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1. (WO2019000015) CELL-FREE MICRORNA SIGNATURES OF PANCREATIC ISLET BETA CELL DEATH
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CLAIMS

1. A method for diagnosing, prognosing, or determining a likelihood of developing a disease or condition associated with or arising from reduced insulin production in a subject, the method comprising:

determining expression levels of ten or more extracellular microRNAs selected from the group consisting of: miR-24, miR-26a, miR-146a, miR-199a-3p, miR-30b, miR-30c, miR-222, miR-20a, miR-26b, miR-223, miR-186, miR-21, miR-16, miR-92a, miR-126, miR-25, miR-30a-5p, and miR-93; in a biological fluid sample obtained from the subject; and

comparing expression levels the microRNAs in the biological fluid sample to control expression level/s of the microRNAs generated from control biological fluid/s equivalent to the sample,

wherein elevated expression levels of the microRNAs in the biological fluid sample compared to the expression level/s of the microRNAs generated from the control biological fluid/s is indicative of a deficient or absent capacity for insulin production in beta-islet cells of the subject, and

the reduced or absent insulin production in the beta-islet cells is:

(i) predictive that the subject will develop the disease or condition; or

(ii) diagnostic of the disease or condition in the subject; or

(hi) prognostic that the disease or condition will progress in the subject.

2. A method for monitoring the response of a subject to a treatment for a disease or condition associated with or arising from reduced insulin production in a subject, the method comprising:

determining expression levels of ten or more extracellular microRNAs selected from the group consisting of: miR-24, miR-26a, miR-146a, mmiR-199a-3p, miR-30b, miR-30c, miR-222, miR-20a, miR-26b, miR-223, miR-186, miR-21, miR-16, miR-92a, miR-126, miR-25, miR-30a-5p, and miR-93; in a biological fluid sample obtained from the subject; and

comparing expression levels the microRNAs in the biological fluid sample to control expression level/s of the microRNAs generated from control biological fluid/s equivalent to the sample,

wherein elevated expression levels of the microRNAs in the biological fluid sample compared to the expression level/s of the microRNAs generated from the control

biological fluid/s is indicative of deficient or absent capacity for insulin production in beta islet cells of the subject, and a deficient or absent response to the treatment by the subject.

3. The method of claim 2, wherein the treatment comprises beta-islet cell transplantation, and/or the administration of a vaccine or therapeutic drug designed to retard loss of beta-islet cells in the subject and/or loss of insulin production capacity in beta-islet cells of the subject.

4. A method for assessing the efficacy of a treatment for inhibiting or preventing loss of beta-islet cell insulin production function and/or beta-islet cell death, the method comprising:

administering the treatment to a subject in need thereof,

determining expression levels of ten or more extracellular microRNAs selected from the group consisting of: miR-24, miR-26a, miR-146a, miR-199a-3p, miR-30b, miR-30c, miR-222, miR-20a, miR-26b, miR-223, miR-186, miR-21 , miR-16, miR-92a, miR-126, miR-25, miR-30a-5p, and miR-93; in a biological fluid sample obtained from the subject; and

comparing expression levels the microRNAs in the biological fluid sample to control expression level/s of the microRNAs generated from control biological fluid/s equivalent to the sample,

wherein equivalent or reduced expression levels of the microRNAs in the biological fluid sample compared to the expression level/s of the microRNAs generated from the control biological fluid/s is indicative that the treatment has efficacy in inhibiting or preventing loss of beta-islet cell insulin production function and/or beta-islet cell death.

5. The method of any one of claims 1 to 4, further comprising removing any cells comprising nuclear material present in the biological fluid prior to determining expression levels of one or more extracellular microRNAs.

6. The method of claim 5, wherein said removing of the cells comprising nuclear material comprises lysing less than: 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2% or 0.1% of the cells.

7. The method of any one of claims 1 to 6, wherein the condition or disease is selected from any of diabetes, pancreatitis, insulinoma, and pancreatic cancer.

8. The method of claim 7, wherein the disease is diabetes.

9. The method of claim 8, wherein the diabetes is Type 1 diabetes.

10. The method of any one of claims 1 to 9, wherein the biological fluid sample is whole blood.

11. The method of any one of claims 1 to 9, wherein the biological fluid sample is serum or plasma.

12. The method of any one of claims 1 to 11, further comprising an initial step of obtaining the biological fluid sample from the subject.

13. The method of any one of claims 1 to 12, wherein the ten or more microRNAs comprise or consist of any: 10, 11, 12, 13, 14, 15, 16, 17, or 18; of said microRNAs.

14. The method of any one of claims 1 to 13, wherein the subject is of Australian ethnicity, of Chinese ethnicity, or of Indian ethnicity.

15. The method of any one of claims 1 to 14, wherein the ten or more microRNAs are selected from the group consisting of: miR- 15b, miR-16, miR-20a, miR-21, miR-22-5p, miR-24, miR-25, miR-26a, miR-26b, miR-29a, miR-30a-5p, miR-30b, miR-30c, miR-30e-3p, miR-92a, miR-93, miR-126, miR-146a, miR-155, miR-186, miR-199a-3p, miR-222, and miR-223 .

16. The method of claim 15, wherein the microRNAs comprise or consist of any: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23; of said microRNAs.

17. The method of claim 15 or claim 16, wherein the subject is of Australian ethnicity or of Chinese ethnicity.

18. The method of any one of claims 1 to 13, wherein the ten or more microRNAs are selected from the group consisting of: miR-16, miR-20a, miR-21 , miR-24, miR-25, miR- 26a, miR-26b, miR-27a, miR-30a-5p, miR-30b, miR-30c, miR-92a, miR-93, miR-126, miR-146a, miR-186, miR-199a-3p, miR-222, miR-223, and miR-340.

19. The method of claim 18, wherein the microRNAs comprise or consist of any: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; of said microRNAs.

20. The method of claim 18 or claim 19, wherein the subject is of Australian ethnicity or of Indian ethnicity.

21. The method of any one of claims 1 to 13, wherein the ten or more microRNAs are selected from the group consisting of: miR-16, miR-20a, miR-21 , miR-24, miR-25, miR-26a, miR-26b, miR-30a-5p, miR-30b, miR-30c, miR-92a, miR-93, miR-126, miR-145, miR-146a, miR-186, miR-199a-3p, miR-222, and miR-223.

22. The method of claim 21 , wherein the microRNAs comprise or consist of any: 10, 1 1, 12, 13, 14, 15, 16, 17, 18, or 19; of said microRNAs.

23. The method of claim 21 or claim 22, wherein the subject is of Chinese ethnicity or of Indian ethnicity.

24. The method of any one of claims 1 to 13, wherein the ten or more microRNAs are selected from the group consisting of: miR-15b, miR-16, miR-20a, miR-21, miR-22-5p, miR-24, miR-25, miR-26a, miR-26b, miR-27b, miR-29a, miR-30a-5p, miR-30b, miR-30c, miR-30e-3p, miR-92a, miR-93, miR-103, miR-126, miR-125a-5p, miR-127, miR-146a, miR-152, miR-155, miR-186, miR-199a-3p, miR-210, miR-222, and miR-223

25. The method of claim 24, wherein the microRNAs comprise or consist of any: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29; of said microRNAs.

26. The method of claim 24 or claim 25, wherein the subject is of Australian ethnicity.

27. The method of any one of claims 1 to 13, wherein the ten or more microRNAs are selected from the group consisting of: let-7e, miR-7, miR-15b, miR-16, miR-20a, miR-21 , miR-22-5p, miR-24, miR-25, miR-26a, miR-26b, miR-29a, miR-30a-5p, miR-30b, miR-

30c, miR-30e-3p, miR-34a, miR-92a, miR-93, miR-126, miR-146a, mlR-148a, miR-155, miR-186, miR-199a-3p, miR-222, miR-223, miR-374, and miR-625.

28. The method of claim 27, wherein the microRNAs comprise or consist of any: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29; of said microRNAs.

29. The method of claim 27 or claim 28, wherein the subject is of Chinese ethnicity.

30. The method of any one of claims 1 to 29, wherein the control biological fluid/s is/are obtained from control subject/s determined not to have a disease or condition associated with aberrant insulin production and/or abnormal insulin metabolism.

31. The method of claim 30, wherein the control subject/s are a population of individuals determined not to have a disease or condition associated with aberrant insulin production and/or abnormal insulin metabolism.

32. The method of claim 31 or claim 32, wherein the control subject/s do not have a disease or condition selected from the group consisting of diabetes, pancreatitis, insulinoma, and pancreatic cancer.

33. A kit comprising primers, probes and/or other binding agents for use in detecting expression of at least ten microRNAs selected from the group consisting of: miR-24, miR-26a, miR-146a, miR-199a-3p, miR-30b, miR-30c, miR-222, miR-20a, miR-26b, miR-223, miR-186, miR-21 , miR-16, miR-92a, miR-126, miR-25, miR-30a-5p, and miR-93; in a biological fluid sample obtained from the subject.

34. A microRNA signature comprising least ten microRNAs selected from the group consisting of: miR-24, miR-26a, miR-146a, miR-199a-3p, miR-30b, miR-30c, miR-222, miR-20a, miR-26b, miR-223, miR-186, miR-21 , miR-16, miR-92a, miR-126, miR-25, miR-30a-5p, and miR-93.

35. The kit of claim 33 or the microRNA signature of claim 34, wherein the at least ten microRNAs comprise or consist of any: 10, 1 1 , 12, 13, 14, 15, 16, 17, or 18; of said microRNAs.

36. The kit of claim 33 or claim 35, or the microRNA signature of claim 34 or claim 35, wherein the at least ten microRNAs are selected from the group consisting of: miR-15b, miR-16, miR-20a, miR-21, miR-22-5p, miR-24, miR-25, miR-26a, miR-26b, miR-29a, miR-30a-5p, miR-30b, miR-30c, miR-30e-3p, miR-92a, miR-93, miR-126, miR-146a, miR-155, miR-186, miR-199a-3p, miR-222, and miR-223 .

37. The kit or microRNA signature of claim 36, wherein the microRNAs comprise or consist of any: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23; of said microRNAs.

38. The kit of claim 33 or claim 35, or the microRNA signature of claim 34 or claim 35, wherein the at least ten microRNAs are selected from the group consisting of: miR-16, miR-20a, miR-21, miR-24, miR-25, miR-26a, miR-26b, miR-30a-5p, miR-30b, miR-30c, miR-92a, miR-93, miR-126, miR-146a, miR-186, miR-199a-3p, miR-222, miR-223, miR-340 and miR-27a.

39. The kit or microRNA signature of claim 38, wherein the microRNAs comprise or consist of any: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; of said microRNAs.

40. The kit of claim 33 or claim 35, or the microRNA signature of claim 34 or claim 35, wherein the at least ten microRNAs are selected from the group consisting of: miR-16, miR-20a, miR-21, miR-24, miR-25, miR-26a, miR-26b, miR-30a-5p, miR-30b, miR-30c, miR-92a, miR-93, miR-126, miR-145, miR-146a, miR-186, miR-199a-3p, miR-222, and miR-223.

41. The kit or microRNA signature of claim 40, wherein the microRNAs comprise or consist of any: 10, 1 1, 12, 13, 14, 15, 16, 17, 18, or 19; of said microRNAs.

42. The kit of claim 33 or claim 35, or the microRNA signature of claim 34 or claim 35, wherein the at least ten microRNAs are selected from the group consisting of: miR-15b, miR-16, miR-20a, miR-21, miR-22-5p, miR-24, miR-25, miR-26a, miR-26b, miR-27b, miR-29a, miR-30a-5p, miR-30b, miR-30c, miR-30e-3p, miR-92a, miR-93, miR-103, miR-126, miR-125a-5p, miR-127, miR-146a, miR-152, miR-155, miR-186, miR-199a-3p, miR-210, miR-222, and miR-223.

43. The kit or microRNA signature of claim 42, wherein the microRNAs comprise or consist of any: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29; of said microRNAs.

44. The kit of claim 33 or claim 35, or the microRNA signature of claim 34 or claim 35, wherein the at least ten microRNAs are selected from the group consisting of: let-7e, miR-7, miR-15b, miR-16, miR-20a, miR-21, miR-22-5p, miR-24, miR-25, miR-26a, miR-26b, miR-29a, miR-30a-5p, miR-30b, miR-30c, miR-30e-3p, miR-34a, miR-92a, miR-93, miR-126, miR-146a, mIR-148a, miR-155, miR-186, miR-199a-3p, miR-222, miR-223, miR-374, and miR-625.

45. The kit or microRNA signature of claim 44, wherein the microRNAs comprise or consist of any: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29; of said microRNAs.

46. The kit of any one of claims 33 or 35 to 45, further comprising any one or more of:

(i) one or more reagents for extracting the biological fluid sample;

(ii) one or more reagents for reverse-transcribing RNA into cDNA;

(iii) one or more reagents for amplifying the cDNA;

(iv) control standards for normalisation of microRNA signature expression data and/or comparison of microRNA signature expression data to determine whether expression of the microRNA signature is increased, reduced, and/or within a standard range;

(v) any one of more of: buffers, washing reagents, RNAse inhibitors, precipitators (e.g. glycogen), nuclease-free water, salt solutions.

47. Use of the kit of any one of claims 33 or 35 to 46, or the microRNA signature of any one of claims claim 34 to 46, for diagnosing, prognosing, or determining a likelihood of developing a disease or condition associated with or arising from reduced insulin production in a subject, wherein the disease or condition is selected from the group consisting of diabetes, pancreatitis, insulinoma, and pancreatic cancer.

48. Use of one or more agents for determining expression levels of ten or more extracellular microRNAs selected from the group consisting of: miR-24, miR-26a, miR-146a, miR-199a-3p, miR-30b, miR-30c, miR-222, miR-20a, miR-26b, miR-223, miR-

186, miR-21 , miR-16, miR-92a, miR-126, miR-25, miR-30a-5p, and miR-93; in a biological fluid sample obtained from a subject, for the preparation of a medicament for diagnosing, prognosing, or determining a likelihood of developing of a disease or condition associated with or arising from reduced insulin production in the subject.

49. Use of one or more agents for determining expression levels of ten or more extracellular microRNAs selected from the group consisting of: miR-24, miR-26a, miR-146a, mmiR-199a-3p, miR-30b, miR-30c, miR-222, miR-20a, miR-26b, miR-223, miR-186, miR-21, miR-16, miR-92a, miR-126, miR-25, miR-30a-5p, and miR-93; in a biological fluid sample obtained from a subject, for the preparation of a medicament for monitoring the response of the subject to a treatment for a disease or condition associated with reduced insulin production.

50. The use of claim 49, wherein the treatment comprises beta-islet cell transplantation, and/or the administration of a vaccine or therapeutic drug designed to retard loss of beta-islet cells in the subject and/or loss of insulin production capacity in beta-islet cells of the subject.

51. Use of one or more agents for determining expression levels of ten or more extracellular microRNAs selected from the group consisting of: miR-24, miR-26a, miR-146a, mmiR-199a-3p, miR-30b, miR-30c, miR-222, miR-20a, miR-26b, miR-223, miR-186, miR-21, miR-16, miR-92a, miR-126, miR-25, miR-30a-5p, and miR-93; in a biological fluid sample obtained from a subject, for the preparation of a medicament for assessing the efficacy of a treatment for inhibiting or preventing loss of beta-islet cell insulin production function and/or beta-islet cell death in the subject.

52. The use of any one of claims 48 to 51, wherein the microRNAs comprise or consist of any: 10, 11, 12, 13, 14, 15, 16, 17, or 18; of said microRNAs.

53. The use of any one of claims 48 to 52, wherein the subject is of Australian ethnicity, of Chinese ethnicity, or of Indian ethnicity.

54. The use of any one of claims 48 to 53, wherein the microRNAs are selected from the group consisting of: miR-15b, miR-16, miR-20a, miR-21 , miR-22-5p, miR-24, miR-

25, miR-26a, miR-26b, miR-29a, miR-30a-5p, miR-30b, miR-30c, miR-30e-3p, miR-92a, miR-93, miR-126, miR-146a, miR-155, miR-186, miR-199a-3p, miR-222, and miR-223 .

55. The use of claim 54, wherein the microRNAs comprise or consist of any: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23; of said microRNAs.

56. The use of claim 54 or claim 55, wherein the subject is of Australian ethnicity or of Chinese ethnicity.

57. The use of any one of claims 48 to 53, wherein the ten or more microRNAs are selected from the group consisting of: miR-16, miR-20a, miR-21 , miR-24, miR-25, miR-26a, miR-26b, miR-30a-5p, miR-30b, miR-30c, miR-92a, miR-93, miR-126, miR-146a, miR-186, miR-199a-3p, miR-222, miR-223, miR-340 and miR-27a.

58. The use of claim 57, wherein the microRNAs comprise or consist of any: 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20; of said microRNAs.

59. The use of claim 57 or claim 58, wherein the subject is of Australian ethnicity or of Indian ethnicity.

60. The use of of any one of claim 48 to 53, wherein the ten or more microRNAs are selected from the group consisting of: miR-16, miR-20a, miR-21, miR-24, miR-25, miR-26a, miR-26b, miR-30a-5p, miR-30b, miR-30c, miR-92a, miR-93, miR-126, miR-145, miR-146a, miR-186, miR-199a-3p, miR-222, and miR-223.

61. The use of claim 60, wherein the microRNAs comprise or consist of any: 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19; of said microRNAs.

62. The use of claim 60 or claim 61, wherein the subject is of Chinese ethnicity or of Indian ethnicity.

63. The use of any one of claims 48 to 53, wherein the ten or more microRNAs are selected from the group consisting of: miR-15b, miR-16, miR-20a, miR-21, miR-22-5p, miR-24, miR-25, miR-26a, miR-26b, miR-27b, miR-29a, miR-30a-5p, miR-30b, miR-

30c, miR-30e-3p, miR-92a, miR-93, miR-103, miR-126, miR-125a-5p, miR-127, miR-146a, miR-152, miR-155, miR-186, miR-199a-3p, miR-210, miR-222, and miR-223

64. The use of claim 63, wherein the microRNAs comprise or consist of any: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29; of said microRNAs.

65. The use of claim 63 or claim 64, wherein the subject is of Australian ethnicity.

66. The use of any one of claims 48 to 53, wherein the ten or more microRNAs are selected from the group consisting of: let-7e, miR-7, miR-15b, miR-16, miR-20a, miR-21 , miR-22-5p, miR-24, miR-25, miR-26a, miR-26b, miR-29a, miR-30a-5p, miR-30b, miR-30c, miR-30e-3p, miR-34a, miR-92a, miR-93, miR-126, miR-146a, mIR-148a, miR-155, miR-186, miR-199a-3p, miR-222, miR-223, miR-374, and miR-625.

67. The use of claim 66, wherein the microRNAs comprise or consist of any: 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29; of said microRNAs.

The use of claim 66 or claim 67, wherein the subject is of Chinese ethnicity.