USE OF MICROVESICLES IN DIAGNOSIS AND PROGNOSIS OF MEDICAL DISEASES AND CONDITIONS

• US 20100196426A1
• Filed: 01/28/2010
• Published: 08/05/2010
• Est. Priority Date: 02/01/2008
• Status: Abandoned Application

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Miranda, Kevin C., Russo, Leileata M., Breakefield, Xandra O., Skog, Johan Karl Olov, Brown, Dennis
• Application Number:
  US12/695,910
• Publication Number:
  US 20100196426A1
• Time in Patent Office: Days
• US Class Current: 424/400
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C12N 15/1017 by filtration, e.g. using filters, frits, membranes
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2600/106 Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• CPC Class Codes: C12Q 2600/112 Disease subtyping, staging or classification
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C12Q 2600/178 miRNA, siRNA or ncRNA

Abstract

The presently disclosed subject matter is directed to methods of aiding diagnosis, prognosis, monitoring and evaluation of a disease or other medical condition in a subject by detecting a biomarker in microvesicles isolated from a biological sample from the subject.

Claims

1. A diagnostic method, wherein said method aids in the detection of a non-infectious disease or condition in a subject, the method comprising the steps of:

(a) isolating a microvesicle fraction from a biological sample from the subject;

(b) detecting the presence or absence of a nucleic acid biomarker within the microvesicle fraction, wherein the biomarker is associated with the disease or other medical condition.

2. A monitoring method, wherein said method aids in monitoring the status of a non-infectious disease or condition in a subject, the method comprising the steps of:

(a) isolating a microvesicle fraction from a biological sample from the subject;

(b) detecting the presence or absence of a nucleic acid biomarker within the microvesicle fraction, wherein the biomarker is associated with the disease or other medical condition.

3. An evaluation method, wherein said method aids in evaluating treatment efficacy in a subject having a disease or other medical condition, the method comprising the steps of:

(a) isolating a microvesicle fraction from a biological sample from the subject;

(b) detecting the presence or absence of a nucleic acid biomarker within the microvesicle fraction, wherein the biomarker is associated with treatment efficacy for the disease or other medical condition.

4. A diagnostic method, wherein said method aids in the detection of a disease or other medical condition in a subject, the method comprising the steps of:

(a) obtaining a biological sample from the subject; and

(b) determining the concentration of microvesicles within the biological sample.

5. A monitoring method, wherein said method aids in monitoring the status of a disease or other medical condition in a subject, the method comprising the steps of:

(a) obtaining a biological sample from the subject; and

(b) determining the concentration of microvesicles within the biological sample.

6. A method for delivering a nucleic acid or protein to a target cell in an individual comprising, administering one or more microvesicles that contain the nucleic acid or protein, or one or more cells that produce such microvesicles, to the individual such that the microvesicles enter the target cell of the individual.

7. A method for performing a body fluid transfusion, comprising the steps of obtaining a fraction of donor body fluid free of all or substantially all microvesicles, or free of all or substantially all microvesicles from a particular cell type, and introducing the microvesicle-free fraction to a patient.

8. A composition of matter comprising a sample of body fluid free of all or substantially all microvesicles, or free of all or substantially all microvesicles from a particular cell type.

9. A method for performing body fluid transfusion, comprising the steps of obtaining a microvesicle-enriched fraction of donor body fluid and introducing the microvesicle-enriched fraction to a patient.

10. A method for diagnosing a cancer in a subject, comprising:

(a) providing a biological sample from a subject;

(b) isolating cancer-derived exosomes comprising microRNAs (mi RNAs) from the biological sample;

(c) determining an amount of one or more of the miRNAs; and

(d) comparing the amount of the one or more miRNAs to one or more miRNA control levels,

wherein the subject is diagnosed as having the cancer if there is a measurable difference in the amount of the one or more miRNAs from the cancer-derived exosomes as compared to the one or more miRNA control levels.

11. The method of claim 10, wherein the cancer is a cancer selected from the group consisting of ovarian cancer, cervical cancer, breast cancer, colon cancer, endometrial cancer, prostate cancer, lung cancer, melanoma, and pancreatic cancer.

12. The method of claim 10, wherein the subject is human.

13. The method of claim 10, wherein the biological sample comprises milk, blood, serum, plasma, ascites, cyst fluid, pleural fluid, cerebral spinal fluid, tears, urine, saliva, sputum, or combinations thereof.

14. The method of claim 10, wherein isolating the cancer-derived exosomes comprises using size exclusion chromatography to isolate the cancer-derived exosomes.

15. The method of claim 10, wherein the exosomes are selectively isolated by immunoabsorbent capture using a cancer specific antigen binding antibody.

16. The method of claim 15, wherein the cancer specific antigen is epithelial-cell adhesion-molecule (EpCAM).

17. The method of claim 16, wherein the cancer specific antigen is epithelial-cell-adhesion-molecule (EpCAM).

18. The method of claim 10, wherein determining an amount of one or more of the miRNAs comprises labeling the one or more miRNAs.

19. The method of claim 10, wherein determining an amount of one or more miRNAs comprises capturing the one or more miRNAs with one or more polynucleotide probes that each selectively bind to the one or more miRNAs.

20. The method of claim 10, wherein determining an amount of the one or more miRNAs comprises using a real-time polymerase chain reaction to quantitate the amount of the one or more miRNAs.

21. The method of claim 10, wherein the one or more miRNAs are one or more of miRNAs let7a, miR-19b, miR-92, miR-93, miR-320, miR-373, miR-324-3p, miR-149, and miR-370.

22. The method of claim 10, wherein, the one or more miRNAs are miR-21 or miR-203.

23. The method of claim 10, further comprising selecting a treatment or modifying a treatment for the cancer based on the amount of the one or more miRNAs determined.

66. The method of claim 10, wherein the cancer is brain cancer.

24. A method for diagnosing a cancer in a subject, comprising:

(a) isolating exosomes from a biological sample from a subject, wherein the exosomes originate from cancer cells and comprise microRNAs (miRNAs);

(b) determining the amount of one or more miRNAs specific to a cancer,

wherein an increase or decrease in the amount of miRNA specific to a cancer is indicative of cancer in the subject.

25. A method for diagnosing a cancer in a subject, comprising:

(a) isolating cancer-derived exosomes from a biological sample from a subject, wherein the exosomes comprise miRNAs;

(b) analyzing the miRNAs for an increase or decrease in levels of miRNAs specific to the cancer;

wherein the presence of an increase or decrease is indicative of cancer in the subject.

26. A method for monitoring progression or regression of a cancer in a subject periodically over time, comprising:

(a) providing a series of biological samples from a subject;

(b) periodically isolating cancer-derived exosomes comprising microRNAs (mi RNAs) from the biological samples;

(c) determining an amount of one or more of the miRNAs for each sample; and

(d) determining any measurable difference in the amount of the one or more miRNAs from the cancer-derived exosomes from the series to thereby monitor progression or regression of the cancer in the subject.

27. The method of claim 26, wherein the cancer is a cancer selected from the group consisting of ovarian cancer, cervical cancer, breast cancer, colon cancer, endometrial cancer, prostate cancer, lung cancer, melanoma, and pancreatic cancer.

28. The method of claim 26, wherein the subject is human.

29. The method of claim 26, wherein the biological sample comprises milk, blood, serum, plasma, ascites, cyst fluid, pleural fluid, cerebral spinal fluid, tears, urine, saliva, sputum, or combinations thereof.

30. The method of claim 26, wherein isolating the exosomes comprises using size exclusion chromatography.

31. The method of claim 26, wherein the exosomes are selectively isolated by immunoabsorbent capture using a cancer specific antigen binding antibody.

32. The method of claim 31, wherein the cancer specific antigen is EpCAM.

33. The method of claim 32, wherein the cancer specific antigen antibody binds the epithelial-cell-adhesion-molecule (EpCAM).

34. The method of claim 26, wherein determining an amount of one or more miRNAs comprises labeling the one or more miRNAs.

35. The method of claim 26, wherein determining an amount of one or more miRNAs comprises capturing the one or more miRNAs with one or more polynucleotide probes that each selectively bind to the one or more miRNAs.

36. The method of claim 26, wherein determining the amount of the one or more miRNAs comprises using a real-time polymerase chain reaction to quantitate the amount of the one or more miRNAs.

37. The method of claim 26, wherein the one or more miRNAs are one or more of miRNAs let7a, miR-19b, miR-92, miR-93, miR-320, miR-373, miR-324-3p, miR-149, and miR-370.

38. The method of claim 26, wherein, the one or more miRNAs are miR-21 or miR-203.

67. The method of claim 26, wherein the cancer is brain cancer.

39. A method for monitoring progression or regression of a cancer in a subject, comprising:

(a) periodically isolating exosomes from a biological sample from a subject, wherein the exosomes originate from cancer cells and comprise microRNAs (miRNAs);

(b) determining the amount of one or more miRNAs specific to a cancer for each period;

wherein an increase or decrease in the amount of miRNA specific to a cancer at each period is indicative of progression or regression of cancer in the subject.

40. A method for monitoring progression or regression of a cancer in a subject, comprising:

(a) periodically isolating cancer-derived exosomes from a biological sample from a subject wherein the exosomes comprise miRNAs;

(b) analyzing the miRNAs for an increase or decrease in levels of miRNAs specific to the cancer for each period;

wherein the presence of an increase or decrease at each period is indicative of progression or regression of cancer in the subject.

41. A method for profiling a cancer in a subject, comprising:

(a) providing a biological sample from a subject;

(b) isolating cancer-derived exosomes comprising microRNAs (mi RNAs) from the biological sample;

(c) determining an amount of one or more of the miRNAs; and

(d) comparing the amount of the one or more miRNAs to one or more miRNA control levels,

wherein the cancer is profiled based on a measurable difference in the amount of the one or more miRNAs from the cancer-derived exosomes as compared to the one or more miRNA control levels.

42. The method of claim 41, wherein the cancer is a cancer selected from the group consisting of ovarian cancer, cervical cancer, breast cancer, colon cancer, endometrial cancer, prostate cancer, lung cancer, melanoma, and pancreatic cancer.

43. The method of claim 41, wherein the subject is human.

44. The method of claim 41, wherein the biological sample comprises milk, blood, serum, plasma, ascites, cyst fluid, pleural fluid, cerebral spinal fluid, tears, urine, saliva, sputum, or combinations thereof.

45. The method of claim 41, wherein isolating the cancer-derived exosomes comprises using size exclusion chromatography to isolate the cancer-derived exosomes.

46. The method of claim 41, wherein the exosomes are selectively isolated by immunoabsorbent capture using a cancer specific antigen binding antibody.

47. The method of claim 46, wherein the cancer specific antigen is epithelial-cell-adhesion-molecule (EpCAM).

48. The method of claim 47, wherein the cancer specific antigen antibody binds the epithelial-cell-adhesion-molecule (EpCAM).

49. The method of claim 41, wherein determining an amount of one or more of the miRNAs comprises labeling the one or more miRNAs.

50. The method of claim 41, wherein determining an amount of one or more miRNAs comprises capturing the one or more miRNAs with one or more polynucleotide probes that each selectively bind to the one or more miRNAs.

51. The method of claim 41, wherein determining an amount of one or more of the miRNAs comprises using a real time polymerase chain reaction to quantitate the amount of the one or more miRNAs.

52. The method of claim 41, wherein the one or more miRNAs are one or more of miRNAs let7a, miR-19b, miR-92, miR-93, miR-320, miR-373, miR-324-3p, miR-149, and miR-370.

53. The method of claim 41, wherein, the one or more miRNAs are miR-21 or miR-203.

54. The method of claim 41, further comprising selecting a treatment for the cancer based on the amount of the one or more miRNAs determined.

68. The method of claim 41, wherein the cancer is brain cancer.

55. A method for identifying a genetic basis that influences treatment effecting a cancer in a subject, comprising:

(a) providing a biological sample from a subject;

(b) isolating cancer-derived exosomes comprising microRNAs (mi RNAs) from the biological sample;

(c) determining an amount of one or more of the miRNAs; and

(d) comparing the amount of the one or more miRNAs to one or more miRNA control levels,

wherein the genetic basis that influences treatment effecting a cancer in a subject is identified based on a measurable difference in the amount of the one or more miRNAs from the cancer-derived exosomes as compared to the one or more miRNA control levels.

69. The method of claim 55, wherein the cancer is brain cancer.

56. A method for profiling a cancer in a subject, comprising:

(a) isolating exosomes originating from a cancer comprising microRNAs (miRNAs) from a biological sample from a subject;

(b) determining the amount of one or more miRNAs specific to a cancer;

wherein the cancer is profiled based on the presence of an increase or decrease in the amount of miRNA specific to the cancer.

57. A method for identifying a genetic basis that influences treatment effecting a cancer in a subject, comprising:

(a) isolating exosomes originating from cancer comprising microRNAs (miRNAs) from a biological sample from a subject;

(b) determining the amount of one or more miRNAs specific to a cancer;

wherein an increase or decrease in the amount of miRNA specific to a cancer is indicative of a genetic basis that influences treatment effecting the cancer in the subject.

58. A method for profiling a cancer in a subject, comprising:

(a) isolating cancer-derived exosomes comprising miRNAs from a biological sample from a subject

(b) analyzing the miRNAs for an increase or decrease in levels of miRNAs specific to the cancer;

wherein the cancer is profiled based on the presence of an increase or decrease in levels of miRNAs specific to the cancer.

59. A method for identifying a genetic basis that influences treatment effecting a cancer in a subject, comprising:

(a) isolating cancer-derived exosomes comprising miRNAs from a biological sample from a subject;

(b) analyzing the miRNAs for an increase or decrease in levels of miRNAs specific to the cancer;

wherein the presence of an increase or decrease is indicative of a genetic basis that influences treatment effecting the cancer in the subject.

60. A method of diagnosing prenatal disease or medical condition, comprising:

(a) providing a biological sample from a subject;

(b) isolating exosomes comprising microRNAs (mi RNAs) from the biological sample;

(c) determining an amount of one or more of the miRNAs; and

(d) comparing the amount of the one or more miRNAs to one or more miRNA control levels,

wherein the subject is diagnosed with the prenatal disease or medical condition if there is a measurable difference in the amount of the one or more miRNAs from the exosomes as compared to the one or more miRNA control levels.

61. The method of claim 60, wherein the prenatal medical condition is abnormal fetal growth.

62. The method of claim 60, wherein the subject is human.

63. The method of claim 60, wherein the biological sample comprises milk, blood, serum, plasma, ascites, cyst fluid, pleural fluid, cerebral spinal fluid, tears, urine, saliva, sputum, or combinations thereof.

64. The method of claim 60, wherein isolating the exosomes comprises using size exclusion chromatography.

65. The method of claim 60, wherein determining an amount of one or more of the miRNAs comprises using a real time polymerase chain reaction to quantitate the amount of the one or more miRNAs.