EXTRACELLULAR VESICLE RIBONUCLEIC ACID (RNA) CARGO AS A BIOMARKER OF HYPERGLYCEMIA AND TYPE 1 DIABETES
Methods and compositions are provided for detecting and/or determining a patient's risk of developing hyperglycemia or type 1 diabetes. The methods are directed to analyzing the miRNA and protein content of extracellular vesicles recovered from a patient.
1 . A method for detecting miR-21-5p associated with circulating extracellular vesicles in a patient, said method comprising
providing a patient's biological sample;
isolating extracellular vesicles from said sample; and
quantitating the miR-21-5p associated with said isolated extracellular vesicles.
2 . The method of claim 1 wherein the biological sample is selected from the group consisting of urine, blood, serum and plasma.
3 . The method of claim 1 wherein the extracellular vesicles are exosomes.
4 . The method of claim 1 wherein the diameter of said exosomes ranges in size from about 40 to about 100 nm.
5 . The method of claim 1 wherein the biological sample is a serum or plasma sample and the extracellular vesicles are exosomes isolated by a precipitation or an ultracentrifugation step.
6 . The method of claim 5 wherein miR-21-5p is quantitated through the use of quantitative real-time PCR.
7 . The method of claim 1 further comprising the step of quantitating additional miRNAs associated with said extracellular vesicles.
8 . The method of claim 7 wherein the additional miRNAs are selected from the group consisting of miR-21-3p, miR-217 and miR-193-3p.
9 . The method of claim 1 , wherein the extracellular vesicle comprises a beta cell extracellular vesicle or an islet cell extracellular vesicle.
10 . The method of claim 1 , wherein the extracellular vesicle isolation step comprises the use of an extracellular vesicle marker selected from the group consisting of CD9 and CD63.
11 . The method of claim 5 , further comprising detecting circulating miR-21-5p in said sample.
12 . The method of claim 1 , further comprising detecting blood glucose levels of said patient.
13 . A method of treating a patient susceptible to hyperglycemia, said method comprising
identifying the patient as susceptible to hyperglycemia, wherein said identification step comprises
obtaining the patient's biological sample;
isolating extracellular vesicles from said sample; and
quantitating the miR-21-5p associated with said isolated extracellular vesicles and identifying the patient with elevated levels of miR-21-5p associated with said isolated extracellular vesicles as being susceptible to hyperglycemia; and
treating said identified patient susceptible to hyperglycemia using standard anti-diabetes therapies.
14 . The method of claim 13 wherein said elevated levels of miR-21-5p associated with said isolated extracellular vesicles are determined based on a comparison of the identified patient's levels of miR-21-5p associated with said isolated extracellular vesicles to levels of miR-21-5p associated with isolated extracellular vesicles in a control patient.
15 . The method of claim 13 wherein said elevated levels of miR-21-5p associated with said isolated extracellular vesicles are determined based on a comparison of the identified patient's levels of miR-21-5p associated with said isolated extracellular vesicles in a first sample relative to levels of miR-21-5p associated with said isolated extracellular vesicles in a second sample recovered from the same patient, wherein there is a temporal spacing between the recovery of said first and second sample, and said second sample has an increase in the levels of miR-21-5p associated with said isolated extracellular vesicles.
16 . The method of claim 15 wherein the temporal spacing is selected from 1 day, 1 week, 1 month, 1 year or 2 years.
17 . A kit for detecting and quantitating the relative concentration of miR-21-5p associated with isolated extracellular vesicles in a biological sample recovered from a patient, said kit comprising
an antibody for isolating extracellular vesicles from a biological sample; and
a reagent for the detection of miRNAs.
18 . The kit of claim 17 wherein the kit comprises a reagent for detection of miR-21-5p and the reagent for detecting miRNAs comprises a PCR primer of SEQ ID NO: 1.
19 . The kit of claim 17 wherein the reagent for isolating extracellular vesicles is an antibody specific for beta-cell specific exosomes.