Analysis of genomic dna, rna, and proteins in exosomes for diagnosis and theranosis

Abstract

The present invention provides that exosomes from human body fluid samples contain double stranded genomic DNA that spans all chromosomes and may be used to determine the mutation status of genes of interest in diseases, such as cancer. Furthermore, the present invention provides the use of exosomes to produce therapeutic proteins and for their use in therapy as well as the detection of cancer cell-derived exosomes to diagnose cancer and monitor therapeutic response.

1 . A method of isolating cancer cell-derived exosomes comprising:
 (a) obtaining a body fluid sample from a cancer patient;   (b) isolating an exosomes fraction of the body fluid sample; and   (c) isolating exosomes comprising glypican 1 from the exosomes fraction, thereby isolating cancer cell-derived exosomes.   
     
     
         2 . The method of  claim 1 , further comprising isolating genomic double-stranded DNA, RNA, or proteins from the cancer cell-derived exosomes. 
     
     
         3 . The method of  claim 2 , further comprising detecting a DNA, RNA, or protein isolated from the cancer cell-derived exosomes. 
     
     
         4 . The method of  claim 1 , wherein the isolating of step (b) or (c) comprises immunomagnetic capture, adhesion-based sorting, magnetic-activated sorting, or fluorescence-activated sorting (FACS). 
     
     
         5 . The method of  claim 1 , further comprising quantifying the number of cancer cell-derived exosomes in the patient. 
     
     
         6 . The method of  claim 1 , further comprising genotyping the cancer cell-derived exosomes. 
     
     
         7 . The method of  claim 1 , wherein the body fluid sample is lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirates, eye exudate/tears, or serum. 
     
     
         8 . 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. 
     
     
         9 . A method of diagnosing cancer in a patient comprising:
 (a) obtaining a body fluid sample from a patient;   (b) isolating an exosomes fraction of the body fluid sample; and   (c) assaying for the presence of glypican 1 in the exosomes fraction, wherein if glypican 1 is present, then the patient is diagnosed as having cancer.   
     
     
         10 . The method of  claim 9 , further comprising quantifying the number of glypican 1-containing exosomes in the patient. 
     
     
         11 . The method of  claim 10 , further defined as a method of monitoring response to therapy in a cancer patient, wherein if the number of glypican 1-containing exosomes decreases over time, then the patient is said to have had a positive response to therapy. 
     
     
         12 . The method of  claim 9 , wherein the patient has not been previously diagnosed with cancer and the method is a method of early cancer detection. 
     
     
         13 . The method of  claim 9 , wherein the patient is in remission and the method is a method of detecting relapse. 
     
     
         14 . The method of  claim 9 , further comprising administering an anti-cancer therapy to the patient. 
     
     
         15 . The method of  claim 9 , wherein the body fluid sample is lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirates, eye exudate/tears, or serum. 
     
     
         16 . The method of  claim 9 , 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. 
     
     
         17 . The method of  claim 9 , further comprising reporting the diagnosis of the patient. 
     
     
         18 . The method of  claim 17 , wherein reporting comprises preparing a written or electronic report. 
     
     
         19 . The method of  claim 18 , further comprising providing the report to the patient, a doctor, a hospital or an insurance company. 
     
     
         20 . A method of isolating genomic double-stranded DNA from a subject comprising:
 (a) obtaining a sample from a patient;   (b) isolating a exosomes fraction of the sample; and   (c) isolating genomic double-stranded DNA from the exosomes fraction.   
     
     
         21 . The method of  claim 20 , wherein step (b) further comprises isolating exosomes comprising glypican 1. 
     
     
         22 . The method of  claim 20 , further comprising performing sequence analysis of the DNA. 
     
     
         23 . The method of  claim 22 , further comprising determining a mutation status of a gene. 
     
     
         24 . The method of  claim 23 , wherein the gene is KRAS or p53. 
     
     
         25 . The method of  claim 23 , wherein the mutation status of the gene is a cancer biomarker. 
     
     
         26 . The method of  claim 25 , wherein the cancer biomarker is used to diagnose the patient as having cancer. 
     
     
         27 . The method of  claim 23 , further comprising reporting the mutation status of the gene. 
     
     
         28 . The method of  claim 27 , wherein reporting comprises preparing a written or electronic report. 
     
     
         29 . The method of  claim 28 , further comprising providing the report to the patient, a doctor, a hospital or an insurance company. 
     
     
         30 . The method of  claim 20 , wherein the sample is lymph, saliva, urine, or serum. 
     
     
         31 . The method of  claim 20 , wherein the sample is essentially free of cells. 
     
     
         32 . The method of  claim 20 , wherein the subject has cancer. 
     
     
         33 . The method of  claim 32 , 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. 
     
     
         34 . The method of  claim 32 , wherein the cancer is pancreatic ductal adenocarcinoma. 
     
     
         35 . The method of  claim 20 , wherein the subject has previously been treated for a cancer. 
     
     
         36 . The method of  claim 20 , wherein the subject has previously had a tumor surgically removed. 
     
     
         37 . A method of identifying a cancer biomarker in a subject comprising:
 (a) isolating genomic DNA in accordance with  claim 20 ;   (b) performing sequence analysis of the genomic DNA;   (c) determining the mutation status of at least one gene, thereby identifying a cancer biomarker.   
     
     
         38 . The method of  claim 37 , wherein step (c) comprises determining the mutation status of at least two genes. 
     
     
         39 . The method of  claim 37 , wherein the presence of the cancer biomarker is used to diagnose the patient has having cancer. 
     
     
         40 . The method of  claim 39 , 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. 
     
     
         41 . The method of  claim 39 , wherein the cancer is pancreatic ductal adenocarcinoma. 
     
     
         42 . The method of  claim 37 , wherein the subject has previously been treated for a cancer. 
     
     
         43 . The method of  claim 37 , wherein the subject has previously had a tumor surgically removed. 
     
     
         44 . The method of  claim 37 , further comprising reporting the mutation status of the gene. 
     
     
         45 . The method of  claim 44 , wherein reporting comprises preparing a written or electronic report. 
     
     
         46 . The method of  claim 45 , further comprising providing the report to the patient, a doctor, a hospital or an insurance company. 
     
     
         47 . A method of treating a cancer in a subject comprising, identifying a subject as having a cancer biomarker in accordance with  claim 37  and administering an anti-cancer therapy to the subject. 
     
     
         48 . The method of  claim 47 , wherein the anti-cancer therapy is a chemotherapy, a radiation therapy, a hormonal therapy, a targeted therapy, an immunotherapy or a surgical therapy. 
     
     
         49 . The method of  claim 47 , wherein the subject is a human. 
     
     
         50 . A method of treating a disease in a patient in need thereof comprising:
 (a) obtaining exosomes from a sample;   (b) transfecting the exosomes with a nucleic acid encoding a therapeutic protein; and   (c) providing the transfected exosomes to a patient, thereby treating the disease in the patient.   
     
     
         51 . The method of  claim 50 , wherein the disease is a cancer. 
     
     
         52 . A method of administering a therapeutic protein to a patient in need thereof comprising:
 (a) obtaining exosomes from a sample;   (b) transfecting the exosomes with a nucleic acid encoding a therapeutic protein;   (c) incubating the exosomes under conditions to allow for expression of the therapeutic protein within the exosomes; and   (d) providing the incubated exosomes to a patient, thereby administering a therapeutic protein to the patient.   
     
     
         53 . The method of  claim 52 , wherein the sample is obtained from the patient. 
     
     
         54 . A method of treating a disease in a subject comprising, identifying a subject as having a cancer biomarker in accordance with  claim 37  and administering a therapeutic protein to the subject in accordance with  claim 52 . 
     
     
         55 . The method of  claim 54 , wherein the cancer biomarker is a p53 mutation and the therapeutic protein is wild-type p53. 
     
     
         56 . A method of producing a recombinant protein comprising:
 (a) obtaining exosomes from a sample;   (b) transfecting the exosomes with a nucleic acid encoding a recombinant protein; and   (c) incubating the exosomes under conditions to allow for expression of the recombinant protein, thereby producing the recombinant protein.   
     
     
         57 . The method of  claim 56 , further comprising purifying the recombinant protein. 
     
     
         58 . The method of  claim 57 , further comprising administering the protein to a patient in need thereof. 
     
     
         59 . The method of  claim 56 , further comprising administering the incubated exosomes to a patient in need thereof. 
     
     
         60 . The method of  claim 59 , wherein the incubated exosomes are autologous to the patient. 
     
     
         61 . The method of  claim 58  or  59 , wherein the patient has been diagnosed with cancer. 
     
     
         62 . The method according to any one of  claim 50 ,  52 , or  57 , wherein the sample is a tissue culture media sample. 
     
     
         63 . The method according to any one of  claim 50 ,  52 , or  57 , wherein the sample is a body fluid sample. 
     
     
         64 . The method of  claim 63 , wherein the body fluid sample is lymph, saliva, urine, cerebrospinal fluid, bone marrow aspirates, eye exudate/tears, or serum. 
     
     
         65 . The method of  claim 63 , wherein the body fluid sample is obtained from the patient. 
     
     
         66 . The method according to any one of  claim 50 ,  52 , or  57 , wherein the nucleic acid is an mRNA. 
     
     
         67 . The method according to any one of  claim 50 ,  52 , or  57 , wherein the nucleic acid is a plasmid. 
     
     
         68 . A purified recombinant protein produced according to the method of  claim 56 . 
     
     
         69 . A composition comprising exosomes transfected with a nucleic acid encoding a therapeutic protein for use in the treatment of a disease in a patient. 
     
     
         70 . The composition of  claim 69 , wherein the disease is a cancer. 
     
     
         71 . The composition of  claim 69 , wherein the exosomes are autologous to the patient. 
     
     
         72 . The composition of  claim 69 , wherein the exosomes have been incubated under conditions to allow for expression of the therapeutic protein within the exosomes. 
     
     
         73 . The composition of  claim 69 , wherein the patient has been identified as having a cancer biomarker in accordance with  claim 37 . 
     
     
         74 . The composition of  claim 73 , wherein the cancer biomarker is a p53 mutation and the therapeutic protein is wild-type p53. 
     
     
         75 . Use of exosomes transfected with a nucleic acid encoding a therapeutic protein in the manufacture of a medicament for the treatment of a disease. 
     
     
         76 . The use of  claim 75 , wherein the disease is a cancer.