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1. WO2017098052 - COMPOSITIONS AND METHODS FOR THE DIAGNOSIS AND TREATMENT OF BONE FRACTURES AND DISORDERS

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CLAIMS

. An in vitro method of diagnosing osteoporosis or determining the risk of osteoporotic fractures or monitoring of treatment in a subject, comprising the steps of:

a) providing a sample from said subject,

b) measuring the level of two or more miRNAs selected from any of miRNAs consisting of hsa-miR- 44-3p, hsa-miR-188-3p, hsa-miR-382-3p, hsa-miR-127-3p, hsa-miR-155-5p, hsa-miR-181 a-3p, hsa-miR-1908, hsa-miR-190a, hsa-miR-199b-5p hsa-miR-203a, hsa-miR-214-3p, hsa-miR-29b-3p, hsa-miR-330-3p, hsa-miR-335-5p, hsa-miR-31 -5p, hsa-miR-342-5p, hsa-miR-369-3p, hsa-miR-377-3p, hsa-miR-378a-5p, hsa-miR-495-3p, hsa-miR-500a-5p, hsa-miR-502-5p, hsa-miR-542-5p, hsa-miR-550a-5p, hsa-miR-576-3p, hsa-miR-582-3p, hsa-miR-642a-5p, hsa-miR-942, hsa-let-7b-5p and hsa-miR-181 d or isoforms or variants thereof, and

c) comparing the level of said miRNAs to a reference level,

wherein the magnitude of difference in said level when compared to the reference level is indicative of osteoporosis or the risk of fractures.

2. The method according to claim 1 , wherein the level of all of hsa-miR-203a, hsa-miR-330-3p, hsa-miR-188-3p, hsa-miR-550a-5p, hsa-miR-335-5p, hsa-miR-29b-3p, hsa-miR-214-3p and hsa-miR-31 -5p is measured.

3. The method according to claim 1 , wherein at least one miRNA selected from the group consisting of hsa-miR-188-3p, hsa-miR-382-3p, hsa-miR-203a, hsa-miR-181 a-3p, hsa-miR-330-3p and hsa-miR-369-3p and at least one miRNA selected from the group consisting of hsa-miR-127-3p, hsa-miR-155-5p, hsa-miR-1908, hsa-miR-190a, hsa-miR-199b-5p, hsa-miR-144-3p, hsa-miR-335-5p, hsa-miR-31 -5p, hsa-miR-214-3p, hsa-miR-29b-4p, hsa-let-7b-5p, hsa-miR-342-5p, hsa-miR-377-3p, hsa-miR-378a-5p, hsa-miR-495-3p, hsa-miR-500a-5p, hsa-miR-502-5p, hsa-miR-542-5p, hsa-miR-550a-5p, hsa-miR-576-3p, hsa-miR-582-3p, hsa-miR-642a-5p, hsa-miR-942, and hsa-miR-181 d are measured.

4. The method according to claim 1 or 3, wherein hsa-miR-188-3p is measured in combination with at least one miRNA selected from the group consisting of hsa-miR- 127-3p, hsa-miR-155-5p, hsa-miR-1908, hsa-miR-190a, hsa-miR-199b-5p, hsa-miR-144-3p, hsa-miR-335-5p, hsa-miR-31 -5p, hsa-miR-2 4-3p, hsa-miR-29b-4p, hsa-let-7b-5p, hsa-miR-203a, hsa-miR-342-5p, hsa-miR-377-3p, hsa-miR-378a-5p, hsa-miR-495-3p, hsa-miR-500a-5p, hsa-miR-502-5p, hsa-miR-542-5p, hsa-miR-550a-5p, hsa-miR-576-3p, hsa-miR-582-3p, hsa-miR-642a-5p, hsa-miR-942 and hsa-miR-181 d.

5. The method according to claim 1 or 3, wherein hsa-miR-382-3p is measured in combination with at least one miRNA selected from the group consisting of hsa-miR-127-3p, hsa-miR-155-5p, hsa-miR-188-3p, hsa-miR-1908, hsa-miR-190a, hsa-miR-199b-5p, hsa-miR-144-3p, hsa-miR-335-5p, hsa-miR-31 -5p, hsa-miR-214-3p, hsa-miR-29b-4p, hsa-let-7b-5p, hsa-miR-203a, hsa-miR-342-5p, hsa-miR-377-3p, hsa-miR-378a-5p, hsa-miR-495-3p, hsa-miR-500a-5p, hsa-miR-502-5p, hsa-miR-542-5p, hsa-miR-550a-5p, hsa-miR-576-3p, hsa-miR-582-3p, hsa-miR-642a-5p, hsa-miR-942 and hsa-miR-181 d.

6. The method according to claim 1 or 3, wherein hsa-miR-181 a-3p is measured in combination with at least one miRNA selected from the group consisting of hsa-miR-127-3p, hsa-miR-155-5p, hsa-miR-188-3p, hsa-miR-1908, hsa-miR-190a, hsa-miR-199b-5p, hsa-miR-144-3p, hsa-miR-335-5p, hsa-miR-31 -5p, hsa-miR-214-3p, hsa-miR-29b-4p, hsa-let-7b-5p, hsa-miR-203a, hsa-miR-342-5p, hsa-miR-377-3p, hsa-miR-378a-5p, hsa-miR-495-3p, hsa-miR-500a-5p, hsa-miR-502-5p, hsa-miR-542-5p, hsa-miR-550a-5p, hsa-miR-576-3p, hsa-miR-582-3p, hsa-miR-642a-5p, hsa-miR-942 and hsa-miR-181 d.

7. The method according to claim 1 or 3, wherein hsa-miR-330-3p is measured in combination with at least one miRNA selected from the group consisting of hsa-miR-127-3p, hsa-miR-155-5p, hsa-miR-188-3p, hsa-miR-1908, hsa-miR-190a, hsa-miR-199b-5p, hsa-miR-144-3p, hsa-miR-335-5p, hsa-miR-31 -5p, hsa-miR-214-3p, hsa-miR-29b-4p, hsa-let-7b-5p, hsa-miR-203a, hsa-miR-342-5p, hsa-miR-377-3p, hsa-miR-378a-5p, hsa-miR-495-3p, hsa-miR-500a-5p, hsa-miR-502-5p, hsa-miR-542-5p, hsa-miR-550a-5p, hsa-miR-576-3p, hsa-miR-582-3p, hsa-miR-642a-5p, hsa-miR-942 and hsa-miR-181 d.

8. The method according to claim 1 or 3, wherein hsa-miR-369-3p is measured in combination with at least one miRNA selected from the group consisting of hsa-miR-127-3p, hsa-miR-155-5p, hsa-miR-188-3p, hsa-miR-1908, hsa-miR-190a, hsa-miR-199b-5p, hsa-miR-144-3p, hsa-miR-335-5p, hsa-miR-31 -δρ, hsa-miR-214-3p, hsa-miR-29b-4p, hsa-let-7b-5p, hsa-miR-203a, hsa-miR-342-5p, hsa-miR-377-3p, hsa-miR-378a-5p, hsa-miR-495-3p, hsa-miR-500a-5p, hsa-miR-502-5p, hsa-miR-542-5p, hsa-miR-550a-5p, hsa-miR-576-3p, hsa-miR-582-3p, hsa-miR-642a-5p, hsa-miR-942 and hsa-miR-181 d.

9. The method according to claim 1 or 3, wherein hsa-miR-203a is measured in combination with at least one miRNA selected from the group consisting of hsa-miR-127-3p, hsa-miR-155-5p, hsa-miR-188-3p, hsa-miR-1908, hsa-miR-190a, hsa-miR-335-5p, hsa-miR-31 -5p, hsa-miR-214-3p, hsa-miR-29b-4p, hsa-let-7b-5p, hsa-miR-342-5p, hsa-miR-377-3p, hsa-miR-378a-5p, hsa-miR-495-3p, hsa-miR-500a-5p, hsa-miR-502-5p, hsa-miR-542-5p, hsa-miR-550a-5p, hsa-miR-576-3p, hsa-miR-582-3p, hsa-miR-642a-5p, hsa-miR-942 and hsa-miR-181d.

10. The method according to any one of claims 1 to 9, wherein the subject is a postmenopausal female or an osteoporosis or osteopenia patient, suffering from or being at risk of developing bone fractures.

1 1 . The method according to any one of claims 1 to 10, wherein the level of at least 3, preferably at least 4, preferably at least 5, specifically up to 30 miRNAs is measured.

12. An in vitro method of diagnosing osteoporosis or determining the risk of osteoporotic fractures or monitoring of treatment in a subject, comprising the steps of: a) providing a sample from said subject,

b) measuring the level of two or more miRNAs selected from any of

miRNAs consisting of hsa-miR-32-3p, hsa-let-7b-5p, hsa-let-7g-5p, hsa-let-7i-5p, hsa-miR-141 -3p, hsa-miR-143-5p, hsa-miR-16-2-3p, hsa-miR-181 c-3p, hsa-miR-181 c-5p, hsa-miR-191 -5p, hsa-miR-19b-1 -5p, hsa-miR-203a, hsa-miR-21 -3p, hsa-miR-323a-3p, hsa-miR-375, hsa-miR-486-5p, hsa-miR-500a-5p, hsa-miR-550a-5p, hsa-

miR-7-5p, hsa-miR-92a-3p, hsa-miR-942, hsa-miR-96-5p, hsa-miR-330-3p, hsa-miR-31 -5p, hsa-miR-144-3p, and hsa-miR-382-3p, and

c) comparing the level of said miRNAs to a reference level,

wherein the magnitude of difference in said level when compared to the reference level is indicative of osteoporosis or the risk of fractures.

13. The method according to claim 12, wherein at least one miRNA selected from the group consisting of hsa-miR-32-3p, hsa-miR-181 c-3p, hsa-miR-323a-3p, hsa-miR-550a-5p and hsa-miR-96-5p and at least one miRNA selected from the group consisting of hsa-let-7b-5p, hsa-let-7g-5p, hsa-let-7i-5p, hsa-miR-141 -3p, hsa-miR-143-5p, hsa-miR-16-2-3p, hsa-miR-181 c-5p, hsa-miR-191 -5p, hsa-miR-19b-1 -5p, hsa-miR-203a, hsa-miR-21 -3p, hsa-miR-375, hsa-miR-486-5p, hsa-miR-500a-5p, hsa-miR-7-5p, hsa-miR-92a-3p, hsa-miR-942, hsa-miR-330-3p, hsa-miR-31 -5p, hsa-miR-144-3p and hsa-miR-382-3p are measured.

14. The method according to claim 12 or 13, wherein hsa-miR-32-3p is measured in combination with at least one miRNA selected from the group consisting of hsa-let-7b-5p, hsa-let-7g-5p, hsa-let-7i-5p, hsa-miR-141 -3p, hsa-miR-143-5p, hsa-miR-16-2-3p, hsa-miR-181 c-5p, hsa-miR-191 -5p, hsa-miR-19b-1 -5p, hsa-miR-203a, hsa-miR-21 -3p, hsa-miR-375, hsa-miR-486-5p, hsa-miR-500a-5p, hsa-miR-7-5p, hsa-miR-92a-3p, hsa-miR-942, hsa-miR-330-3p, hsa-miR-31 -δρ, hsa-miR-144-3p and hsa-miR-382-3p.

15. The method according to claim 12 or 13, wherein hsa-miR-181 c-3p is measured in combination with at least one miRNA selected from the group consisting of hsa-let-7b-5p, hsa-let-7g-5p, hsa-let-7i-5p, hsa-miR-141 -3p, hsa-miR-143-5p, hsa-miR-16-2-3p, hsa-miR-181 c-5p, hsa-miR-191 -5p, hsa-miR-19b-1 -5p, hsa-miR-203a, hsa-miR-21 -3p, hsa-miR-375, hsa-miR-486-5p, hsa-miR-500a-5p, hsa-miR-7-5p, hsa-miR-92a-3p, hsa-miR-942, hsa-miR-330-3p, hsa-miR-31 -5p, hsa-miR-144-3p and hsa-miR-382-3p.

16. The method according to claim 12 or 13, wherein hsa-miR-323a-3p is measured in combination with at least one miRNA selected from the group consisting of hsa-let-7b-5p, hsa-let-7g-5p, hsa-let-7i-5p, hsa-miR-141 -3p, hsa-miR-143-5p, hsa- miR-16-2-3p, hsa-miR-181 c-5p, hsa-miR-191 -5p, hsa-miR-19b-1 -5p, hsa-miR-203a, hsa-miR-21 -3p, hsa-miR-375, hsa-miR-486-5p, hsa-miR-500a-5p, hsa-miR-7-5p, hsa-miR-92a-3p, hsa-miR-942, hsa-miR-330-3p, hsa-miR-31 -5p, hsa-miR- 44-3p and hsa-miR-382-3p.

17. The method according to claim 12 or 13, wherein hsa-miR-550a-5p is measured in combination with at least one miRNA selected from the group consisting of hsa-let-7b-5p, hsa-let-7g-5p, hsa-let-7i-5p, hsa-miR-141 -3p, hsa-miR-143-5p, hsa-miR-16-2-3p, hsa-miR-181 c-5p, hsa-miR-191 -5p, hsa-miR-19b-1 -5p, hsa-miR-203a, hsa-miR-21 -3p, hsa-miR-375, hsa-miR-486-5p, hsa-miR-500a-5p, hsa-miR-7-5p, hsa-miR-92a-3p, hsa-miR-942, hsa-miR-330-3p, hsa-miR-31 -5p, hsa-miR-144-3p and hsa-miR382-3p.

18. The method according to claim 12 or 13, wherein hsa-miR-96-5p is measured in combination with at least one miRNA selected from the group consisting of hsa-let-7b-5p, hsa-let-7g-5p, hsa-let-7i-5p, hsa-miR-141 -3p, hsa-miR-143-5p, hsa-miR-16-2-3p, hsa-miR-181 c-5p, hsa-miR-191 -5p, hsa-miR-19b-1 -5p, hsa-miR-203a, hsa-miR-21 -3p, hsa-miR-375, hsa-miR-486-5p, hsa-miR-500a-5p, hsa-miR-7-5p, hsa-miR-92a-3p, hsa-miR-942, , hsa-miR-330-3p, hsa-miR-31 -5p, hsa-miR-144-3p and hsa-miR-382-3p.

19. The method according to any one of claims 12 to 18, wherein the subject is a patient being at risk of or suffering from type 2 diabetes mellitus.

20. The method according to any one of claims 12 to 19, wherein the sample is a blood sample.

21 . The method according to any one of claims 12 to 20, wherein the level of at least 3, preferably at least 4, preferably at least 5, specifically up to 26 miRNAs is measured.

22. The method according to any one of claims 1 to 21 , wherein the level of said miRNAs is compared with the average level of corresponding miRNAs in healthy

subjects, wherein a difference by more than one standard deviations is indicative of osteoporosis with increased risk of future fractures.

23. The method according to any one of claims 1 to 22, wherein the level of all miRNAs is measured.

24. The method according to any one of claims 1 to 23, wherein one or more further miRNAs are detected, wherein said miRNAs are

a) differentially regulated in osteoporotic individuals as compared to healthy individuals and are

b) involved in osteogenic differentiation and/or in osteoclastogenic activation.

25. Use of a method according to any one of claims 1 to 24 for monitoring a subject, specifically for the prognosis of bone fraction.

26. Use of a method according to any one of claims 1 to 24 for monitoring a subject, specifically for measuring the response of a subject to a nti -osteoporotic treatments.

27. The method according to any one of claims 1 to 25, wherein the difference in miRNA levels is determined by quantitative or digital PGR, sequencing, microarray, Luminex nucleic acid assays, or other hybridization-based techniques.

28. Composition for use in treating or preventing osteoporosis or fractures comprising

a) at least two synthetic human miRNAs from miRNAs of claims 1 and/or 12 and/or

b) an antagonist/inhibitor of at least two of miRNAs of claimsl and/or 12 that i. decreases the level of said miRNAs; and/or

ii. inhibits or down-regulates expression of the sequences coding for said miRNAs or degrades or cleaves said miRNAs.

29. Use of a composition comprising at least one, specifically at least two synthetic human miRNAs of claims 1 and/or 12 and/or an antagonist/inhibitor of at least two of miRNAs of claims 1 and/or 12 that decreases the level of said miRNAs; and/or inhibits or down-regulates expression of the sequences coding for said miRNAs or degrades or cleaves said miRNAsfor preparing a medicament.