Biomarkers of traumatic brain injury

Abstract

To provide a method of diagnosing and / or monitoring traumatic brain injury (TBI) in a subject.SOLUTION: A method comprises determining a level of at least one miRNA in a fluid sample from the subject. The miRNA may be selected from miR-425-5p, miR-502, miR-21 and miR-335. The method may involve determining whether a subject is suffering from mild-TBI or moderate to severe TBI. Also, provided are a sensor element, a detection system and a kit for diagnosing and / or monitoring TBI, and a method of determining an appropriate treatment for a subject with suspected TBI.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to compositions, kits, systems and methods for diagnosing and / or monitoring traumatic brain injury (TBI), more particularly, the present invention relates to the diagnosis and monitoring of TBI using miRNA biomarkers. [Background technology]

[0002] Traumatic brain injury (TBI) is the leading cause of death and disability in Western countries in people under 45. Its medical and societal costs are expected to continue to rise, with the World Health Organization estimating that by 2020, TBI will be the third leading cause of disability worldwide.

[0003] Despite numerous studies, no reliable biomarkers have been found to assess the severity of TBI and predict recovery. This is especially true for mild TBI (mTBI), which is currently difficult to assess in clinical practice. TBI patients are initially assessed by Glasgow Coma Score (GCS) and neuroimaging techniques, which require expensive equipment and current diagnostic tools lack the ability to precisely define and quantify the actual severity of brain injury, meaning that severe TBI is easily detected, but mTBI, which accounts for the majority of cases (75-90%), is not.

[0004] Accurate diagnosis of mTBI is especially important in patients such as athletes, military personnel, and children who are at high risk for repeated mTBI and a dramatic form of brain injury known as Second Impact Syndrome (SIS), in which the synergistic effects of repeated TBI can cause severe damage and even death. Thus, early diagnosis of TBI and assessment of its severity becomes critical for the patient's well-being and ultimately to save their life.

[0005] The need for TBI biomarkers has received a significant impetus from the increasing attention given to sports concussion in the mass media. In recent years, numerous studies have focused on biomarkers that can support clinical decision-making at the sidelines or in sports clinics. However, protein biomarkers reported in the literature lack specificity or sensitivity, or are not detectable for some time after injury. This may be due to the fact that after concussion, a form of TBI, only very small amounts of brain-derived compounds are released and the blood-brain barrier remains mostly closed.

[0006] MicroRNAs (miRNAs) are a large class of highly conserved non-coding RNA molecules, approximately 22 nucleotides long, that trigger mRNA degradation, translational repression, or both through pairing with partially complementary sites in the 3'UTR of target genes. The human genome encodes over 2000 miRNAs, which may target approximately 60% of all genes. However, despite the abundance of miRNAs, their biomolecular functions and involvement in pathogenesis are not fully clear. miRNAs play a central role in numerous biological processes, including cell cycle, cellular metabolism, apoptosis, and immune response, and have attracted increasing attention in clinical research as potential biomarkers for the detection, identification, and classification of cancer and other disease states, including neurodegenerative diseases. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been devised with these considerations in mind.

[0008] The present invention provides methods for diagnosing or monitoring traumatic brain injury (TBI) in a subject. [Means for solving the problem]

[0009] According to a first aspect of the present invention, there is provided a method for diagnosing and / or monitoring traumatic brain injury (TBI) in a subject, comprising detecting the presence and / or determining the level of at least one miRNA in a sample from the subject.

[0010] At least one miRNA (also referred to herein as "miR") is selected from the group consisting of miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * , miR-671-3p, hsa-let-7c-5p, hsa-let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b -5p, miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, miR-424-5p, miR-30a-5p;miR-107;miR-135b-5p;miR-199b-5p;mi The miRNAs may be selected from the group consisting of miR-324-5p; miR-652-3p; miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p and miR-629. These miRNAs may be referred to herein as the miRNA of interest or target miRNA.

[0011] In some embodiments, the at least one miRNA is selected from the group consisting of miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, hsa-miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * These microRNAs have been found to be biomarkers expressed in all TBI patients (mild or severe).

[0012] In some embodiments, the at least one miRNA is selected from the group consisting of miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, and miR-335.

[0013] In some embodiments, the at least one miRNA is selected from the group consisting of let-7c-5p, let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, and miR-424-5p; miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p.

[0014] In some embodiments, the at least one miRNA is selected from the group consisting of miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-194, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p, and miR-629.

[0015] For the avoidance of doubt, "at least one miRNA is selected from a group of miRNAs," as used herein, will be understood to mean that the method in question, whether performed for diagnostic, prognostic, or therapeutic purposes, may be performed with any one of the listed miRNAs or with any multiple of the listed miRNAs (e.g., two, three, four, or more of the listed miRNAs). As a result, any one or more of the listed miRNAs may be expressly excluded. For example, if the at least one miRNA is selected from the group consisting of miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, and miR-335, the method may include detecting and / or assessing the levels of any combination of miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, and miR-let-7g to the exclusion of miR-335.

[0016] According to one aspect of the present invention, there is provided a method of diagnosing and / or monitoring traumatic brain injury (TBI) in a subject, comprising determining a level of at least one miRNA in a sample from the subject, wherein the miRNA is selected from the group consisting of miR-425-5p; miR-502; miR-21; and miR-335. [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 shows miR-425-5p and miR-502 expression in three different classifications of trauma and HV. [Diagram 2] FIG. 1 shows miR-21 and miR-335 expression in three different classifications of trauma and HV. [Figure 3A] FIG. 1 shows the time course of miR-425-5p expression in three different classes of trauma and HV. [Figure 3B] FIG. 1 shows the time course of miR-502 expression in three different classes of trauma and HV. [Figure 4A] FIG. 1 shows the time course of miR-21 expression in three different classes of trauma and HV. [Figure 4B] FIG. 1 shows the time course of miR-335 expression in three different classes of trauma and HV.

[0018] Traumatic brain injury occurs when an external force traumatically injures the brain. There are various systems for classifying TBI, for example, based on severity, type of injury, and prognosis. The most commonly used system for classifying TBI is the Glasgow Coma Scale (GCS), which grades a person's level of consciousness on a scale of 3 to 15 based on verbal, motor, and eye-opening responses to stimuli. Generally, TBI with a GCS score of 13 or higher is defined as mild, 9 to 12 as moderate, and 8 or less as severe. Another system, the Mayo Classification System, has three main classifications, including definitive moderate-severe TBI, probable mild TBI, and probable TBI. Each diagnosis uses multiple criteria, including signs of loss of consciousness, post-traumatic amnesia, skull fracture, and neuroradiological abnormalities, including subdural hematoma, cerebral contusion, and hemorrhagic contusion. Classification of TBI using the GCS or Mayo systems will be known to those of skill in the art.

[0019] As used herein, references to "mild," "moderate," and "severe" TBI are made according to the GCS. References herein to "moderate-severe" TBI include both moderate and severe TBI according to the GCS.

[0020] Diagnosis and / or monitoring of TBI using the biomarkers of the present invention is expected to aid in clinical decision making and treatment regimens in a variety of contexts, including as part of the initial paramedical evaluation to determine whether the patient should be transported to a facility with neurosurgical expertise, a major trauma center or a local trauma unit; in hospital emergency departments to determine appropriate treatment, including the need for a CT brain scan; at the sidelines of stadiums to aid in the decision to remove an athlete from play and the assessment of the need to transport the athlete to a hospital; in sports outpatient clinics to confirm a concussive event and enable decision making regarding return to play; and in combat situations to determine the need to dispatch rescue teams and evacuate the victim. Thus, subjects to whom the present invention provides particular benefit include accident victims, athletes and military personnel.

[0021] In either case, particularly where the subject is perhaps at relatively high risk for TBI (e.g., if the subject is a professional athlete or enlisted in the military), a sample can be obtained from the subject prior to any known or recent trauma (e.g., near the beginning of a sports career or prior to military deployment), and any miRNA of interest can be assessed at or after the subject may have suffered a TBI. Such a sample can thereby provide an internal reference standard.

[0022] In some embodiments, the subject is a human.

[0023] The TBI can be mild TBI (mTBI), moderate TBI or severe TBI (sTBI). In some embodiments, the TBI is moderate-severe TBI (m-sTBI).

[0024] The level of the miRNA or each miRNA in the sample can be determined quantitatively or semi-quantitatively. By "quantitative" it will be understood that the absolute amount or concentration of the miRNA or each miRNA in the sample is determined. The absolute amount of the miRNA or each miRNA in the sample can then be compared to a predefined threshold (e.g., published literature value for expected normal level), to the known level of the same or reference miRNA in a control sample taken from a healthy subject, or to the amount of the reference miRNA in a sample taken from the subject. In some embodiments, the subject is diagnosed as having TBI if the level of the miRNA is below a predefined threshold or is reduced compared to a reference or control sample. In other embodiments, the subject is diagnosed as having TBI if the level of the miRNA is increased compared to a predefined threshold.

[0025] By "semi-quantitative" it will be understood that the level of the or each miRNA of interest is measured relative to a reference.

[0026] The reference can be an invariant miRNA, i.e., a miRNA whose expression level remains substantially unchanged between healthy subjects and subjects with TBI. A subject can be diagnosed as suffering from TBI if the level of the or each miRNA of interest is increased or decreased compared to that of the invariant miRNA. Suitable invariant miRNAs include miR-331, miR-223, miR-411, miR-421, miR-431, miR-441, miR-451, miR-461, miR-471, miR-481, miR-491, miR-501, miR-511, miR-521, miR-531, miR-541, miR-551, miR-601, miR-611, miR-621, miR-701, miR-801, miR-811, miR-901, miR-1001, miR-110, miR-120, miR-130, miR-140, miR-150, miR-160, miR-170, miR-180, miR-190, miR-200, miR-210, miR-220, miR-230, miR-240, miR-250, miR-260, miR-270, miR-280, miR-290, miR-300, miR-310, miR-320, miR-330, miR-340, miR-350, miR-360, miR-370, miR-380, miR-410, miR-420, miR-430, miR-440, miR-450, miR-510, miR-520, miR-530, miR-54 * , miR-23a-3p and miR148b-3p. miR-23a-3p and miR148b-3p are unchanged only in saliva.

[0027] In some embodiments, the level of the or each miRNA in a sample obtained from a subject can be about 0.01-fold to about 100-fold, about 0.05-fold to about 50-fold, about 0.1-fold to about 10-fold, about 0.5-fold to about 5-fold, about 1.0-fold to about 3-fold, or about 1.5-fold to about 2.0-fold lower or higher than the level in a control sample, a reference level, or a published value.

[0028] When a device or method is used to generate a value, the inventors may qualify the value with the term "about" to capture the stated value and any amount of variation in that value inherent to the device or method used. When a value or range of values ​​is specifically disclosed, "about" can mean ±10% of the stated value or range. For example, about 10 minutes can mean 9 to 11 minutes.

[0029] The level of the or each miRNA of interest can be determined using methods known to those skilled in the art. In some embodiments, determining the level of the or each miRNA of interest comprises amplifying the miRNA. In some embodiments, total miRNA can be first isolated from the sample using standard techniques, for example, using miRNeasy mini kit (Qiagen). The amount of the miRNA of interest can then be determined. In some embodiments, the level of the or each miRNA of interest in the sample is determined using PCR (polymerase chain reaction). For example, quantitative PCR can be used to quantitatively determine the level of the or each miRNA of interest. PCR can also be used to semi-quantitatively determine the level of the or each miRNA of interest in the sample by comparing the level of the or each miRNA of interest in the sample with that of a reference (e.g., an invariant miRNA).

[0030] Suitable techniques for miRNA detection and / or quantification include qPCR, miRNA assays, next generation sequencing (NGS), and multi-miRNA profiling assays, as will be known to those of skill in the art.

[0031] In some embodiments, the level of the or each miRNA of interest is determined using in situ hybridization, for example, using a probe specific for the miRNA (eg, a labeled probe).

[0032] The levels of miRNA can be determined in samples obtained from a subject immediately after injury (i.e., less than an hour after injury) and / or at one or more time points hours or days after injury. That is, changes in miRNA levels can be detected over time to allow monitoring of TBI. In cases where miRNA levels change over time, the methods for monitoring TBI described herein can be extended to include maintaining or adjusting the subject's treatment regimen accordingly.

[0033] Depending on the specific miRNA and type of TBI, the level of miRNA in a subject may change significantly over time.In some embodiments, therefore, it may be advantageous to measure miRNA relatively soon after injury to allow accurate diagnosis.In some embodiments, the level of miRNA is determined in a sample obtained from a subject 72 hours or less, 48 ​​hours or less, 36 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, 4 hours or less, 2 hours or less, or 1 hour or less after injury.

[0034] The levels of some miRNAs are substantially stable over time, allowing diagnosis to be made hours, days, or even weeks after the injury. In some embodiments, the levels of the miRNAs are determined in samples obtained from the subject up to 20, 18, 15, 12, 10, 8, 5, or 2 days after the injury.

[0035] In some embodiments, the level of the miRNA is determined in a sample obtained from the subject immediately after injury (e.g., T=0 h), 4-12 hours after injury, 48-72 hours after injury, or 15 days after injury.

[0036] In some embodiments, the TBI is mild TBI (mTBI) or moderate-severe TBI (m-sTBI) and the at least one miRNA is selected from the group consisting of miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, hsa-miR-126, * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * , and miR-671-3p.

[0037] In some embodiments, the TBI is mild TBI (mTBI) and the miRNA is selected from the group consisting of miR-425-5p and miR-502. A subject can be diagnosed as having mTBI when the level of miR-425-5p and / or miR-502 is determined to be below a predetermined threshold or is decreased compared to a reference.

[0038] In some embodiments, levels of miR-425-5p and / or miR-502 that are below a predetermined threshold or that are reduced compared to a reference are diagnostic of mTBI when the levels are determined in a sample obtained less than 48 hours after injury.

[0039] In some embodiments, the TBI is moderate-severe TBI (m-sTBI) and the miRNA is selected from the group consisting of miR-21 and miR-335. If the levels of miR-21 and / or miR-335 are determined to be above a predetermined threshold or are increased compared to a reference, the subject can be diagnosed as having moderate-severe TBI.

[0040] In some embodiments, levels of miR-21 and / or miR-335 above a predetermined threshold or that are increased compared to a reference are diagnostic of moderate to severe TBI when levels are measured in samples obtained up to 15 days after injury.

[0041] In some embodiments, the TBI is moderate-severe TBI (m-sTBI) and the at least one miRNA is selected from the group consisting of miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-194, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p, and miR-629.

[0042] A subject can be diagnosed as having m-sTBI if the level of miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-618, miR-95, miR-130a, miR-152, miR-194, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642 and / or miR-99a is determined to be above a predetermined threshold or is increased compared to a reference.

[0043] If the levels of miR-192, miR-429, miR-520D-3p and / or miR-629 are determined to be below a predetermined threshold or are decreased compared to a reference, the subject can be diagnosed as having m-sTBI.

[0044] miRNAs can be used individually to diagnose TBI: for example, in sports concussion, miR-502 or miR-425-5p could be used to confirm that traumatic brain injury has occurred.

[0045] That is, a further aspect of the present invention is a method for determining the severity of TBI, and the steps of the method can be repeated to monitor the subject over time.It will be understood that a positive result for a single miRNA (e.g., the level of a single miRNA is determined to be above / below a predefined threshold or increased / decreased compared to a reference) is sufficient to determine the severity of TBI.For example, if the level of miR-425-5p is below a predefined threshold or decreased compared to a reference, the severity of TBI is determined to be mild (mTBI).However, it may be convenient to combine different miRNAs (e.g., in a test panel) to facilitate the assessment of TBI severity.

[0046] In some embodiments, the method includes determining the levels of multiple (e.g., two or more) miRNAs in the sample. In some embodiments, the two or more miRNAs are selected from the group consisting of miR-425-5p; miR-502; miR-21; and miR-335.

[0047] In some embodiments, the method comprises the steps of: (i) a first miRNA selected from miR-425-5p and miR-502; and (ii) a second miRNA selected from miR-21 and miR-335; The step of determining the level of

[0048] If the level of miR-425-5p or miR-502 is determined to be below a predetermined threshold or is decreased compared to a reference, or the level of miR-21 or miR-335 is determined to be above a predetermined threshold or is increased compared to a reference, the subject can be diagnosed as having TBI.

[0049] In some embodiments, the TBI is mild TBI (mTBI) and the at least one miRNA is selected from the group consisting of let-7c-5p, let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miRmmiR-29a-3p, miR-29c-3p, miR-424-5p, miR-30a-5p, miR-107, miR-135b-5p, miR-199b-5p, miR-324-5p, and miR-652-3p, or a combination thereof. A subject can be diagnosed with mTBI if the fold change in the level of the microRNA compared to the reference is at least 0.5, at least 1.0, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, or at least 4.0. In some embodiments, a subject is diagnosed with mTBI if the level of the microRNA is increased compared to the reference.

[0050] The sequences and accession numbers for the miRNAs described herein are provided in Table 1: [Table 1] JPEG0007654769000002.jpg224153JPEG0007654769000003.jpg224151JPEG0007654769000004.jpg125153

[0051] Conveniently, the sample can be any suitable body fluid or tissue sample obtained from a subject. For example, the biological sample can comprise at least one of the group consisting of the following samples: urine, saliva, whole blood, plasma, serum, sputum, semen, faeces, nasal swab, tear, vaginal swab, rectal swab, cervical smear, tissue biopsy, and urethral swab. In some embodiments, the sample is a body fluid sample. Suitably, the sample is one that can be easily obtained from an individual, such as urine, saliva, blood and sputum. In some embodiments, the sample comprises saliva, blood, plasma or serum. It will be understood that in some embodiments, the process of obtaining the sample does not form part of the invention described herein.

[0052] In some embodiments, the sample comprises or is composed of serum.Serum not only has practical advantages, but also does not contain anticoagulant components such as heparin, which is a potential inhibitor of PCR reaction.Serum may also be less susceptible to hemolysis compared to plasma.

[0053] In some embodiments, the sample is saliva, which can be easily obtained from a patient (e.g., at the sidelines of a stadium or in the field) without specialist training or medical equipment.

[0054] miRNAs found in saliva to be indicative of mTBI include: hsa-let-7ca-5p, hsa-let-7i-5p, hsa-miR-1421-3p, hsa-miR-148a-3p, hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miR-181a-5p, hsa-miR-20a-5p, hsa-miR-20b-5p, hsa-miR- 221-3p, hsa-miR-24-3p, hsa-miR-27b-3p, hsa-miR-29a-3p, hsa-miR-29c-3p, hsa-miR-340-5p, hsa-miR-424-5p; miR-30a-5p; miR-107; miR-135b-5p; miR-199b, -5p; miR-324-5p; and miR-652-3p.

[0055] The levels of miRNA can be used to track a subject's recovery from injury, thus the invention encompasses monitoring a subject's recovery from TBI as an alternative to, or in addition to, early diagnosis.

[0056] In some embodiments, the method includes monitoring TBI, and the level of at least one miRNA is determined in a sample obtained from the subject at least 2 days, at least 3 days, at least 5 days, at least 7 days, at least 10 days, or at least 14 days after injury. In some embodiments, the level of at least one miRNA is determined in a sample obtained from the subject 15 days after injury. In some embodiments, the level of at least one miRNA is determined in at least two samples obtained at different time intervals after injury, thus allowing recovery to be monitored. For example, the miRNA level could be determined 7 days and 14 days after injury, or 5 days, 10 days, and 15 days after injury. Return of the miRNA level to normal may be indicative of the subject's recovery from TBI.

[0057] In some embodiments, a subject is determined to have recovered from mTBI when the levels of miR-425-5p and / or miR-502 are no longer below a predetermined threshold or no longer decreased compared to a reference.

[0058] In some embodiments, a subject is determined to have recovered from moderate to severe TBI when levels of miR-21 and / or miR-335 no longer exceed a predetermined threshold or are no longer increased compared to a reference.

[0059] The diagnosis of a subject suffering from TBI, particularly a diagnosis of mild TBI or moderate to severe TBI, may facilitate the determination of appropriate treatment. That is, the present invention provides a test that allows medical personnel such as surgeons, clinicians, paramedicals, and even non-medical personnel (e.g., teachers, sports coaches, military personnel) to determine the appropriate action for a subject suspected of having TBI. A subject determined to have TBI can thus receive the most appropriate treatment as a result of the diagnosis made. That is, the method of the present invention can further include a step of subjecting a subject diagnosed with TBI to an appropriate therapy.

[0060] Subjects diagnosed with TBI can be further evaluated, for example, by CT scan. In some embodiments, the subject is admitted to a hospital. In some embodiments, if moderate to severe TBI can be excluded, the subject may not need to be admitted to a hospital for evaluation. Subjects diagnosed with moderate to severe TBI can be admitted to a hospital or specialized center with neurotrauma expertise.

[0061] A subject diagnosed with a TBI (especially an mTBI) outside of a hospital setting, for example during an athletic event, combat or competition, can be promptly removed from competition or combat, followed by a gradual return to competition or combat.

[0062] In a further aspect, the method comprises the steps of: determining the level of at least one miRNA in a sample from the subject; and determining whether or not it is appropriate to administer a therapy to reduce TBI based on the level of the at least one miRNA; Provided is a method for determining whether a subject is appropriate to receive a therapy to reduce TBI, comprising:

[0063] It will be understood that the step of administering a therapy to a subject does not form part of the claimed method unless specifically stated.

[0064] In some embodiments, the method can further include administering a suitable treatment to the subject. In some embodiments, the treatment can include a therapy to reduce TBI. Thus, the present invention features a method for diagnosing and treating TBI in a subject, comprising the steps of: (a) obtaining a sample (e.g., a blood, plasma, serum, urine, or saliva sample) from the subject; (b) detecting one or more miRNAs (selected from those described herein); diagnosing the patient as having TBI if the level of the miRNA is different from the reference standard (as described herein); and administering a treatment for TBI.

[0065] In a further aspect, the present invention provides a method for determining an appropriate treatment for a subject suspected of suffering from TBI, comprising the step of identifying whether the subject has TBI by determining the level of at least one miRNA in a sample from the subject.

[0066] If a subject is identified as having a TBI, appropriate treatment can include one or more of the following treatments: further evaluating the subject, for example with additional testing (e.g., language, cognitive, motor and / or vision testing), CT and / or MRI scans; removing the subject from activities (e.g., the activities during which the TBI occurred); admitting the subject to a hospital or specialized outpatient clinic; surgery; and administering therapy to the subject to reduce the TBI.

[0067] Therapies to mitigate TBI include neuroprotective agents, e.g., drugs to treat brain swelling such as mannitol and hypertonic saline, and / or other neuroprotective measures such as avoidance of hypertensive resuscitation and use of sedatives.

[0068] In some embodiments, the subject can subsequently be monitored, for example in a hospital or clinical setting, to follow their recovery.

[0069] According to a further aspect of the invention, there is provided a method of detecting and / or determining the level of a target miRNA in a subject, comprising the steps of: (a) obtaining a sample from the subject; and (b) detecting and / or determining the level of a target miRNA in the sample by contacting the sample with a probe specific for the target miRNA.

[0070] The sample may be any suitable body fluid or tissue sample obtained from a subject, as defined above, hi some embodiments, the sample is blood, serum, plasma, urine or saliva.

[0071] In some embodiments, the method may comprise determining the levels of two or more target miRNAs in the sample.

[0072] According to a further aspect of the present invention, there is provided a therapy for reducing TBI for use in a method of treating a subject in need thereof, wherein the subject has been identified as having TBI by determining the level of at least one miRNA in a sample from the subject.

[0073] Determining the level of the target miRNA may include contacting the sample with a substrate functionalized with probes, e.g., a chip equipped with the probes. The substrate or chip may conveniently include a plurality of probes, each specific for a different target miRNA.

[0074] The subject can be injured, particularly head injury.The subject may be suspected of having TBI.In some embodiments, the sample is obtained 72 hours or less, 48 ​​hours or less, 36 hours or less, 24 hours or less, 12 hours or less, 6 hours or less, 4 hours or less, 2 hours or less or 1 hour or less after injury.

[0075] In some embodiments, the method further comprises treating the subject. The treatment includes one or more of the following: further evaluating the subject, for example, by further examination (e.g., language test, cognitive test, motor test, and / or vision test), CT and / or MRI scan; removing the subject from the activity (e.g., the activity during which the TBI occurred); admitting the subject to a hospital or specialized outpatient clinic; and administering a therapy to the subject to reduce the TBI. In some embodiments, the treatment comprises administering an effective amount of a neuroprotective drug.

[0076] Thus, in a still further aspect, the present invention provides a method for producing a method for preparing a pharmaceutical composition comprising the steps of: determining the level of at least one miRNA in a sample from the subject; and administering an appropriate treatment for mTBI if the level of the at least one miRNA is indicative of mTBI; or administering an appropriate treatment for m-sTBI if the level of the at least one miRNA is indicative of m-sTBI. The present invention provides a method for treating TBI, comprising:

[0077] It will be appreciated by those skilled in the art that different treatment routes may be used for mTBI and m-sTBI.

[0078] Appropriate treatment for mTBI includes the following: removing the subject from activity; treatment on-site or in the community; further evaluation of the subject in hospital without overnight hospitalization (typically, mTBI patients are promptly discharged with head injury notice); or admission to hospital for an observation period (typically 1-2 days). Subjects can be further evaluated with tests (e.g., language, cognitive, motor and / or vision tests). CT scans are generally only required if certain signs are present, according to NICE guidelines, including suspected skull fracture, post-traumatic seizures, focal neurological deficits, repeated vomiting, and a GCS score of less than 13 (less than 14 for children or less than 15 for infants under 1 year old) on initial evaluation.

[0079] Appropriate treatment for m-sTBI includes the following procedures: MRI or CT scan (especially within one hour of injury); hospital admission (which may include intensive care unit admission and / or transfer to a major trauma center with specialized outpatient or neurosurgical facilities); neuromonitoring; surgery; and administration of therapies to mitigate the TBI, such as administration of neuroprotective agents (e.g., drugs to treat brain swelling, such as mannitol and hypertonic saline), and / or other neuroprotective measures, such as avoidance of hypertensive resuscitation and use of sedatives.

[0080] That is, the present invention allows a subject with a TBI to be rapidly classified as having mTBI or m-sTBI, thereby allowing the subject to receive the most appropriate treatment.

[0081] According to a further aspect of the invention, there is provided a detection system for diagnosing and / or monitoring TBI comprising a sensor element comprising a substrate functionalized with a probe specific for a target miRNA. The detection system may further comprise a detection device capable of detecting binding of the target miRNA to the probe.

[0082] According to yet a further aspect of the present invention, there is provided a sensor element for use in a detection system for diagnosing and / or monitoring TBI comprising a substrate functionalized with a probe specific for a target miRNA.

[0083] The sensor element may further include a sample application zone thereon for receiving a sample (eg, a bodily fluid sample).

[0084] The probe can selectively bind to the miRNA of interest. The substrate can be functionalized with multiple probes. The probes can be all identical, or two or more different probes can be provided. For example, in some embodiments, the substrate can be functionalized with a first probe specific to a first miRNA and a second probe specific to a second miRNA. The first and second probes can be grouped together, for example, on different parts of the sensor element.

[0085] In a further aspect of the invention, a composition is provided for use in a method of diagnosing and / or monitoring traumatic brain injury (TBI) in a subject, comprising a probe specific for a target miRNA. The composition can comprise any one of the listed miRNAs, or any multiple of the listed miRNAs (e.g., two, three, four, or more of the listed miRNAs).

[0086] In some embodiments, the target miRNA is selected from the group consisting of miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * , miR-671-3p, hsa-let-7c-5p, hsa-let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p, miR-424-5p, miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p and miR-629

[0087] In some embodiments, the target miRNA is selected from the group consisting of miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, hsa-miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 *These microRNAs have been found to be biomarkers expressed in all TBI patients (mild or severe).

[0088] In some embodiments, the target miRNA is selected from the group consisting of the following miRNAs: miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, and miR-335.

[0089] In some embodiments, the target miRNA is selected from the group consisting of let-7c-5p, let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, and miR-424-5p; miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p.

[0090] In some embodiments, the target miRNA is selected from the group consisting of miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-194, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p, and miR-629.

[0091] In some embodiments, the target miRNA is selected from the group consisting of the following miRNAs: miR-425-5p, miR-502, miR-21, and miR-335.

[0092] The probe may comprise a biological molecule such as a protein (e.g., an antibody) or a nucleic acid. In some embodiments, the probe comprises a nucleic acid. The nucleic acid may comprise a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identical to a sequence that is the complement of the full-length sequence of the target miRNA. In some embodiments, the nucleic acid comprises a sequence that is 100% identical to a sequence that is the complement of the sequence of the target miRNA (i.e., the receptor comprises a nucleic acid sequence that is the exact complement of the target miRNA sequence).

[0093] The probes can be linked to the surface of the substrate by any suitable means, such as by coupling chemistry known to those skilled in the art. In some embodiments, each probe is linked to the surface of the substrate via a linker. In some embodiments, the probe comprises a moiety for immobilizing the probe on the substrate or for linking the probe to a linker immobilized on the substrate.

[0094] Alternatively, or in addition, the probe can include a detectable label, which can be, for example, radioactive, fluorescent, luminescent, or antibody-based (e.g., can comprise a conventional tetrameric antibody or a detectable fragment thereof).

[0095] The substrate of the sensor element can be formed from any suitable material. In some embodiments, the substrate comprises or is formed from metal, plastic, glass, silica, silicon, graphite, graphene, or any combination thereof. In some embodiments, the substrate comprises multiple layers. For example, the substrate can be made by forming a graphene surface or layer on a silicon carbide or silica layer. The graphene surface can be chemically modified, for example, to graphene oxide (GO) or graphene-amine (GA). Methods for forming graphene layers, such as epitaxial growth and sublimation growth, will be known to those skilled in the art.

[0096] Conventionally, a probe containing or consisting of a nucleic acid can be linked to the GO surface via a linker using an amino coupling reagent (e.g., (O-(7-azabenzotriazol-1-yl)-N,N,N,N'-tetramethyluronium hexafluorophosphate (HATU)). The sensor element comprising a surface functionalized with a nucleic acid probe can then be used to selectively detect the miRNA complementary thereto.

[0097] Suitable linkers may include an aniline moiety (or a derivative thereof), a benzoic acid moiety (or a derivative thereof) or an ethenediamine moiety (or a derivative thereof). An aniline linker may be formed by linking a nitrobenzene molecule (or a derivative) to the graphene surface (e.g., using a diazonium salt) and reducing the nitrobenzene to aniline. The amine group of the aniline may then be used to link to a probe. Similarly, a diazonium salt (e.g., 4-benzoic acid diazonium tetrafluoroborate) may be used to link benzoic acid or a benzoic acid derivative to the graphene surface. An ethanediamine moiety may be linked to carboxylated graphene or graphene oxide.

[0098] The sensor element may be contained within a test strip. The test strip may be disposable.

[0099] The detection device can be configured to detect binding of the target miRNA to the receptor by any suitable means known to those of skill in the art, for example, by detecting changes in electrical impedance, hydrogen ion concentration, or conformational changes caused by hybridization.

[0100] The detection device may further comprise a user interface for outputting data to a user.

[0101] In some embodiments, the detection device comprises a database of treatment information. The device may be capable of identifying suitable treatment options from the database depending on the level of the or each miRNA of interest. The treatment information may be provided to the user via a user interface.

[0102] Conveniently, the detection device can be portable, for example handheld. The detection device can include a data storage unit for storing miRNA levels and other information about the subject. In some embodiments, the device includes a data communication means for communicating data to other devices. For example, the device can communicate data wirelessly via WiFi, 3G, 4G, Bluetooth, or other mobile apps. This can conveniently allow medical professionals to easily access the data if necessary.

[0103] Thus, it is anticipated that the detection device of the present invention will provide an affordable, portable, non-invasive point of care means for diagnosing and monitoring TBI. The device can be used by emergency personnel, the military, schools, sports clubs and medical professionals, thereby enabling accurate assessment and triage of patients suspected of having TBI.

[0104] In a further aspect, a kit is provided for use in the method. The kit can include at least a probe (e.g., a protein such as an antibody, or a nucleic acid) that can selectively bind to the miRNA of interest. In some embodiments, the kit includes an array that includes a plurality of probes. In some embodiments, at least one probe is a primer for performing PCR. The kit can further include instructions for use, for example, instructions for use in diagnosing and / or monitoring TBI. The kit can further include suitable buffers and reagents, such as amplification primers and enzymes (e.g., DNA polymerase, reverse transcriptase for converting miRNA to cDNA).

[0105] It will be understood that statements made herein in relation to any aspect of the invention may, where appropriate, be equally applicable to any other aspect of the invention. The present invention includes, for example, the following embodiments: [Embodiment 1] A method for diagnosing and / or monitoring traumatic brain injury (TBI) in a subject, comprising determining the level of at least one miRNA in a body fluid sample from the subject. [Embodiment 2] The at least one miRNA is one of the following miRNAs: miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 *, miR-671-3p, hsa-let-7c-5p, hsa-let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20 b-5p, miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, miR-424-5p, miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p; miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p and miR-629, or any combination thereof. 2. The method of embodiment 1, wherein the compound is selected from the group consisting of: [Embodiment 3] The at least one miRNA is selected from the group consisting of the following miRNAs: miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, and hsa-miR-126. * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * 3. The method of embodiment 1 or 2, wherein the IL-161A is selected from the group consisting of miR-671-3p, and miR-671-3p, or any combination thereof. [Embodiment 4] The method according to any one of embodiments 1 to 3, wherein the at least one miRNA is selected from the group consisting of the following miRNAs: miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, and miR-335, or any combination thereof. [Embodiment 5] The method according to embodiment 1 or 2, wherein the at least one miRNA is selected from the group consisting of the following miRNAs: let-7c-5p, let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, and miR-424-5p; miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p, or any combination thereof. [Embodiment 6] The method according to embodiment 1 or 2, wherein the at least one miRNA is selected from the group consisting of the following miRNAs: miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-194, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p and miR-629, or any combination thereof. [Embodiment 7] The method according to any one of embodiments 1 to 4, wherein the at least one miRNA is selected from the group consisting of the following miRNAs: miR-425-5p; miR-502; miR-21; and miR-335, or any combination thereof. [Embodiment 8] The method of embodiment 7, wherein the TBI is mild TBI (mTBI) and the miRNA is selected from the group consisting of the following miRNAs: miR-425-5p and miR-502. [Embodiment 9] The method of embodiment 8, wherein the subject is diagnosed with mTBI if the level of miR-425-5p and / or miR-502 is determined to be below a predetermined threshold or is decreased compared to a reference. [Embodiment 10] The method of embodiment 7, wherein the TBI is severe TBI (sTBI) and the miRNA is selected from the group consisting of the following miRNAs: miR-21 and miR-335. [Embodiment 11] The method of embodiment 10, wherein the subject is diagnosed with sTBI if the levels of miR-21 and miR-335 are determined to exceed a predetermined threshold or are increased compared to a reference. [Embodiment 12] The following miRNAs: (i) a first miRNA selected from miR-425-5p and miR-502; and (ii) a second miRNA selected from miR-21 and miR-335; 8. The method of embodiment 7, comprising determining the level of [Embodiment 13] The method of embodiment 12, wherein the subject is diagnosed as having any severity of TBI if the level of miR-425-5p or miR-502 is determined to be below a predetermined threshold or is decreased compared to the reference, and the level of miR-21 or miR-335 is determined to be above a predetermined threshold or is increased compared to the reference. [Embodiment 14] The method of any of embodiments 1 to 13, wherein the method is for diagnosing TBI, and the level of at least one miRNA is determined in a body fluid sample obtained from the subject 48 hours or less after injury. [Embodiment 15] The method described in any one of embodiments 1 to 14, wherein the bodily fluid sample comprises saliva, blood, plasma or serum. [Embodiment 16] The method described in embodiment 15, wherein the bodily fluid sample is saliva. [Embodiment 17] A sensor element for a detection system for diagnosing and / or monitoring TBI, comprising a substrate functionalized with a probe specific for a target miRNA. [Embodiment 18] The sensor element of embodiment 17, wherein the probe comprises a nucleic acid. [Embodiment 19] A sensor element described in embodiment 18, wherein the nucleic acid comprises a sequence that is at least 80% identical to a sequence that is a complement of the sequence of the target miRNA. [Embodiment 20] A sensor element described in any one of embodiments 17 to 19, wherein the substrate is formed from metal, plastic, glass, silica, silicon, graphite or graphene, or any combination thereof. [Embodiment 21] The following components: a sensor element according to any one of embodiments 17 to 20; and A detection system for diagnosing and / or monitoring TBI, comprising a detection device capable of detecting binding of a target miRNA to said probe. [Embodiment 22] A kit for use in a method for diagnosing and / or monitoring traumatic brain injury (TBI) in a body fluid sample from a subject, comprising at least one probe specific for a target miRNA. [Embodiment 23] The steps are as follows: determining the level of at least one miRNA in a bodily fluid sample from the subject; and determining whether or not it is appropriate to administer a therapy to reduce TBI based on the level of the at least one miRNA; The present invention relates to a method for determining whether it is appropriate to administer to a subject a therapy to reduce TBI, comprising: [Embodiment 24] A method for determining an appropriate treatment for a subject suspected of having TBI, comprising a step of identifying whether the subject has TBI by determining the level of at least one miRNA in a bodily fluid sample from the subject. [Embodiment 25] The method of embodiment 24, wherein if the subject is identified as having TBI, the appropriate treatment is determined to be at least one of the following treatments: further evaluating the subject; removing the subject from activity; admitting the subject to a hospital or outpatient facility; and administering a therapy to the subject to reduce the TBI. [Embodiment 26] A therapy for reducing TBI for use in a method of treating a subject in need thereof, wherein the subject is identified as having TBI by determining the level of at least one miRNA in a bodily fluid sample from the subject. [Embodiment 27] The method of any of embodiments 24 to 26, wherein the therapy for reducing TBI is selected from at least one of the following therapies: neuroprotective drugs, sedatives; and avoidance of the use of hypertensive resuscitation. [Embodiment 28] A composition for use in a method for diagnosing and / or monitoring traumatic brain injury (TBI) in a subject, comprising a probe specific for a target miRNA. [Embodiment 29] The composition described in embodiment 28, comprising multiple probes. [Embodiment 30] The miRNA is one of the following miRNAs: miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-335, miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 *, miR-671-3p, hsa-let-7c-5p, hsa-let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p , miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, miR-30a-5p;miR-107;m iR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p, miR-424-5p, miR-10a, The sensor element, detection system, kit, method or composition according to any one of embodiments 17 to 29, wherein the nucleic acid sequence is selected from the group consisting of miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p and miR-629, or any combination thereof. [Embodiment 31] The sensor element, detection system, kit, method or composition described in embodiment 30, wherein the miRNA is selected from the group consisting of the following miRNAs: miR-425-5p; miR-502; miR-21; and miR-335, or any combination thereof.

[0106] Detailed Description of the Invention Embodiments of the present invention will now be described, by way of example only, and with reference to the accompanying drawings, in which:

[0107] Figure 1: miR-425-5p and miR-502 expression in three different classifications of trauma and HV. miR-425-5p and miR-502 expression in 10 HV, 10 mTBI+EC (1 day), 10 mTBI+EC (15 days), 10 EC (1 day), 10 EC (15 days), 10 sTBI+EC (1 day) and 10 sTBI+EC (15 days) patients detected by qRT-PCR analysis. miR-425-5p expression was found to be significantly decreased in mTBI+EC (1 day) compared to HV (p<0.01), mTBI+EC (15 days) (p<0.001) and sTBI+EC (1 day) (p<0.01) (Figure 1A). miR-502 expression was found to be significantly decreased in mTBI+EC (1 day) compared to HV (p<0.05), mTBI+EC (15 days) (p<0.01), and sTBI+EC (1 day) (p<0.05) (Figure 1B).

[0108] Figure 2: miR-21 and miR-335 expression in three different classifications of trauma and HV. miR-21 and miR-335 expression in 10 HV, 10 mTBI+EC (1 day), 10 mTBI+EC (15 days), 10 EC (1 day), 10 EC (15 days), 10 sTBI+EC (1 day) and 10 sTBI+EC (15 days) patients detected by qRT-PCR analysis. miR-21 expression was found to be significantly upregulated in sTBI+EC (1 day and 15 days) compared to HV (p<0.01) (Figure 2A). miR-335 expression was found to be significantly upregulated in sTBI+EC (1 day) compared to HV (p<0.001), EC (15 days) (p<0.001), and mTBI+EC (1 day) (p<0.05) (Figure 2B).

[0109] Figure 3A: Time course of miR-425-5p expression in three different classifications of trauma and HV. miR-425-5p expression at various time points from injury (T0, T4~12h, T48~72h, 15 days) in 30 HV, 30 mTBI+EC, 30 EC, and 30 sTBI+EC patients detected by qRT-PCR analysis. miR-425-5p expression was found to be significantly decreased in mTBI+EC (T0 and T4~12h) compared to HV, sTBI+EC, and EC (p<0.05). p-values ​​were determined by Tukey's post-hoc test. *: significantly different from HV.

[0110] Figure 3B: Time course of miR-502 expression in three different classifications of trauma and HV. miR-502 expression at various time points from injury (T0, T4-12h, T48-72h, 15 days) in 30 HV, 30 mTBI+EC, 30 EC, and 30 sTBI+EC patients detected by qRT-PCR analysis. miR-502 expression was found to be significantly decreased in mTBI+EC (T0 and T4-12h) compared to HV, sTBI+EC, and EC (p<0.05). p-values ​​were determined by Tukey's post-hoc test. *: significantly different from HV.

[0111] Figure 4A: Time course of miR-21 expression in three different classifications of trauma and HV. miR-21 expression at various time points from injury (T0, T4~12h, T48~72h, 15 days) in 30 HV, 30 mTBI+EC, 30 EC, and 30 sTBI+EC patients detected by qRT-PCR analysis. miR-21 expression was found to be significantly upregulated in sTBI+EC (T4~12h, T48~72h, and 15 days) compared to HV (p<0.01). p-values ​​were determined by Tukey's post-hoc test. *: significantly different from HV.

[0112] Figure 4B: Time course of miR-335 expression in three different classifications of trauma and HV. miR-335 expression at various time points from injury (T0, T4-12h, T48-72h, 15 days) in 30 HV, 30 mTBI+EC, 30 EC, and 30 sTBI+EC patients detected by qRT-PCR analysis. miR-335 expression was found to be significantly upregulated in sTBI+EC (T0, T4-12h, T48-72h, and 15 days) compared to HV and mTBI+EC (p<0.001), but not significantly upregulated compared to EC alone. p values ​​were determined by Tukey's post-hoc test. *: significantly different from HV. EXAMPLES

[0113] As miRNAs have emerged as promising biomarkers in a range of different pathological conditions, we sought to explore their role in TBI.

[0114] Example 1 material and method Patients and sample collection Study participants were recruited from the Surgical Reconstruction and Microbiology Research Centre (SRMRC) at the Queen Elizabeth Hospital Birmingham (UK) as part of the Post-Traumatic Brain Biomarkers (Golden Hour Study) study (Ethics Ref. 13 / WA / 0399).

[0115] First, we performed a screening of 754 miRNAs in 5 mTBI patients with extracranial injury (EC), 5 sTBI+EC injury patients and healthy volunteers (HV) at 1 and 15 days after injury with the aim of selecting specific candidate biomarkers that could differentiate between mild and severe TBI and predict recovery from mTBI after 15 days. Based on this information (Table 2), it was then possible to confirm the results of the study in an extended cohort of 40 individual patients divided into 4 different classifications: HV (n=10), EC (n=10), mTBI+EC (n=10), sTBI+EC (n=10). The healthy volunteers consented and participated in the RECOS study. The EC injury patients had radiologically confirmed fractures, no head trauma, no infections, no history of neurological or psychiatric disorders, and no alcohol or drug dependence. Mild TBI with EC included patients with non-penetrating head injury and a Glasgow Coma Scale (GCS) score > 13. Severe TBI with EC included patients with a GCS score ≤ 8. All patients were sex- and age-matched for HV.

[0116] Sample processing Peripheral blood samples were obtained for each patient at 1 and 15 days after injury. Blood samples were processed for serum isolation within 2 hours of collection. Whole blood was allowed to stand at room temperature for 30 minutes, followed by centrifugation at 3000 rpm for 10 minutes at 4°C. Serum was aliquoted and stored at -80°C until analysis.

[0117] RNA isolation, reverse transcription and miRNA profiling by TaqMan Low Density Arrays (TLDA) An initial screening (discovery set) was performed on 5 mTBI+EC and 5 sTBI+EC patients compared to HV at two different time points (1 and 15 days after injury). Serum from these patients was used to generate a transcriptome profile of 754 miRNAs. Serum samples were pelleted at 2000 rpm for 10 min to remove circulating cells or debris. miRNAs were extracted from 400 μL serum samples using the Qiagen miRNeasy mini kit (Qiagen GmbH, Hilden, Germany) according to Qiagen's supplementary protocol for purification of small RNAs from serum and plasma, and finally eluted in a volume of 30 μL RNase-free water. The concentration and purity of the resulting RNA were determined using an ND-1000 UV-Vis spectrophotometer (NanoDrop). 20 ng of serum RNA was retrotranscribed and pre-amplified according to the manufacturer's instructions. The preliminary amplification products were loaded onto the TLDA (TaqMan Human MicroRNA Array v3.0 A and B (Applied Biosystems LifeTechnologies TM )) PCR in TLDA was performed using the 7900HT Fast RealTime PCR System (Applied Biosystems, LifeTechnologies TM ) was used.

[0118] 4. Data Analysis To obtain accurate miRNA profiles, we used the global median normalization method. Similar to microarray analysis, the Ct values ​​from each sample were normalized to the median Ct of the array. Furthermore, by calculating the median and mean Ct of each array and the Pearson correction between the Ct of each miRNA, we found that two miRNAs, namely miR-331 and miR-223, showed expression profiles similar to the median and mean of TLDA. *These miRNAs were identified using two different methods: DataAssist v.3 software (Applied Biosystem Life Technologies TM ) and geNorm algorithm, they were also confirmed to be among the most stable in TLDA. Thus, miR-331 and miR-223 * were used as reference genes for validation by single TaqMan assay. Expression fold change was calculated by the 2-ΔΔCT method. Differentially expressed miRNAs (DE miRNAs) were identified by Significance of Microarrays Analysis (SAM) with Multi experiment viewer v4.8.1, applying a two-class unpaired test between ΔCt and using a p-value based on 100 permutations; imputation engine: K-nearest neighbor (10 neighbors); false discovery rate <0.15 was used as correction for multiple comparisons. We found only DE miRNAs to be reliable, consistent with using all endogenous controls.

[0119] Single TaqMan Assay Ten differentially expressed miRNAs were selected from the array as potential candidate biomarkers for distinguishing between mild and severe TBI and monitoring recovery from mild TBI. These candidates were used to measure the expression of miRNAs in a single TaqMan assay (Applied Biosystems, Life Technologies). TM ) to validate the data in an expanded cohort of 30 patients (validation set) and 10 controls (HV) divided into three different classifications (mTBI+EC, sTBI+EC, and EC only) at two different time points (1 day and 15 days after injury). Samples were extracted and retrotranscribed as described above and subjected to RT-qPCR analysis on a Bio-Rad iQ5 Real-time PCR Detection system (Bio-Rad, CA, USA). Fold expression changes were calculated by the 2-ΔΔCT method.

[0120] statistical analysis Data were checked for normal distribution and transformed to perform parametric tests. Comparisons between groups at each time point and within groups over time were performed on transformed data by one-way analysis of variance and Tukey's post-hoc test. Receiver operating characteristic analysis was used to calculate the sensitivity and specificity of each biomarker for diagnosing either mTBI or sTBI, expressed as the area under the curve (AUC). All analyses were performed using SPSS v.20 (IBM). Differences were considered statistically significant when p-value was less than 0.05.

[0121] result Expression profiling by TaqMan Low Density Array (TLDA) From the 754 screenable miRNAs in TLDA, we identified 10 circulating miRNAs (1 day) and 13 (15 days) as differentially expressed in mTBI+ECs, and 19 (1 day) and 22 (15 days) in sTBI+ECs (Table 2). From this list, hsa-miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * and miR-671-3p were excluded from further analysis because they were expressed in the majority of patients and therefore are not suitable candidate biomarkers for only mild or severe trauma. However, the above microRNAs can be identified in TBI of any severity and are therefore useful TBI biomarkers. Meanwhile, miR-184, miR-301b, miR-502 and miR-505 were uniquely and differentially expressed in mTBI+EC (1 day) and were selected as early candidate biomarkers for mTBI. In addition, miR-203, miR-425-5p, miR-654-3p and miR-655, which are differentially expressed 15 days after mTBI+EC, were selected as candidate biomarkers that can track the recovery of mTBI.

[0122] Finally, two miRNAs that were stably expressed at both time points in sTBI+ECs, miR-21 and miR-335, were selected for further study.

[0123] [Table 2]

[0124] A single TaqMan assay for candidate biomarkers of mTBI To validate these findings, we subsequently examined the expression of selected miRNAs in three separate and independent groups (10 mTBI+EC, 10 sTBI+EC, 10 EC) at two selected time points (1 day and 15 days after injury) using a single TaqMan assay. Results were compared to 10 HV. Fold changes were observed for miR-331 and miR-223. * was used as a reference gene and calculated by the 2-ΔΔCT method.

[0125] Among the candidate biomarkers of mTBI at both time points (miR-184, miR-301b, miR-502, miR-505, miR-203, miR-425-5p, miR-654-3p and miR-655), two showed interesting results and were significantly and differentially expressed in three different categories compared to HV. In particular, miR-425-5p and miR-502 showed similar trends (Figure 1). They were both significantly downregulated (mean 0.387 ± 0.201 and 0.314 ± 0.146) in mTBI+EC at 1 day after injury compared to HV (p<0.001), EC (p<0.001) and sTBI+EC (p<0.001), respectively. At 15 days after mild injury, miR-425-5p and miR-502 returned to normal levels (0.886±0.310 and 1.157±0.258). Expression of miR-425-5p and miR-502 was also found to be similar to HV in EC samples at 1 and 15 days after injury, suggesting that these two biomarkers are differentially expressed only in brain injury patients. Moreover, none of them showed significant differences in sTBI+EC compared to HV at both time points. Therefore, miR-425-5p and miR-502 could be considered as the most promising candidate biomarkers for early diagnosis and monitoring of mTBI at 15 days after trauma. The AUCs for these biomarkers are shown in Table 3.

[0126] A single TaqMan assay for candidate biomarkers of sTBI miR-21 and miR-335 were analyzed as possible biomarkers of sTBI, as both of them were found to be upregulated at both time points in sTBI+EC in the initial screening. They were shown to be strong candidates in the second patient dataset as well (Figure 2). miR-21 was significantly upregulated (7.106±4.192 and 4.012±1.577) at both time points in sTBI with EC versus HV (p,0.001), EC (p<0.001) and mTBI (p<0.001). No significant differences were found in the residual classification compared to HV. miR-335 showed upregulation in sTBI+EC and at both time points (16.824±14.195 and 12.324±8.931, respectively). At day 1, these groups were significantly different from controls (p=0.001) and mTBI+EC (p=0.031), but not from EC. Interestingly, a significant upregulation in EC patients was seen at day 1 (7.951±4.870), but not at 15 days after injury (1.260±0.531). For this reason, at day 15, miR335 was significantly higher in the sTBI+EC group versus HV (p=0.002), EC (p=0.007), and mTBI+EC (p=0.001). miR-335 did not show any significant difference in mTBI+EC at both time points compared to HV. The AUCs for these biomarkers are also shown in Table 3.

[0127] [Table 3]

[0128] Consideration In this study, we investigated whether changes at the levels of miRNAs could be applied to the diagnosis of TBI and the assessment of its severity.Four miRNAs were identified as differentially expressed in TBI: miR-425-5p, miR 502, miR-21, and miR-335.

[0129] miR-425-5p showed significant results at day 1 in mTBI+EC compared to HV, and similar results were obtained in all other categories. Its downregulation within the first 24 hours after mild injury and return to normal levels after 15 days make miR-425-5p a good candidate biomarker for mild trauma.

[0130] miR-502 was also found to be differentially expressed in mTBI+ECs. The trend of this miRNA was very similar to that of miR-425-5p. This miRNA showed specificity for brain injury patients and returned to normal levels 15 days after mild injury, so it could also be used to track recovery.

[0131] Following sTBI, two miRNAs (miR-21, miR-335) were noted to be expressed at both 1 and 15 days and were therefore selected as possible biomarkers for sTBI. miR-21 and miR-335 were significantly upregulated at both time points in sTBI+ECs compared to controls. Thus, overexpression confirmed the array results and demonstrated the potential of these molecules as biomarkers for sTBI.

[0132] A selected panel of miRNAs has the potential to accurately diagnose TBI and allows for stratification of patients according to their severity, thus allowing for the provision of the most appropriate treatment.

[0133] Example 2 Patients and sample collection Study participants were recruited from the Surgical Reconstruction and Microbiology Research Centre (SRMRC) at the Queen Elizabeth Hospital Birmingham (UK) as part of the SIR (Steroids and Immunity from injury through to Rehabilitation) study (Ethics Ref. 11 / SW / 0177), ReCoS (REpetitive CONcussion in Sport) study (Ethics Ref. 11-0429AP28) and the Golden Hour study (Ethics Ref. 13 / WA / 0399). Written informed consent was received from participants or a legally valid power of attorney (from a family member or professional not directly involved in the study) prior to inclusion in the study.

[0134] The second sample dataset used serum samples from a total of 120 individuals divided into four different classifications: HV (n = 30), EC (n = 30), mTBI + EC (n = 30), and sTBI + EC (n = 30). For each patient, blood was collected at different time points (T0-1 h, T4-12 h, T48-72 h, and 15 days). Healthy volunteers consented and participated in the ReCoS study. EC injury patients had radiologically confirmed orthopedic fractures, no head trauma, no infections, no history of neurological or psychiatric disorders, and no alcohol or drug dependence. Mild TBI with EC included patients with non-penetrating head trauma and Glasgow Coma Scale (GCS) score ≥ 13. Severe TBI with EC included patients with GCS ≤ 8.

[0135] Sample processing Blood samples were processed for serum isolation within 2 hours of collection. Whole blood was allowed to stand at room temperature for approximately 30 minutes, followed by centrifugation at 3000 rpm for 10 minutes at 4° C. Serum was divided into aliquots and stored at −80° C. until analysis.

[0136] RNA isolation, data analysis, assays and statistical analysis were performed as described in Example 1.

[0137] result A single TaqMan assay for candidate biomarkers of mTBI To validate the findings in Example 1, expression of selected miRNAs in three separate and independent groups (30 mTBI+EC, 30 sTBI+EC, 30 EC) was measured at various time points (T0, T4-12h, T48-72h and 15 days after injury) using a single TaqMan assay. Results were compared to 10 HV. Fold changes were observed for miR-331 and miR-223. * was used as a reference and calculated by the 2-ΔΔCT method.

[0138] Among the candidate biomarkers for mTBI at both time points (miR-184, miR-502, miR-505, miR-301b, miR-203, miR-425-5p, miR-654-3p, and miR-655), only two showed interesting results and were significantly and differentially expressed in three different categories compared to HV. Specifically, miR-425-5p and miR-502 showed similar trends (Figure 3). Both of these were downregulated in mTBI+EC, miR-425-5p at T0-1h (p=0.01845) and T4-12h (p=0.01962), respectively, compared to HV or EC and sTBI+EC (p<0.05), and miR-502 at T0-1h and T4-12h (p=0.02538 and p=0.03718, respectively) compared to HV, or EC and sTBI+EC (p<0.01). At 48 hours after mild injury, miR-425-5p and miR-502 returned to normal levels. Expression of miR-425-5p and miR-502 was also found at comparable levels in the EC group compared to HV, suggesting that these two biomarkers are downregulated only in brain injury patients. Moreover, none of these showed significant downregulation in sTBI+EC compared to HV at all time points. Therefore, miR-425-5p and miR-502 could be considered the most promising candidate biomarkers for early diagnosis and monitoring of mTBI. The AUCs for these biomarkers at the most important time points are shown in Table 4.

[0139] A single TaqMan assay for candidate biomarkers of sTBI miR-21 and miR-335 were analyzed as possible biomarkers of sTBI, as they were both found to be upregulated at both time points in sTBI+EC in the initial screening. They were also shown to be strong candidates in the second patient dataset (Figure 4). miR-21 was significantly upregulated in sTBI with EC at all time points from 4 hours after injury versus HV, EC and mTBI+EC (p=0.00306 (T4~12h), p=0.00844 (T48~72h) and p=0.00077 (15 days)). No significant differences were found in the residual classification compared to HV. miR-335 showed upregulation at all time points in sTBI+EC compared to HV (p=0.00109 (T0-1h), p=0.00284 (T4-12h), p=0.00012 (T48-72h) and p=0.01284 (15 days)) and in mTBI+EC, but no significant upregulation was found compared to EC. The AUCs for these biomarkers are also shown in Table 4.

[0140] [Table 4]

[0141] Consideration In this study, we verified previous findings that changes in miRNA levels could be applied to the diagnosis of TBI and the assessment of its severity.This study identified four miRNAs to be differentially expressed in TBI: miR-425-5p, miR 502, miR-21, and miR-335.

[0142] miR-425-5p showed significant results at T0 and T4-12h in mTBI+EC compared to HV, and similar results were obtained in all other classifications. Its downregulation returned to normal levels after T48-72h, confirming miR-425-5p as a suitable candidate biomarker for mild trauma.

[0143] miR-502 was also identified as differentially expressed in mTBI+ECs. The trend of this miRNA was very similar to that of miR-425-5p. This miRNA showed specificity for brain injury patients and returned to normal levels 48-72 hours after mild injury, so it could be used to track recovery.

[0144] Following sTBI, two miRNAs (miR-21, miR-335) were noted to be expressed at all time points analyzed and thus were confirmed as possible biomarkers for sTBI. miR-21 and miR-335 were significantly upregulated in sTBI+ECs when compared to controls. Thus, overexpression confirmed the potential of these molecules as biomarkers for sTBI.

[0145] Example 3 Saliva samples were collected from professional athletes who had sustained a concussion 2–3 days after injury and analyzed for microRNAs present in the saliva. Athletes were clinically diagnosed with mTBI.

[0146] material and method MicroRNAs are analyzed using nCounter technology (nanoString Technologies®), which uses molecular "barcodes" and microscopic imaging to detect and count up to hundreds of unique transcripts in a single hybridization reaction. Each color-coded barcode is linked to a single target-specific probe that corresponds to the microRNA of interest.

[0147] The analysis was performed according to the manufacturer's protocol, which included the following steps:

[0148] HybridizationThe technology utilizes two ~20-base probes per microRNA that hybridize in solution: the reporter probe retains the signal, and the capture probe allows the complex to be immobilized for data collection. Purification and immobilization After hybridization, excess probe is removed and the probe / target complexes are aligned and immobilized in the nCounter cartridge. Data collection The sample cartridge is placed into a digital analyzer device for data collection. The color codes on the surface of the cartridge are counted and tabulated for each target molecule.

[0149] result Table 5 below lists the microRNAs that were found to be significantly and differentially expressed in concussed athletes compared to healthy volunteers. The table shows the fold change in expression of the microRNAs in patients compared to the control group. The fold change was calculated using miR-23a-3p and miR-148b-3p as reference genes.

[0150] [Table 5]

[0151] Consideration This study shows that microRNAs present in saliva are indicators of concussion / mTBI. This is important because saliva is more easily obtained than blood, meaning that detection of microRNAs in saliva provides a rapid and convenient means to diagnose TBI, especially at the sidelines of sports fields.

[0152] [Sequence table] SEQUENCE LISTING <110> The University of Birmingham <120> Biomarkers of traumatic brain injury <130> PA23-676 <150> GB1603967.9 <151> 2016-03-08 <160> 69 <170> PatentIn version 3.5 <210> 1 <211> 22 <212> RNA <213> Homo sapiens <400> 1 uagcuuauca gacugauguu ga 22 <210> 2 <211> 23 <212> RNA <213> Homo sapiens <400> 2 aaugacacga ucacucccgu uga 23 <210> 3 <211> 21 <212> RNA <213> Homo sapiens <400> 3 auccuugcua ucugggugcu a 21 <210> 4 <211> 23 <212> RNA <213> Homo sapiens <400> 4 ucaagagcaa uaacgaaaaa ugu 23 <210> 5 <211> 23 <212> RNA <213> Homo sapiens <400> 5 cagugcaaug auugucaa agc 23 <210> 6 <211> 22 <212> RNA <213> Homo sapiens <400> 6 uggacggaga acugauaagg gu 22 <210> 7 <211> 22 <212> RNA <213> Homo sapiens <400> 7 cgucaacacu ugcugguuuc cu 22 <210> 8 <211> 22 <212> RNA <213> Homo sapiens <400> 8 gugaaauguu uaggaccacu ag 22 <210> 9 <211> 22 <212> RNA <213> Homo sapiens <400> 9 uaugucugcu gaccaucacc uu 22 <210> 10 <211> 22 <212> RNA <213> Homo sapiens <400> 10 auaauacaug guuaaccucu uu 22 <210> 11 <211> 21 <212> RNA <213> Homo sapiens <400> 11 gccccugggc cuauccuaga a 21 <210> 12 <211> 22 <212> RNA <213> Homo sapiens <400> 12 cguguauuug acaagcugag uu 22 <210> 13 <211> 22 <212> RNA <213> Homo sapiens <400> 13 ugagguagua guuuguacag uu 22 <210> 14 <211> 21 <212> RNA <213> Homo sapiens <400> 14 cauuauuacu uuugguacgc g 21 <210> 15 <211> 22 <212> RNA <213> Homo sapiens <400> 15 ugggucuuuug cgggcgagau ga 22 <210> 16 <211> 22 <212> RNA <213> Homo sapiens <400> 16 ggauaucauc auauacugua ag 22 <210> 17 <211> 21 <212> RNA <213> Homo sapiens <400> 17 cuauauauca aacauauucc u 21 <210> 18 <211> 22 <212> RNA <213> Homo sapiens <400> 18 uguaacagca acuccaugug ga 22 <210> 19 <211> 22 <212> RNA <213> Homo sapiens <400> 19 uaaugcccccu aaaaauccuu au 22 <210> 20 <211> 21 <212> RNA <213> Homo sapiens <400> 20 uaauuuuaug uauaagcuag u 21 <210> 21 <211> 22 <212> RNA <213> Homo sapiens <400> 21 uaguaccagu accuuguguu ca 22 <210> 22 <211> 22 <212> RNA <213> Homo sapiens <400> 22 gacuauagaa cuuucccccu ca 22 <210> 23 <211> 21 <212> RNA <213> Homo sapiens <400> 23 uccgguucuc agggcuccac c 21 <210> 24 <211> 22 <212> RNA <213> Homo sapiens <400> 24 ugagguagua gguuguaugg uu 22 <210> 25 <211> 22 <212> RNA <213> Homo sapiens <400> 25 ugagguagua guuugugcug uu 22 <210> 26 <211> 23 <212> RNA <213> Homo sapiens <400> 26 uguaguguuu ccuacuuuau gga 23 <210> 27 <211> 22 <212> RNA <213> Homo sapiens <400> 27 ucagugcacu acagaacuuu gu 22 <210> 28 <211> 22 <212> RNA <213> Homo sapiens <400> 28 uagcagcaca ucaugguuua ca 22 <210> 29 <211> 22 <212> RNA <213> Homo sapiens <400> 29 uagcagcacg uaauauugg cg 22 <210> 30 <211> 23 <212> RNA <213> Homo sapiens <400> 30 aacauucaac gcugucggug agu 23 <210> 31 <211> 23 <212> RNA <213> Homo sapiens <400> 31 uaaagugcuu auagugcagg uag 23 <210> 32 <211> 23 <212> RNA <213> Homo sapiens <400> 32 caaagugcuc auagugcagg uag 23 <210> 33 <211> 23 <212> RNA <213> Homo sapiens <400> 33 agcuacauug ucugcugggu uuc 23 <210> 34 <211> 22 <212> RNA <213> Homo sapiens <400> 34 uggcucaguu cagcaggaac ag 22 <210> 35 <211> 21 <212> RNA <213> Homo sapiens <400> 35 uucacagugg cuaaguucug c 21 <210> 36 <211> 22 <212> RNA <213> Homo sapiens <400> 36 uagcaccauc ugaaaucggu ua 22 <210> 37 <211> 22 <212> RNA <213> Homo sapiens <400> 37 uagcaccauu ugaaaucggu ua 22 <210> 38 <211> 22 <212> RNA <213> Homo sapiens <400> 38 uguaaacauc cucgacugga ag 22 <210> 39 <211> 23 <212> RNA <213> Homo sapiens <400> 39 agcagcauug uacagggcua uca 23 <210> 40 <211> 23 <212> RNA <213> Homo sapiens <400> 40 uauggcuuuuu cauuccuaug uga 23 <210> 41 <211> 23 <212> RNA <213> Homo sapiens <400> 41 cccaguguuu agacuaucug uuc 23 <210> 42 <211> 23 <212> RNA <213> Homo sapiens <400> 42 cgcaucccccu agggcauugg ugu 23 <210> 43 <211> 21 <212> RNA <213> Homo sapiens <400> 43 aauggcgcca cuaggguugu g 21 <210> 44 <211> 22 <212> RNA <213> Homo sapiens <400> 44 cagcagcaau ucauguuuug aa 22 <210> 45 <211> 23 <212> RNA <213> Homo sapiens <400> 45 uacccuguag auccgaauuu gug 23 <210> 46 <211> 22 <212> RNA <213> Homo sapiens <400> 46 uaacagucua cagccauggu cg 22 <210> 47 <211> 22 <212> RNA <213> Homo sapiens <400> 47 ugucaguuug ucaaauaccc ca 22 <210> 48 <211> 21 <212> RNA <213> Homo sapiens <400> 48 ugagaugaag cacuguagcu c 21 <210> 49 <211> 22 <212> RNA <213> Homo sapiens <400> 49 ucagugcauc acagaacuuu gu 22 <210> 50 <211> 23 <212> RNA <213> Homo sapiens <400> 50 uaaggugcau cuagugcaga uag 23 <210> 51 <211> 21 <212> RNA <213> Homo sapiens <400> 51 cugaccuaug aauugacagc c 21 <210> 52 <211> 22 <212> RNA <213> Homo sapiens <400> 52 uaauacuguc ugguaaaacc gu 22 <210> 53 <211> 23 <212> RNA <213> Homo sapiens <400> 53 aaacucuacu uguccuucug agu 23 <210> 54 <211> 21 <212> RNA <213> Homo sapiens <400> 54 ucaauaaug ucuguugaau u 21 <210> 55 <211> 22 <212> RNA <213> Homo sapiens <400> 55 cagugcaaug uuaaaagggc au 22 <210> 56 <211> 23 <212> RNA <213> Homo sapiens <400> 56 agguucugug auacacuccg acu 23 <210> 57 <211> 21 <212> RNA <213> Homo sapiens <400> 57 uucacagugg cuaaguucug c 21 <210> 58 <211> 23 <212> RNA <213> Homo sapiens <400> 58 cagugcaaua guauugucaa agc 23 <210> 59 <211> 20 <212> RNA <213> Homo sapiens <400> 59 ccucuggggcc cuuccuccag 20 <210> 60 <211> 22 <212> RNA <213> Homo sapiens <400> 60 gcugacuccu aguccagggc uc 22 <210> 61 <211> 22 <212> RNA <213> Homo sapiens <400> 61 uuaucagaau cuccagggggu ac 22 <210> 62 <211> 22 <212> RNA <213> Homo sapiens <400> 62 acuggacuua gggucagaag gc 22 <210> 63 <211> 23 <212> RNA <213> Homo sapiens <400> 63 uucauuuggu auaaaccgcg auu 23 <210> 64 <211> 22 <212> RNA <213> Homo sapiens <400> 64 gucccucucc aaaugugucu ug 22 <210> 65 <211> 22 <212> RNA <213> Homo sapiens <400> 65 aacccguaga uccgaucuug ug 22 <210> 66 <211> 22 <212> RNA <213> Homo sapiens <400> 66 aaagugcuuc ucuuuggugg gu <210> 67 <211> 21 <212> RNA <213> Homo sapiens <400> 67 uggguuuacg uugggagaac u <210> 68 <211> 21 <212> RNA <213> Homo sapiens <400> 68 aucacauugc cagggauuuc c <210> 69 <211> 22 <212> RNA <213> Homo sapiens <400> 69 ucagugcauc acagacuuu 22nd

Claims

1. A method for detecting the level of at least one miRNA in a sample from a subject for diagnosing and / or monitoring traumatic brain injury (TBI) in the subject, comprising determining the level of at least one miRNA in a serum sample obtained from the subject up to 15 days after injury and comparing the level of the at least one miRNA to a predetermined threshold or reference level, wherein the at least one miRNA comprises miR-335, and the reference level is the level of a reference miRNA in a control sample taken from a healthy subject; The above method, wherein the presence of severe TBI (sTBI) in the subject is detected when the level of miR-335 is determined to exceed a predetermined threshold or is increased compared to a reference level in a serum sample obtained up to 15 days after injury.

2. An miRNA comprising: (a) miR-21, (b) miR-425-5p, miR-21, and miR-502, or any combination thereof; (c) miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-126 * , miR-193a-5p, miR-144 * , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * , miR-671-3p, hsa-let-7c-5p, hsa-let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20b-5p, miR-221-3p, miR-24-3p, miR-27b-3p, miR-29a-3p, miR-29c-3p, miR-424-5p, miR-30a-5p; miR-107; miR-135b-5p; miR-199b-5p; miR-324-5p; miR-652-3p; miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p and miR-629, or any combination thereof, (d) miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, hsa-miR-126*, miR-193a-5p, miR-144*, miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625*, and miR-671-3p, or any combination thereof; (e) miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21 and miR-let-7g, or any combination thereof.

2. The method of claim 1, further comprising determining a level of at least one additional miRNA selected from the group consisting of:

3. The method of claim 2, comprising determining the levels of two, three, four or more miRNAs.

4. The method comprises administering to a subject a miRNA comprising: (i) a first miRNA selected from miR-425-5p and miR-502; and (ii) miR-335 determining a level of The method of any one of claims 1 to 3, wherein the presence of mild TBI (mTBI) in the subject is detected when the level of miR-335 in a serum sample obtained up to 15 days after injury is determined to not exceed a pre-defined threshold or is not increased compared to a reference, and the level of miR-425-5p and / or miR-502 in a serum sample obtained up to 48 hours after injury is determined to be below a pre-defined threshold or is decreased compared to a reference.

5. A sensor element for a detection system for diagnosing and / or monitoring TBI, comprising a substrate functionalized with a probe specific for a target miRNA, said target miRNA comprising miR-335, said probe comprising a nucleic acid, said nucleic acid comprising a sequence that is at least 90% identical to a sequence that is the complement of the sequence of the target miRNA; The above sensor element, wherein the subject is diagnosed as having severe TBI (sTBI) if the level of miR-335 in a serum sample obtained from the subject up to 15 days after injury is determined to exceed a predetermined threshold or is increased compared to the level of a reference miRNA in a control sample taken from a healthy subject.

6. A miRNA comprising: (a) miR-21, (b) miR-425-5p, miR-21, and miR-502, or any combination thereof; or (c) miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-126*, miR-193a-5p, miR-144* , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * , miR-671-3p, hsa-let-7c-5p, hsa-let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20 b-5p; miR-221-3p; miR-24-3p; miR-27b-3p; miR-29a-3p; miR-29c-3p; miR-30a-5p; miR-107; miR-652-3p, miR-424-5p; miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p and miR-629, or any combination thereof. The sensor element of claim 5, further comprising at least one additional probe specific for a target miRNA selected from the group consisting of:

7. Components: - a sensor element according to claim 5 or 6; and A detection system for diagnosing and / or monitoring TBI, comprising a detection device capable of detecting binding of a target miRNA to said probe, the target miRNA comprises miR-335; The above detection system diagnoses a subject as having severe TBI (sTBI) if the level of miR-335 in a serum sample obtained from the subject by 15 days after injury is determined to exceed a predetermined threshold or is increased compared to the level of a reference miRNA in a control sample taken from a healthy subject.

8. 1. A kit for diagnosing and / or monitoring traumatic brain injury (TBI) in a serum sample from a subject, comprising at least one probe specific for a target miRNA, wherein the target miRNA comprises miR-335, and wherein the probe comprises a nucleic acid, the nucleic acid comprising a sequence that is at least 90% identical to a sequence that is the complement of a sequence of the target miRNA; the diagnosis and / or monitoring comprises determining a level of miR-335 in a serum sample obtained from the subject, and comparing the level of miR-335 with a predefined threshold or reference level, the reference level being the level of a reference miRNA in a control sample taken from a healthy subject; The above kit, wherein the subject is diagnosed as having severe TBI (sTBI) if the level of miR-335 in a serum sample obtained from the subject up to 15 days after injury is determined to exceed a predetermined threshold or is increased compared to the level of a reference miRNA in a control sample taken from a healthy subject.

9. The target miRNA is the following miRNA: (a) miR-21, (b) miR-425-5p, miR-21, and miR-502, or any combination thereof; or (c) miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-126*, miR-193a-5p, miR-144* , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * , miR-671-3p, hsa-let-7c-5p, hsa-let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20 b-5p; miR-221-3p; miR-24-3p; miR-27b-3p; miR-29a-3p; miR-29c-3p; miR-30a-5p; miR-107; miR-652-3p, miR-424-5p; miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p and miR-629, or any combination thereof. The kit of claim 8, further comprising at least one miRNA selected from the group consisting of:

10. A composition for diagnosing and / or monitoring traumatic brain injury (TBI) in a subject, comprising a probe specific for a target miRNA, the target miRNA comprising miR-335, the probe comprising a nucleic acid, the nucleic acid comprising a sequence that is at least 90% identical to a sequence that is a complement of a sequence of the target miRNA; the diagnosis and / or monitoring comprises determining a level of miR-335 in a serum sample obtained from the subject, and comparing the level of miR-335 with a predefined threshold or reference level, the reference level being the level of a reference miRNA in a control sample taken from a healthy subject; The composition, wherein the subject is diagnosed as having severe TBI (sTBI) if the level of miR-335 in a serum sample obtained from the subject by 15 days after injury is determined to exceed a predetermined threshold or is increased compared to the level of a reference miRNA in a control sample taken from a healthy subject.

11. A miRNA comprising: (a) miR-21, (b) miR-425-5p, miR-21, and miR-502, or any combination thereof; or (c) miR-505, miR-203, miR-654-3p, miR-655, miR-184, miR-301b, miR-425-5p, miR-502, miR-21, miR-let-7g, miR-126*, miR-193a-5p, miR-144* , miR-190, miR-194, miR-365, miR-590-3p, miR-624, miR-625 * , miR-671-3p, hsa-let-7c-5p, hsa-let-7i-5p, miR-142-3p, miR-148a-3p, miR-15b-5p, miR-16-5p, miR-181a-5p, miR-20a-5p, miR-20 b-5p; miR-221-3p; miR-24-3p; miR-27b-3p; miR-29a-3p; miR-29c-3p; miR-30a-5p; miR-107; miR-652-3p, miR-424-5p; miR-10a, miR-132, miR-223, miR-143, miR-148b, miR-18a, miR-192, miR-429, miR-618, miR-95, miR-130a, miR-152, miR-27b, miR-301, miR-326, miR-345, miR-361, miR-422a, miR-579, miR-642, miR-99a, miR-520D-3p and miR-629, or any combination thereof. The composition of claim 10, further comprising at least one additional probe specific for a target miRNA selected from the group consisting of:

Images