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1. WO2015183667 - HYBRID tRNA/pre-miRNA MOLECULES AND METHODS OF USE

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CLAIMS

What is claimed is:

1. A polynucleotide comprising a tRNA operably linked to a pre-microRNA (pre-miRNA) or short hairpin RNA (shRNA).

2. The polynucleotide of claim 1 , wherein the tRNA is a methionyl tRNA.

3. The polynucleotide of claim 2, wherein the tRNA has a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:l.

4. The polynucleotide of any one of claims 1 to 3, wherein the pre-miRNA is naturally or artificially derived.

5. The polynucleotide of any one of claims 1 to 4, wherein the pre-miRNA is selected from pre-miRNA-1291, pre-miRNA-34a, pre-miRNA-125b, pre-miRNA-124, pre-miRNA-27b, and pre-miRNA-22.

6. The polynucleotide of claim 5, wherein:

a) the pre-miRNA-1291 comprises a nucleic acid sequence having at least

90%) sequence identity to the miRBase Accession No. MI0006353;

b) the pre-miRNA-34a comprises a nucleic acid sequence having at least 90%o sequence identity to miRBase Accession No. MI0000268;

c) the pre-miRNA-125b-1 comprises a nucleic acid sequence having at least 90% sequence identity to miRBase Accession No. MI0000446;

d) the pre-miRNA-124 comprises a nucleic acid sequence having at least 90%o sequence identity to miRBase Accession No. MI0000443;

e) the pre-miRNA-27b comprises a nucleic acid sequence having at least 90%) sequence identity to miRBase Accession No. MI0000440; and/or

f) the pre-miRNA-22 comprises a nucleic acid sequence having at least 90% sequence identity to miRBase Accession No. MI0000078.

7. The polynucleotide of any one of claims 5 to 6, wherein:

a) the pre-miRNA-34a comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:2;

b) the pre-miRNA-1291 comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:9;

c) the pre-miRNA- 125-1 comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 17;

d) the pre-miRNA-124 comprises a nucleic acid sequence having at least

90% sequence identity to SEQ ID NO:26;

e) the pre-miRNA-27b comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:29; and/or

f) the pre-miRNA-22 comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:32.

8. The polynucleotide of any one of claims 1 to 7, comprising a methionyl tRNA operably linked to a pre-miRNA, or mutants or variants thereof, selected from the group consisting of pre-miRNA-1291, pre-miRNA-34a, pre-miRNA- 125b, pre-miRNA-124, pre-miRNA-27b and pre-miRNA-22.

9. The polynucleotide of claim 8, wherein:

a) the methionyl tRNA operably linked to the pre-miRNA-34a has a nucleic acid sequence having at least 90% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6; SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:39 and SEQ ID NO:40;

b) the methionyl tRNA operably linked to the pre-miRNA-1291 has a nucleic acid sequence having at least 90% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13;

SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16;

c) the methionyl tRNA operably linked to the pre-miRNA- 125 has a nucleic acid sequence having at least 90% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21;

SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25;

d) the methionyl tRNA operably linked to the pre-miRNA- 124 has a nucleic acid sequence having at least 90% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO:27 and SEQ ID NO:28;

e) the methionyl tRNA operably linked to the pre-miRNA-27b has a nucleic acid sequence having at least 90% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO:30 and SEQ ID NO:31; and/or

f) the methionyl tRNA operably linked to the pre-miRNA-155 has a nucleic acid sequence having at least 90% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO:33 and SEQ ID NO:34.

10. The polynucleotide of any one of claims 1 to 9, wherein all or part of the stem-loop anticodon of the tRNA is replaced with the pre-miRNA.

11. The polynucleotide of any one of claims 1 to 3, wherein the tRNA operably linked to the short hairpin RNA (shRNA) comprises a nucleic acid sequence having at least 90% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NOs:41-45.

12. The polynucleotide of any one of claims 1 to 11, the tRNA and/or pre-miRNA or shRNA are further operably linked to one or more inserted RNA molecules.

13. The polynucleotide of claim 12, wherein the inserted RNA molecule is inserted at, abutted with or operably linked to:

a) the 5' end of the pre-miRNA;

b) the 3 ' end of the pre-miRNA;

c) 5' of a dicer cleavage site of the pre-miRNA; or

d) 3' of a dicer cleavage site of the pre-miRNA.

14. The polynucleotide of any one of claims 12 to 13, wherein the inserted RNA has at least about 18 nucleotides and up to about 200 nucleotides.

15. The polynucleotide of claim 14, wherein the inserted RNA has at least about 18 nucleotides and up to about 50 nucleotides.

16. The polynucleotide of any one of claims 12 to 15, wherein the inserted RNA is selected from the group consisting of a noncoding RNA (ncRNA), mature micro RNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), a guide RNA (gRNA), a catalytic RNA, a riboswitch, and an RNA aptamer.

17. The polynucleotide of any one of claims 12 to 16, wherein the inserted RNA is a noncoding RNA.

18. The polynucleotide of claim 17, wherein the noncoding RNA is HOX antisense intergenic RNA (HOT AIR).

19. The polynucleotide of any one of claims 12 to 17, wherein the inserted RNA is a mature miRNA selected from the group consisting of miR-21, miR-22, miR-27b, miR-33, miR-34a, miR-122, miR-124-1, miR-125-1, miR-1291 and let-7a.

20. The polynucleotide of any one of claims 12 to 19, wherein the inserted RNA prevents, reduces or inhibits the expression of a target polypeptide.

21. The polynucleotide of any one of claims 12 to 15, wherein the inserted RNA is an aptamer that binds to a target molecule or a target polypeptide.

22. The polynucleotide of any one of claims 20 to 21, wherein the target polypeptide is selected from the group consisting of a fluorescent protein, a cytokine, a growth factor, a hormone, an enzyme, an ion channel, a kinase, a nuclear receptor, a

G protein-coupled receptor, an epigenetic regulator, a transcription factor.

23. The polynucleotide of claim 22, wherein the fluorescent protein is selected from the group consisting of a violet fluorescent protein, a blue fluorescent protein (BFP), a cyan fluorescent protein, a green fluorescent protein (GFP), a yellow fluorescent protein (YFP), an orange fluorescent protein (OFP), a red fluorescent protein (RFP) and a sapphire-type protein.

24. The polynucleotide of claim 22, wherein the cytokine is selected from the group consisting of IL-1α, IL-Ιβ, TNFα, IFNα, IFNβ, IFNγ, TGFβ1, IL-5, IL-6, IL-8, IL-10, IL-12, IL-17, IL-18, IL-22, IL-23 and MIF.

25. The polynucleotide of claim 22, wherein the nuclear receptor is Peroxisome proliferator-activated receptor gamma (PPAR-γ or PPARG).

26. The polynucleotide of claim 22, wherein the growth factor is vascular endothelial growth factor (VEGF).

27. The polynucleotide of claim 22, wherein the kinase is epidermal growth factor receptor (EGFR).

28. The polynucleotide of any one of claims 1 to 27, wherein the polynucleotide has a nucleic acid sequence having at least 90% sequence identity to a polynucleotide selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6; SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:l l, SEQ ID NO:12, SEQ ID NO:13; SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16; SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21; SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25; SEQ ID NO:27, SEQ ID NO:28; SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39 and SEQ ID NO:40.

29. An expression cassette comprising the polynucleotide of any one of claims 1 to 28.

30. A liposome, a polymer or a nanoparticle comprising the polynucleotide of any one of claims 1 to 27 or the expression cassette of claim 29.

31. A viral vector comprising the polynucleotide of any one of claims 1 to 28 or the expression cassette of claim 29.

32. A host cell transfected or transformed with a polynucleotide of any one of claims 1 to 28 or the expression cassette of claim 29 or the liposome, a polymer or a nanoparticle of claim 30 or the viral vector of claim 31.

33. The host cell of claim 32, wherein the host cell is a prokaryotic cell or a eukaryotic cell.

34. The host cell of any one of claims 32 to 33, wherein the host cell is selected from a bacterial cell, a mammalian cell, an insect cell or a plant cell.

35. A method of producing a hybrid tRNA/pre-microRNA or tRNA/shRNA molecule, comprising expressing in a population of host cells the tRNA/pre-microRNA or tRNA/shRNA polynucleotide of any one of claims 1 to 28, the expression cassette of claim 29 or the viral vector of claim 31.

36. The method of claim 35, wherein at least 1 mg of the hybrid tRNA/pre-microRNA or tRNA/shRNA molecule are produced from a 1 liter culture comprising the population of host cells.

37. The method of any one of claims 35 to 36, wherein the host cell is selected from a bacterial cell, a mammalian cell, an insect cell or a plant cell.

38. The method of any one of claims 35 to 37, wherein at least 1 mg of the hybrid tRNA/pre-microRNA or tRNA/shRNA molecule are produced from a 1 liter culture of E. coli host cells over a period of about 24 hours.

39. The method of any one of claims 35 to 37, wherein at least 1 mg of the hybrid tRNA/pre-microRNA or tRNA/shRNA molecule is produced from a 1 liter culture of yeast host cells.

40. The method of any one of claims 35 to 39, wherein the tRNA/pre-microRNA molecule produced comprises at least about 5% of the total RNA.

41. A method of producing an RNA molecule, comprising expressing in a population of host cells the RNA molecule from the tRNA/pre-microRNA or tRNA/shRNA polynucleotide of any one of claims 1 to 28, the expression cassette of claim 29 or the viral vector of claim 31.

42. The method of claim 41, wherein at least 1 mg of the RNA molecule are produced from a 1 liter culture comprising the population of host cells.

43. The method of any one of claims 41 to 42, wherein the host cell is selected from a bacterial cell, a mammalian cell, an insect cell or a plant cell.

44. The method of any one of claims 41 to 43, wherein at least 1 mg of the RNA molecule are produced from a 1 liter culture of E. coli host cells over a period of about 24 hours.

45. The method of any one of claims 41 to 43, wherein at least 1 mg of the RNA molecule is produced from a 1 liter culture of yeast host cells.

46. A method of preventing, reducing or inhibiting the expression of a target polynucleotide in a subject in need thereof, comprising administering to the subject the polynucleotide of any one of claims 12 to 28, the expression cassette of claim 29, the liposome or nanoparticle of claim 30, or the viral vector of claim 31.

47. A method of preventing, mitigating, reducing and/or inhibiting the growth, proliferation, and/or progression of a cancer in a subject in need thereof, comprising administering to the subject the polynucleotide of any one of claims 12 to 28, the expression cassette of claim 29, the liposome or nanoparticle of claim 30, or the viral vector of claim 31.

48. The method of claim 47, wherein the cancer is selected from the group consisting of breast cancer, lymphoma, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, prostate cancer, and lung cancer.

49. A method of identifying the presence of a target molecule in a sample, comprising contacting a sample suspected of containing the target molecule under conditions that allow binding an inserted RNA expressed from the polynucleotide of any one of claims 12 to 28, the expression cassette of claim 29, the liposome or nanoparticle of claim 30, or the viral vector of claim 31 , wherein binding of the inserted RNA to the target molecule in the sample identifies its presence.

50. A kit comprising the polynucleotide of any one of claims 1 to 28, the expression cassette of claim 29, the liposome or nanoparticle of claim 30, or the viral vector of claim 31.