miRNA BIOGENESIS IN EXOSOMES FOR DIAGNOSIS AND THERAPY

Abstract

Methods for diagnosis and treatment of cancers by use of exosomes comprising miRNAs and precursors thereof. For example, in some aspects, a cancer may be diagnosed or evaluated by determining the miRNA content of exosomes in a sample from a subject or by detecting miRNA processing in exosomes.

1 . An in vitro method of detecting cancer biomarker in a subject comprising:
 (a) obtaining a biological sample from the subject;   (b) measuring the level of:
 (i) a RISC protein in an exosome fraction of the sample; 
 (ii) a precursor miRNA; 
 (iii) one or more miRNA(s) selected from the miRNAs provided in Table 5 in an exosome fraction of the sample; and/or 
 (iv) a primary miRNA or precursor miRNA processing activity in an exosome fraction of the sample; and 
   (c) identifying the subject having or not having a cancer biomarker based on the measured level of said miRNA(s), precursor miRNA, RISC protein or miRNA processing activity.   
     
     
         2 . The method of  claim 1 , wherein the sample is essentially free of cells. 
     
     
         3 . The method of  claim 1 , wherein the sample is a lymph, saliva, urine or plasma sample. 
     
     
         4 . The method of  claim 1 , further comprising purifying an exosome fraction of the sample or increasing production of an exosome fraction of the sample. 
     
     
         5 . The method of  claim 1 , wherein the cancer is a breast cancer, lung cancer, head & neck cancer, prostate cancer, esophageal cancer, tracheal cancer, brain cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer or skin cancer. 
     
     
         6 . The method of  claim 5 , wherein the cancer is a breast cancer. 
     
     
         7 . The method of  claim 1 , further comprising measuring the level of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 of said miRNAs. 
     
     
         8 . The method of  claim 1 , further comprising measuring the level of DICER, AGO2, or TRBP. 
     
     
         9 . The method of  claim 1 , wherein measuring the level of a precursor miRNA comprises measuring the level of a precursor of one of the miRNA(s) of Table 5. 
     
     
         10 . The method of  claim 1 , wherein the subject has previously been treated for a cancer. 
     
     
         11 . The method of  claim 10 , wherein the subject has previously had a tumor surgically removed. 
     
     
         12 . The method of  claim 1 , wherein identifying the subject as having or not having a cancer biomarker further comprises correlating the measured miRNA level(s), precursor miRNA level; RISC level or miRNA processing activity with a risk for cancer. 
     
     
         13 . The method of  claim 1 , wherein identifying the subject as having or not having a cancer biomarker further comprises analysis of the measured miRNA level(s), precursor miRNA level; RISC level or miRNA processing activity using an algorithm. 
     
     
         14 . The method of  claim 13 , wherein said analysis is performed by a computer. 
     
     
         15 . The method of  claim 1 , further comprising:
 b) measuring the level of:
 (i) a RISC protein in an exosome fraction of the sample and a reference sample; 
 (ii) a precursor miRNA in an exosome fraction of the sample and a reference sample; 
 (iii) one or more miRNA(s) selected from the miRNAs provided in Table 5 in an exosome fraction of the sample and a reference sample; and/or 
 (iv) a miRNA processing activity in an exosome fraction of the sample and a reference sample; and 
   (c) identifying the subject as having or not having a cancer biomarker by comparing the level of RISC, precursor miRNA, miRNA(s), or miRNA processing activity in the sample from the subject to the level of miRNA(s), precursor miRNA, RISC or miRNA processing activity in the reference sample.   
     
     
         16 . The method of  claim 1 , wherein measuring RISC protein levels comprises performing a Western blot, an ELISA or binding to an antibody array. 
     
     
         17 . The method of  claim 1 , wherein measuring miRNA levels comprises measuring processed miRNA levels. 
     
     
         18 . The method of  claim 1 , wherein measuring miRNA levels comprises performing RT-PCR, Northern blot or an array hybridization. 
     
     
         19 . The method of  claim 1 , further comprising reporting whether the subject has or does not have a cancer biomarker. 
     
     
         20 . The method of  claim 19 , wherein reporting comprises preparing a written or electronic report. 
     
     
         21 . The method of  claim 19 , further comprising providing the report to the patient, a doctor, a hospital or an insurance company. 
     
     
         22 . A method of treating a subject comprising:
 selecting a subject identified as having a cancer biomarker in accordance with  claim 1 ; and   administering an anti-cancer therapy the subject.   
     
     
         23 . The method of  claim 22 , wherein the anti-cancer therapy is a chemotherapy, a radiation therapy, a hormonal therapy, a targeted therapy, an immunotherapy or a surgical therapy. 
     
     
         24 . The method of  claim 22 , wherein the anti-cancer therapy is targeted to the brain. 
     
     
         25 . A method of treating a subject comprising:
 (a) obtaining the level of (i) one or more miRNA(s) selected from the miRNAs provided in Table 5; (ii) a precursor miRNA level; (iii) a RISC protein; or (iv) a miRNA processing activity, in an exosome fraction of a sample from the subject;   (b) selecting a subject having a cancer biomarker based on the level of said mRNA(s), precursor miRNA; RISC protein or miRNA processing activity; and   (c) treating the selected subject with an anti-cancer therapy.   
     
     
         26 . The method of  claim 25 , wherein the anti-cancer therapy is a chemotherapy, a radiation therapy, a hormonal therapy, a targeted therapy, an immunotherapy or a surgical therapy. 
     
     
         27 . A method of selecting a subject for a diagnostic procedure comprising:
 (a) obtaining the level of (i) one or more miRNA(s) selected from the miRNAs provided in Table 5; (ii) a precursor miRNA; (iii) a RISC protein; or (iv) a miRNA processing activity, in an exosome fraction of a sample from the subject;   (b) selecting a subject having a cancer biomarker based on the level of said mRNA(s), precursor miRNA, RISC protein or miRNA processing activity; and   (c) performing a diagnostic procedure on the selected on the subject.   
     
     
         28 . The method of  claim 27 , wherein the diagnostic procedure comprises diagnostic imaging. 
     
     
         29 . The method of  claim 28 , wherein the imaging is a X-ray, CT, MRI or PET imaging. 
     
     
         30 . A tangible computer-readable medium comprising computer-readable code that, when executed by a computer, causes the computer to perform operations comprising:
 a) receiving information corresponding to a level of (i) one or more miRNA(s) selected from the miRNAs provided in Table 5; (ii) a precursor miRNA; (iii) a RISC protein; or (iv) a miRNA processing activity, in an exosome fraction of a sample from the subject; and   b) determining a relative level of one ore more of said miRNAs or RISC proteins compared to a reference level, wherein altered level compared to a reference level indicates that the subject has a cancer biomarker.   
     
     
         31 . The tangible computer-readable medium of  claim 30 , further comprising receiving information corresponding to a reference level of (i) one or more miRNA(s) selected from the miRNAs provided in Table 5; (ii) a precursor miRNA (iii) a RISC protein; or (iv) a miRNA processing activity, in an exosome fraction of a subject no having a cancer. 
     
     
         32 . The tangible computer-readable medium of  claim 30 , wherein the reference level is stored in said tangible computer-readable medium. 
     
     
         33 . The tangible computer-readable medium of  claim 30 , wherein the receiving information comprises receiving from a tangible data storage device information corresponding to a level of miRNA; a precursor miRNA; RISC protein or miRNA processing activity, in a sample from a subject. 
     
     
         34 . The tangible computer-readable medium of  claim 30 , further comprising computer-readable code that, when executed by a computer, causes the computer to perform one or more additional operations comprising: sending information corresponding to the relative level of miRNA; a precursor miRNA; RISC protein or miRNA processing activity, to a tangible data storage device. 
     
     
         35 . The tangible computer-readable medium of  claim 30 , wherein the receiving information further comprises receiving information corresponding to a level of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 of said miRNAs in a sample from a subject. 
     
     
         36 . The tangible computer-readable medium of  claim 30 , wherein the computer-readable code, when executed by a computer, causes the computer to perform operations further comprising: c) calculating a diagnostic score for the sample, wherein the diagnostic score is indicative of the probability that the sample is from a subject having a cancer. 
     
     
         37 . An in vitro method of detecting cancer biomarker in a subject comprising:
 (a) obtaining a biological sample from the subject;   (b) measuring the level of one or more miRNA(s) in the sample selected from the miRNAs provided in Table 5; and   (c) identifying the subject having or not having a cancer biomarker based on the measured level of said miRNA(s).   
     
     
         38 . The method of  claim 37 , wherein the sample is essentially free of cells. 
     
     
         39 . The method of  claim 37 , wherein the sample is an exosome fraction of a body fluid. 
     
     
         40 . The method of  claim 37 , wherein the sample is a lymph, saliva, urine or plasma sample. 
     
     
         41 . An in vitro method for delivery of active inhibitory RNA comprising contacting a cell with an inhibitory RNA that is provided in association with a RISC protein complex. 
     
     
         42 . The method of  claim 41 , wherein the RISC protein complex comprises TRBP, DICER and AGO2. 
     
     
         43 . The method of  claim 41 , wherein the inhibitory RNA is a siRNA or shRNA. 
     
     
         44 . The method of  claim 41 , wherein the inhibitory RNA is a human miRNA. 
     
     
         45 . The method of  claim 41 , wherein the inhibitory RNA and RISC protein complex are comprises in a liposome, a nanoparticle or a microcapsule comprising a lipid bilayer. 
     
     
         46 . The method of  claim 46 , wherein the microcapsule is an exosome. 
     
     
         47 . The method of  claim 41 , wherein contacting a cell comprises transfecting a cell with the inhibitory RNA and RISC protein complex. 
     
     
         48 . The method of  claim 41 , further comprising administering the inhibitory RNA and RISC protein complex to a subject. 
     
     
         49 . A composition comprising a recombinant or synthetic inhibitory RNA in association with a RISC protein complex, said complex comprised in a liposome, a nanoparticle or a microcapsule. 
     
     
         50 . The composition of  claim 49 , wherein the RISC protein complex comprises TRBP, DICER and AGO2. 
     
     
         51 . The composition of  claim 49 , wherein the inhibitory RNA is a siRNA or shRNA. 
     
     
         52 . The composition of  claim 49 , wherein the inhibitory RNA is a human miRNA. 
     
     
         53 . The composition of  claim 49 , wherein the complex is comprised in a synthetic liposome, a nanoparticle or a microcapsule. 
     
     
         54 . The composition of  claim 49 , wherein the microcapsule is an exosome.