRNA inhibitor that is between 17 and 25 nucleotides in length and comprises a 5′

to 3′

sequence that is at least 90% complementary to the 5′

to 3′

sequence of a mature miRNA.

51. The method of claim 50, further comprising:

b) comparing one or more characteristics of a cell having the miRNA inhibitor with a cell not having the miRNA inhibitor.

52. The method of claim 50, further comprising assaying the cell for expression of the mature miRNA.

53. The method of claim 50, further comprising synthesizing the miRNA inhibitor.

54. The method of claim 51, wherein the miRNA inhibitor further comprises one or more of the following:

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

terminus of the miRNA inhibitor;

or

ii) one or more sugar modifications.

55. The method of claim 54, wherein the miRNA inhibitor comprises a replacement group for phosphate or hydroxyl of the nucleotide at the 5′

terminus of the miRNA inhibitor.

56-66. -66. (canceled)

67. A method for reducing or inhibiting cell proliferation in a cell comprising introducing into the cell an effective amount of at least one

i) miRNA inhibitor molecule that is between 17 and 25 nucleotides in length and comprises a 5′

to 3′

sequence that is at least 90% complementary to the 5′

to 3′

sequence of a mature miRNA selected from the group consisting of;

mir-3 1, mir-92, mir-99a, mir-100, mir-125a, mir-129, mir-130a, mir-150, mir-187, miR-190, miR-191, miR-193, miR 204, mir-210, mir-21 1, mir-212, mir-213, mir-215, mir-216, mir-217, miR 218, mir-224, mir-292, mir-294, mir-320, mir-324, mir-325, mir-326, mir-330, mir-331, mir-338, mir-341, mir-369, and mir-370;

or

ii) synthetic miRNA molecule of between 17 and 125 residues in length comprising an miRNA region whose sequence from 5′

to 3′

is identical to a mature miRNA sequence, and a complementary region whose sequence from 5′

to 3′

is between 60% and 100% complementary to the mature miRNA sequence, wherein the mature miRNA sequence is selected from the group consisting of;

miR-15a, miR-16, miR 21, miR 24, miR-96, miR-101, miR-105, miR-124, miR-126, miR-142, miR-147, miR-192, miR-194, miR-206, miR-215, or miR-346.

68. The method of claim 67, further comprising identifying a cell or a patient having the cell as in need of reducing or inhibiting cell proliferation.

69. A method for inducing or increasing cell proliferation in a cell comprising introducing into the cell an effective amount of at least one

i) miRNA inhibitor molecule that is between 17 and 25 nucleotides in length and comprises a 5′

to 3′

sequence that is at least 90% complementary to the 5′

to 3′

sequence of a mature miRNA selected from the group consisting of let7a-1, Let-7a, Let-7b, let7b-1, let7c, let7d, Let-7g, mir-9, mir-10a, mir-10b, mir-15a, mir-16, mir-21, mir-23a, mir-23b, mir-24, mir-25, mir-92, mir-95, mir-133a, mir-133a-2, mir-133b, mir-142, mir-152, mir-153, mir-155, mir-181a, mir-182, mir-183, mir-184, mir-186, mir-187, mir-191, mir-193, mir-194, mir-196, mir-199a-1, mir-200b, mir-204, mir-206, mir-211, mir-222, mir-223, mir-298, mir-328, mir-342, mir-371, and mir-412;

or

ii) synthetic miRNA molecule of between 17 and 125 residues in length comprising an miRNA region whose sequence from 5′

to 3′

is identical to a mature miRNA sequence, and a complementary region whose sequence from 5′

to 3′

is between 60% and 100% complementary to the miRNA sequence selected from the group consisting of;

Let-7a, Let-7b, Let-7c, Let-7d, Let-7g, miR-7, mir-9, miR-10a, miR-10b, miR-15a, miR-18, miR-19a, miR-17-3p, miR-20, miR-23b, mir-25, miR-26a, miR-26a, mir-30e-5p, mir-31, mir-32, mir-92, mir-93, miR-100, miR-125a, miR-125b, miR-126, mir-127, miR-128, miR-129, mir-130a, mir-135, mir-138, mir-139, miR-140, mir-141, mir-143, mir-145, mir-146, miR-150, mir-154, mir-155, mir-181a, miR-182, mir-186, miR-187, miR-188, mir-190, mir-191, mir-193, mir-194, mir-196, mir-197, mir-198, mir-199, mir-201, mir-204, mir-216, mir-218, miR-223, mir-293, miR-291-3p, miR-294, miR-295, miR-322, mir-333, mir-335, mir-338, mir-341, mir-350, mir-369, miR-373, mir-410, and mir-412.

70. The method of claim 69, further comprising identifying a cell or a patient having the cell as in need of inducing or increasing cell proliferation.

71-78. -78. (canceled)

79. A method for treating cancer in a subject comprising administering to the subject an effective amount of one or more synthetic miRNA molecules and/or miRNA inhibitors corresponding to miR-21, miR-126, miR-143, miR-145, miR-188, miR-200b, miR-219, or miR-331.

80. The method of claim 79, wherein the cancer is breast cancer and the subject is administered 1) one or more miRNA inhibitors corresponding to miR-21, miR-15a, miR-16, miR-24, and/or miR-25, and/or 2) one or more miRNAs corresponding to miR-99, miR-100, miR-205, miR-197, miR-126, miR-143, miR-145 and/or miR-321.

81. The method of claim 79, wherein the cancer is colon cancer and the subject is administered 1) one or more miRNA inhibitors corresponding to miR-2 1, miR-106, miR-189, miR-200b, miR-223, miR-224, miR-31, and/or miR-17;

and/or

     2) one or more miRNAs corresponding to miR-145, miR-143, miR-133, miR-342, miR-125a, miR-195, miR-30a, miR-10a, miR-130, miR-130a, miR-192, miR-194, miR-215, miR-144, miR-23, miR-26a, miR-126, miR-199a, miR-188, miR-331, and/or miR-21.

82. The method of claim 79, wherein the cancer is thyroid cancer and the subject is administered 1) one or more miRNA inhibitors corresponding to miR-21 miR-125, miR-24, miR-200b, miR-29b, miR-221, miR-222, miR-224, miR-10a, and/or miR-183;

and/or

     2) one or more miRNAs corresponding to miR-145, miR-22, miR-331, miR-126, miR-30a, miR-199a, miR-223, and/or miR-321.

83. The method of claim 79, wherein the cancer is lung cancer and the subject is administered 1) one or more miRNA inhibitors corresponding to miR-223, miR-106, miR-21, miR-200b, miR-321, miR-182, miR-183, miR-17, and/or miR-205;

and/or

     2) one or more miRNAs corresponding to miR-130a, miR-145, miR-126, miR-331, miR-342, miR-143, Let-7, miR-30a, miR-16, miR-26a, miR-125a, miR-29b, miR-24, miR-328, miR-201, miR-195, miR-22, miR-181a, miR-331, and/or miR-321.

84. A method for treating cancer in a subject comprising administering to the subject an effective amount of one or more synthetic miRNA molecules and/or miRNA inhibitors corresponding to let-7, miR-10a, miR-16, miR-17, miR-21, miR-22, miR-23, miR-24, miR-26a, miR-29b, miR-30a, miR-106, miR-125a, miR-126, miR-130, miR130a, miR-133, miR-143, miR-144, miR-145, miR-181a, miR-182, miR-183, miR-188, miR-189, miR-219, miR-192, miR-194, miR-195, miR-199a, miR-200b, miR-201, miR-205, miR-215, miR-223, miR-224, miR-321, miR-328, miR-331, or miR-342.

85-98. -98. (canceled)

100. (canceled)

101. A kit comprising, in suitable containers:

a) at least one synthetic miRNA molecule of between 17 and 125 residues in length comprising an miRNA region whose sequence from 5′

to 3′

is identical to a mature miRNA sequence, and a complementary region whose sequence from 5′

to 3′

is between 60% and 100% complementary to the miRNA sequence; and

b) at least one miRNA inhibitor between 17 and 25 nucleotides in length and comprising a 5′

to 3′

sequence that is at least 90% complementary to the 5′

to 3′

sequence of a mature miRNA.