RNA biomarkers for hereditary angioedema

Abstract

To provide methods for identifying a subject having, suspected of having, or being at risk for a disease associated with the contact activation system.SOLUTION: A method for analyzing a sample includes measuring the level of a RNA biomarker set including at least one RNA biomarker disclosed herein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 395,811, filed September 16, 2016, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] background The plasma contact activation system (CPAS) is a pro-inflammatory and pro-coagulant system involving a group of plasma proteases. It is activated by factor XIIa upon exposure to foreign or negatively charged surfaces or by prolylcarboxypeptidase on the surface of endothelial cells (Sainz IM et al., Thromb. Haemost. (2007) 98, 77-83). Inappropriate or unregulated activation of the contact system is involved in various diseases, including hereditary angioedema (HAE).

[0003] HAE is a disease that causes sudden attacks of swelling that can affect multiple parts of the body (such as the face, limbs, genitals, gastrointestinal tract, and upper respiratory tract). Because HAE symptoms often resemble those of allergies or intestinal colic, patients with HAE are often difficult to identify until they exhibit severe or life-threatening symptoms. Early diagnosis would allow for better management of emergency situations involving acute HAE attacks, and early diagnosis would also help manage HAE patients to prevent or attenuate acute HAE episodes (e.g., by having HAE patients avoid exposure to stimuli that may trigger HAE episodes).

[0004] Therefore, it is of great interest to identify biomarkers for HAE and develop reliable diagnostic and prognostic methods to identify subjects with specific types of HAE or at risk of suffering from acute HAE attacks. Such biomarkers would also be beneficial for research into the mechanisms of the disease, which may facilitate the development of effective new treatments for this disease. Summary of the Invention

[0005] Summary of the Disclosure The present disclosure is based on the identification of RNA biomarkers that are differentially present in biological samples obtained from subjects with diseases associated with the contact activation system compared to healthy individuals and / or that are differentially present in biological samples obtained from subjects with different disease states (e.g., seizures versus basal conditions).

[0006] Accordingly, one aspect of the present disclosure provides a method of analyzing a sample, comprising: (i) providing a biological sample (e.g., a serum sample or plasma sample) obtained from a subject (such as a human subject) having, suspected of having, or at risk for having a disease associated with the contact activation system; and (ii) measuring the level of a set of RNA biomarkers comprising at least one RNA biomarker selected from Table 1, wherein when the set of biomarkers consists of one RNA biomarker, the RNA biomarker is not any of hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-19a-3p, and hsa-miR-20a-5p. In some embodiments, the disease associated with the contact activation system is hereditary angioedema (HAE), such as type I HAE or type II HAE.

[0007] In some embodiments, the set of biomarkers consists of 2 to 10 RNA biomarkers selected from Table 1. In some embodiments, the RNA biomarkers are messenger RNAs encoding mitochondrial proteins, which may be mitochondrially encoded cytochrome C oxidase III (MT-CO3) or mitochondrially encoded oxidoreductase core subunit (MT-ND3). In some embodiments, the RNA biomarkers are microRNAs (e.g., hsa-miR-423-3p, hsa-miR-1307-3p, hsa-miR-355-3p, hsa-miR-485-5p, hsa-miR-16-5p, hsa-miR-19a-3p, hsa-miR-20a-5p, hsa-miR-17-5p, hsa-miR-885-5p, hsa-miR-335-3p, hsa-miR-485-5p).

[0008] In some embodiments, the levels of a set of RNA biomarkers may be measured by a process involving polymerase chain reaction and / or nucleic acid hybridization.

[0009] In some embodiments, the method further comprises: identifying the subject as having a disease associated with the contact system if the level of the set of RNA biomarkers in the subject deviates from the level of the same set of RNA biomarkers in a control subject. In some embodiments, the method further comprises: administering to the subject an effective amount of a therapeutic agent for treating the disease (such as a plasma kallikrein (pKal) inhibitor, a bradykinin 2 receptor inhibitor, and / or a C1 esterase inhibitor) if the subject is identified as having the disease. In some embodiments, the pKal inhibitor is an anti-pKal antibody (e.g., lanadelumab) or an inhibitory peptide (e.g., ecallantide). In some examples, the bradykinin 2 receptor inhibitor is an inhibitory peptide (e.g., icatibant). In some examples, the C1 esterase inhibitor is human plasma-derived C1 esterase inhibitor.

[0010] In some embodiments, the subject is a human patient undergoing treatment for a disease, wherein the method further comprises assessing the effectiveness of the treatment based on the levels of the set of RNA biomarkers, wherein a deviation in the level of the set of RNA biomarkers in the subject from that of a control subject indicates the effectiveness of the treatment. In some embodiments, the method further comprises identifying a treatment suitable for the subject based on the levels of the set of RNA biomarkers. In some embodiments, the method further comprises identifying the subject as a candidate for treatment for the disease based on the levels of the set of RNA biomarkers.

[0011] In some embodiments, the set of RNA biomarkers comprises one or more RNA biomarkers selected from the group consisting of hsa-miR-1307-3p, hsa-miR-335-3p, and hsa-miR-485-5p. In some embodiments, the method further comprises assessing the risk of the disease attack in the subject based on the level of the set of RNA biomarkers, wherein a deviation in the level of the set of RNA biomarkers in the subject from that of a control subject indicates a risk of the disease attack.

[0012] The present disclosure provides RNA biomarkers that can identify patients with diseases associated with the contact activation system (e.g., HAE). Measuring the levels of a set of biomarkers can also be useful in the assessment and treatment of such diseases.

[0013] In another aspect, a kit for analyzing a sample from a subject having, suspected of having, or at risk of having a disease associated with the contact system is provided, the kit comprising a first binding substance specific for a first RNA biomarker selected from Table 1 and a second binding substance specific for a second RNA biomarker selected from Table 1, wherein the first and second RNA biomarkers are different. In some examples, the first binding substance is an oligonucleotide complementary (fully or partially) to the first RNA biomarker, and / or the second binding substance is an oligonucleotide complementary (fully or partially) to the second RNA biomarker. The first binding substance and the second binding substance may be immobilized on a support member. In some examples, the first binding substance and / or the second binding substance are conjugated to a label (e.g., a fluorescent label).

[0014] The details of one or more embodiments of the present disclosure are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the following drawings and detailed description of certain embodiments, as well as from the appended claims.

[0015] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the disclosure that can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1 is a graph showing increased transcript levels of mitochondrial cytochrome oxidase (MT-CO3) and mitochondrial NADH dehydrogenase (MT-ND3) during both quiescent HAE (HAE during basal conditions) and seizures. The fold change of each transcript measured by sequencing and by RT-qPCR is shown. [Figure 2]Figure 2 presents graphs showing the levels of selected microRNAs in plasma samples obtained from healthy individuals and HAE patients. A: Fold change in RNA transcripts in a quiescent state (basal HAE) compared to a healthy individual ("HV"). B: Fold change in RNA transcripts in an HAE attack compared to a healthy individual ("HV"). C: Fold change in RNA transcripts in an HAE attack compared to a quiescent state (basal HAE). DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description The contact activation system initiates the intrinsic pathway of blood coagulation and promotes inflammation through the release of the pro-inflammatory peptide bradykinin. Factor XII (FXII), also known as Hageman factor, is a serine protease that plays a role in activating the intrinsic pathway of blood coagulation and the kallikrein-kinin system. FXII is activated by negatively charged surfaces (e.g., polyanionic surfaces, glass, polyphosphate, ellagic acid) to produce its active form, FXIIa. Activated FXIIa has the ability to cleave prekallikrein, generating activated pKal. Activated pKal can then cleave FXII to FXIIa, which in turn generates more pKal, which further activates FXII to FXIIa, creating a positive feedback loop. Activated pKal can also cleave high-molecular-weight kininogen (HMWK) to release bradykinin. In diseases associated with activation of the contact system, such as HAE, increased levels of bradykinin can induce vasodilation and inflammation, leading to edematous HAE attacks. It is desirable to identify novel biomarkers that can be used to identify diseases, such as those mediated by the contact activation system, and to identify subjects who have or are at risk for having such diseases.

[0018] The present disclosure is based, at least in part, on the identification, by transcriptome analysis, of nucleic acids (RNAs; e.g., microRNAs and protein-coding RNA transcripts) that are differentially present in biological samples obtained from subjects with a disease associated with the contact activation system (e.g., basal state or seizures) compared to healthy individuals. Furthermore, several RNAs (e.g., microRNA biomarkers) have been identified as being differentially present in biological samples obtained from subjects with a disease of the contact activation system during a seizure compared to subjects with the disease in a quiescent (basal) state.

[0019] Thus, provided herein is a method for analyzing a biological sample from a subject who has, is suspected of having, or is at risk for a disease associated with the contact activation system (e.g., HAE) by detecting the presence or measuring the level of a set of RNA biomarkers. Such a method may be useful, for example, to identify patients at risk for a disease associated with the contact activation system (e.g., HAE), to select treatment candidates, to monitor disease progression or disease state, to evaluate the effectiveness of treatment for the disease, to determine the course of treatment, to assess whether a subject is at risk for disease onset, to identify whether a disease or disorder is associated with the contact activation system, and / or for research purposes (including, for example, studying the mechanisms of the disease and / or the biological pathways / processes involved in the disease, which may be used to develop new therapies).

[0020] RNA biomarkers of the contact activation system The methods and kits described herein are based, at least in part, on the identification of RNAs that are found to be differentially present in samples from subjects with HAE compared to samples from healthy subjects, and / or to be differentially present in samples at different stages of such disease (e.g., basal state vs. attack). As used herein, the term "RNA biomarker" or "set of RNA biomarkers" refers to an RNA or set of RNAs that are present at different levels in samples from different groups of subjects (e.g., subjects with a disease associated with the contact system vs. healthy subjects (e.g., subjects without the disease), or subjects with the disease but in a quiescent stage vs. subjects in the midst of an attack of the disease). Such biomarkers / sets of biomarkers can be used for both diagnostic / prognostic purposes and non-clinical purposes (e.g., for research purposes).

[0021] In some embodiments, an RNA biomarker may be present at an elevated level in a sample from a subject with a disease associated with the contact activation system (e.g., HAE) compared to the level of the same RNA biomarker in a sample from a healthy subject. In some embodiments, an RNA biomarker may be present at a reduced level in a sample from a subject with a disease associated with the contact activation system (e.g., HAE) compared to the level of the biomarker in a sample from a healthy subject. In yet another example, an RNA biomarker may be present at an elevated level in a sample obtained from a subject during an attack of a disease as described herein compared to a subject during a quiescent state of the disease. Alternatively, an RNA biomarker may be present at a reduced level in a sample obtained from a subject during an attack of a disease as described herein compared to a subject during a quiescent state of the disease.

[0022] In some embodiments, a set of RNA biomarkers comprising one or more biomarkers can be analyzed by the method described herein.When a set of RNA biomarkers comprises two or more biomarkers, all of these biomarkers can be present at elevated or reduced levels in subjects with disease compared to healthy subjects.Alternatively, a set of RNA biomarkers can include at least one biomarker that is elevated in subjects with disease compared to healthy subjects, and at least one biomarker that is reduced in subjects with disease compared to healthy subjects.

[0023] Similarly, a set of RNA biomarkers for distinguishing between subjects undergoing a disease attack and those in a quiescent state of the disease, the set of biomarkers may include multiple biomarkers all of which are elevated or decreased in a first disease stage (e.g., a stroke) relative to a second disease stage (e.g., a quiescent state). Alternatively, the set of biomarkers may include at least one biomarker that is elevated in the first disease stage relative to the second disease stage and at least one biomarker that is decreased in the first disease stage relative to the second disease stage.

[0024] Table 1 below provides RNA biomarkers that can be assessed by the methods described herein for assessing a subject or a biological sample from a subject for a disease associated with the contact activation system.

[0025] [Table 1]

[0026] In some embodiments, the set of biomarkers measured and analyzed by any of the methods described herein includes at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more) RNAs selected from Table 1. When the set of biomarkers includes a single RNA biomarker, the RNA biomarker may be none of hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-19a-3p, and hsa-miR-20a-5p. In some examples, the set of RNA biomarkers measured and analyzed by the methods described herein does not include any combination of hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-19a-3p, and hsa-miR-20a-5p.

[0027] As described in Example 1, several RNAs encoding proteins involved in similar cellular processes and pathways were unexpectedly found to be differentially present in samples from subjects with HAE compared to healthy subjects. This data indicates that the RNAs shown in Table 1 may play a role in or be affected by diseases associated with the contact system.

[0028] The RNA biomarkers described herein may be characterized as "involved in" or "associated with" a particular pathway or activity. As used herein, the term "involved in" or "associated with" refers to RNA that contributes to a pathway. For example, an RNA (such as a protein-coding RNA or microRNA) that is involved in or associated with a pathway may perform a function within the pathway (e.g., regulate the expression of another nucleic acid or the activity of a protein) or may encode a molecule (e.g., a protein) that performs a function within the pathway or cellular process.

[0029] In some embodiments, the set of RNA biomarkers comprises one or more RNAs encoding mitochondrial proteins, such as those listed in Table 1.

[0030] In some embodiments, the set of RNA biomarkers comprises one or more microRNAs (eg, one or more microRNAs selected from Table 1).

[0031] Also, as described in Example 1, some RNAs have been found to be differentially present in samples from subjects with HAE during an HAE attack compared to subjects with HAE during a resting (basal) state. In some embodiments, the set of biomarkers includes one or more microRNAs (e.g., one or more microRNAs selected from Table 1).

[0032] The utility of RNA biomarkers One aspect of the present disclosure relates to a method for analyzing a sample obtained from a subject (e.g., a human patient) having, suspected of having, or at risk for a disease associated with the contact activation system by measuring the levels of a set of biomarkers as described herein in the sample. The results obtained from such an assay may be useful for diagnostic and / or prognostic applications, as well as other non-clinical applications (such as research applications).

[0033] (i) Analysis of biological samples The methods described herein involve providing a biological sample obtained from a subject. As used herein, "biological sample" refers to a composition containing tissue (e.g., blood, plasma, or protein) from a subject. Samples include both the initial, unprocessed sample collected from a subject as well as subsequently processed (e.g., partially purified or preserved) forms. Exemplary samples include blood, plasma, tears, or mucus. In some embodiments, the sample is a bodily fluid sample, such as a serum sample or plasma sample. In some embodiments, multiple (e.g., at least two, three, four, five, or more) biological samples may be collected from a subject over time or at specific time intervals, for example, to assess disease progression or the effectiveness of a treatment.

[0034] A biological sample can be obtained from a subject using any means known in the art. In some embodiments, a sample is obtained from a subject by collecting a sample (e.g., a blood sample) in an evacuated collection tube (e.g., a vacuum collection tube). In some embodiments, the evacuated collection tube contains one or more protease inhibitors, for example, to reduce or prevent activation of the ex vivo contact system during sample collection. Such protease inhibitors may be included in a liquid formulation. In some embodiments, the protease inhibitors include at least one serine protease inhibitor and at least one cysteine protease inhibitor. Such evacuated collection tubes are known in the art. See, for example, PCT Application No. US2016 / 046681. Optionally, the evacuated collection tube may further contain one or more anticoagulants.

[0035] The terms "patient," "subject," or "individual" may be used interchangeably and refer to a subject in need of analysis as described herein. In some embodiments, the subject is a human or a non-human mammal. In some embodiments, the subject is suspected of or at risk for a disease or disorder associated with the contact activation system (e.g., HAE). Such a subject may exhibit one or more symptoms associated with the disease. Alternatively, or in addition, such a subject may possess one or more risk factors for the disease (e.g., a genetic factor associated with the disease (e.g., a genetic defect in CI-INH)).

[0036] Alternatively, the subject who needs the analysis described herein may be a patient with the disease. Such a subject may currently be suffering from an attack of the disease, or may have previously suffered from the disease (e.g., currently in a quiescent state). In some cases, the subject is a human patient who may be undergoing treatment for the disease (e.g., treatment with C1 esterase inhibitor (C1-INH), plasma kallikrein inhibitor, or bradykinin inhibitor). In other cases, such a human patient may not be undergoing such treatment.

[0037] Examples of diseases associated with the contact activation system include, but are not limited to, kallikrein-mediated disorders, e.g., bradykinin-mediated disorders (such as hereditary angioedema (HAE)), non-histamine-dependent idiopathic angioedema, rheumatoid arthritis, Crohn's disease, lupus, Alzheimer's disease, septic shock, burns, cerebral ischemia / reperfusion injury, cerebral edema, diabetic retinopathy, diabetic nephropathy, macular edema, vasculitis, arterial or venous thrombosis, thrombosis associated with ventricular assist devices or stents, heparin-induced thrombocytopenia with thrombosis, thromboembolic disease, and coronary heart disease with unstable angina, edema, eye disease, gout, intestinal bowel disease, and the like. disease), oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis - degenerative spinal disease, postoperative ileus, aortic aneurysm, osteoarthritis, hereditary angioedema, pulmonary embolism, stroke, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, autoimmune diseases, inflammatory diseases, cardiovascular diseases, neurological diseases, diseases associated with protein misfolding, diseases associated with angiogenesis, hypertensive and diabetic nephropathy, allergic and respiratory diseases (e.g., anaphylaxis, asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, persistent rhinitis), and tissue injury (e.g., burns or chemical injuries).

[0038] In some embodiments, the disease or condition associated with the contact activation system is hereditary angioedema (HAE).Hereditary angioedema (HAE) is also known as "Quincke's edema," C1 esterase inhibitor deficiency, C1 inhibitor deficiency, and hereditary angioneurotic edema (HANE).HAE is characterized by recurrent episodes of severe swelling (angioedema), which can affect, for example, the extremities, face, genitals, gastrointestinal tract, and airway.Symptoms of HAE include, for example, swelling in the arms, legs, lips, eyes, tongue, and / or throat; airway obstruction, which may be accompanied by throat swelling and sudden hoarseness; repeated episodes of abdominal cramps without apparent cause; and / or intestinal swelling, which may become severe and lead to abdominal cramps, vomiting, dehydration, diarrhea, pain, and / or shock.Approximately one-third of individuals with HAE develop a non-itchy rash called erythema marginatum during an attack.

[0039] Airway swelling can be life-threatening and, in some patients, leads to death. The mortality rate is estimated at 15–33%. HAE results in approximately 15,000–30,000 emergency room visits per year. Trauma or stress (e.g., dental procedures, illness (e.g., viral illnesses such as colds and influenza), menstruation, and surgery) can trigger attacks of angioedema. To prevent acute attacks of HAE, patients can try to avoid specific stimuli that have previously triggered attacks. However, attacks often occur without a known trigger. Typically, HAE symptoms first appear in childhood and worsen during adolescence. On average, untreated individuals experience attacks every 1–2 weeks, with most episodes lasting approximately 3–4 days (ghr.nlm.nih.gov / condition / hereditary-angioedema). The frequency and duration of attacks vary widely among people with hereditary angioedema, even within the same family.

[0040] There are three types of HAE, known as types I, II, and III. It is estimated that 1 in 50,000 people suffer from HAE, with type I accounting for approximately 85% of cases, type II accounting for approximately 15% of cases, and type III being extremely rare. Type III is the most recently described form and was initially thought to occur only in women, although families with affected men have been identified.

[0041] HAE is inherited in an autosomal dominant pattern, so affected individuals may inherit the mutation from one affected parent. De novo mutations in the gene can also occur, so HAE can occur in people with no family history of the disorder. It is estimated that 20-25% of cases arise from de novo spontaneous mutations.

[0042] Mutations in the SERPING1 gene cause type I and type II hereditary angioedema. The SERPING1 gene provides instructions for producing the C1 inhibitor protein, which is important for suppressing inflammation. C1 inhibitor blocks the activity of certain proteins that promote inflammation. Mutations that cause type I hereditary angioedema reduce levels of C1 inhibitor in the blood. In contrast, mutations that cause type II hereditary angioedema result in the production of abnormally functioning C1 inhibitor. When functional C1 inhibitor levels are inadequate, excessive amounts of bradykinin are produced. Bradykinin promotes inflammation by increasing fluid leakage through blood vessel walls into body tissues. Excessive accumulation of fluid in body tissues causes the swelling episodes seen in individuals with type I and type II hereditary angioedema.

[0043] Mutations in the F12 gene are associated with some cases of type III hereditary angioedema. The F12 gene provides instructions for producing clotting factor XII. In addition to playing a critical role in blood clotting (clotting), factor XII is also an important stimulator of inflammation and is involved in the production of bradykinin. Specific mutations in the F12 gene result in the production of increased activity of factor XII. As a result, more bradykinin is produced, increasing the leakiness of blood vessel walls, which leads to episodes of swelling. The cause of other cases of type III hereditary angioedema remains unknown. In these cases, mutations in one or more yet-to-be-identified genes may be responsible for the disorder.

[0044] Although HAE can manifest similarly to other forms of angioedema caused by allergies or other medical conditions, HAE differs significantly in terms of cause and treatment. When HAE is misdiagnosed as an allergy, it is most commonly treated with antihistamines, steroids, and / or epinephrine, which are usually ineffective for HAE (although epinephrine can be used for life-threatening reactions). Misdiagnosis has also led to unnecessary exploratory laparotomy in patients with abdominal swelling, and in some HAE patients, abdominal pain has been incorrectly diagnosed as being psychogenic.

[0045] C1 inhibitor therapy as well as other therapies for HAE are described in Kaplan, AP, J Allergy Clin Immunol, 2010, 126(5):918-925.

[0046] Acute treatment of an HAE attack is administered to halt the progression of edema as quickly as possible. Intravenously administered C1 inhibitor concentrates derived from donor blood are one type of acute treatment. However, this treatment is not available in many countries. In emergency situations where C1 inhibitor concentrates are unavailable, fresh frozen plasma (FFP) can be used as an alternative because it also contains C1 inhibitor.

[0047] Purified C1 inhibitor derived from human blood has been used in Europe since 1979. Several C1 inhibitor treatments are currently available in the United States, and two C1 inhibitor products are currently available in Canada. Verinert (CSL Behring), which is pasteurized, was approved by the FDA in 2009 for acute attacks. Synrise®, which is nanofiltered, was approved by the FDA in 2008 for prophylaxis. Rutin / Ruconest (Pharming) is an investigational recombinant C1 inhibitor that does not carry the risk of infectious disease transmission due to human bloodborne pathogens.

[0048] Treatment for acute HAE attacks may also include medication and / or intravenous fluids for pain relief.

[0049] Other treatments may stimulate C1 inhibitor synthesis or reduce C1 inhibitor consumption. Androgenic drugs such as danazol can reduce the frequency and severity of attacks by stimulating C1 inhibitor production.

[0050] Helicobacter pylori can cause abdominal attacks. Antibiotics to treat H. pylori may reduce abdominal attacks.

[0051] Newer treatments attack the contact cascade. Ecallantide (KALBITOR®) inhibits plasma kallikrein and is approved in the United States. Icatibant (Firasil®, Shire) inhibits the bradykinin B2 receptor and is approved in Europe and the United States.

[0052] Diagnosis of HAE can rely, for example, on family medical history and / or blood tests. Laboratory findings associated with types I, II, and III HAE are described, for example, in Kaplan, AP, J Allergy Clin Immunol, 2010, 126(5):918-925. In type I HAE, C1 inhibitor levels are decreased, as are C4 levels, but C1q levels are normal. In type II HAE, C1 inhibitor levels are normal or increased, but C1 inhibitor function is abnormal. C4 levels are decreased and C1q levels are normal. In type III, C1 inhibitor, C4, and C1q levels may all be normal. The present disclosure is based, at least in part, on the identification of RNAs (Table 1) that have different levels in samples from HAE patients compared to healthy individuals. Measuring the levels of a set of these RNA biomarkers can be used to identify whether a subject has a disease (such as HAE). In some embodiments, the methods may be utilized to determine whether a patient has had an HAE attack or is experiencing an HAE attack.

[0053] Symptoms of HAE can be evaluated, for example, by using a questionnaire (for example, a questionnaire that is answered by the patient, clinician, or family). Such questionnaires are known in the art, and include, for example, visual analog scales. For example, see McMillan, CV et al. Patient. 2012;5(2):113-26.

[0054] A biological sample described herein may be subjected to analysis by measuring the level of a set of RNA biomarkers as described herein in the biological sample. The level (e.g., amount) of a biomarker disclosed herein or a change in the level of a biomarker may be assessed using an assay described herein and / or an assay known in the art. One or more of the biomarkers described herein may be analyzed using conventional methods (e.g., PCR, nucleic acid hybridization, or microarray). In some embodiments, the level of a biomarker is assessed or measured by directly detecting the RNA biomarker in the biological sample. In some embodiments, the RNA biomarker may be amplified, for example, by PCR, prior to detection.

[0055] The type of detection assay used to detect and / or quantify biomarkers of the contact activation system (such as those presented herein) will depend on the particular context in which the assay will be used (e.g., clinical or research use), the type and number of biomarkers to be detected, and the type and number of patient samples to be run in parallel, to name a few parameters.

[0056] In some embodiments, biomarkers are measured directly in a biological sample, for example, by contacting the sample with a binding agent that selectively binds to one or more RNA biomarkers (e.g., any one of the biomarkers presented in Table 1). In some embodiments, the biomarkers (RNA, or cDNA corresponding to the RNA biomarkers) are detected using a hybridization method (such as contacting the sample with a nucleic acid probe that specifically binds to the biomarker, such as Southern or Northern blotting).

[0057] In some embodiments, the binding agent is an oligonucleotide complementary to an RNA biomarker. Oligonucleotides for use in the methods described herein are oligonucleotides (single-stranded DNA or RNA molecules) that are complementary (partially or fully) to a region of an RNA biomarker (or a cDNA corresponding to the RNA biomarker).

[0058] As used herein, "complementary" refers to nucleic acid base complementarity, as commonly known in the art. For example, adenine is complementary to thymine (in DNA) or uracil in RNA, and guanine is complementary to cytosine. As used herein, "sequence complementarity" or "complementary nucleic acid sequences" means that when two nucleic acid molecules are aligned antiparallel to each other, the nucleotide bases at each position, or at most positions in the sequence, are complementary, and the two nucleic acid molecules can hybridize to form a duplex under appropriate conditions (e.g., hybridization temperature). As known in the art, 100% sequence complementarity is not required for two nucleic acid molecules to hybridize to form a duplex. The sequence complementarity between an oligonucleotide and an RNA biomarker (or a cDNA corresponding to the RNA biomarker) may be at least 80% complementary to the corresponding region in the RNA biomarker. In some embodiments, the oligonucleotides comprise fragments that are at least 80% (e.g., 85%, 90%, 95%, 98% or 100%) complementary to a portion of the RNA biomarker (or cDNA corresponding to the RNA biomarker). In some cases, the oligonucleotides comprise fragments that are fully complementary (100% complementary) to a portion of the RNA biomarker (or cDNA corresponding to the RNA biomarker). Such oligonucleotides may be used to distinguish RNA biomarkers from substantially similar nucleic acids (e.g., nucleic acids with 1, 2 or 3 base differences from the target nucleic acid).

[0059] An oligonucleotide may contain 100 or fewer nucleotides (e.g., 80 nt, 60 nt, 50 nt, or 30 nt or fewer). In some embodiments, an oligonucleotide may be 8 to 50 nucleotides in length (e.g., 8 to 40, 8 to 30, 10 to 30, 15 to 30, or 15 to 20 nucleotides in length). In some examples, an oligonucleotide may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some examples, the entire oligonucleotide molecule is complementary to a portion of an RNA biomarker. In other examples, a fragment of an oligonucleotide is complementary to a portion of an RNA biomarker. For example, an oligonucleotide may include a linker (e.g., a polyA linker or a polyT linker) for attachment to a support member. Alternatively, or in addition, a detection probe may include such a linker for conjugation to a label. The fragment of the oligonucleotide that is complementary to a portion of the RNA biomarker may be located at the 5' end of the oligonucleotide, the 3' end of the oligonucleotide, or in the middle of the oligonucleotide. In some embodiments, the fragment of the oligonucleotide that is complementary to the RNA biomarker may be at least 10 nucleotides in length (e.g., at least 12, 15, 18, 20, or 25 nucleotides in length).

[0060] In some embodiments, the oligonucleotide contains one or more modified nucleotides, including, for example, nucleotides modified with a 2'-O-methoxyl group, a 2'-O-methoxylethyl group, and / or a phosphorothioate group. In some examples, the oligonucleotide contains one or more locked nucleic acids (LNAs). LNAs, often referred to as inaccessible RNAs, are modified RNA nucleotides in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and the 4' carbon. This bridge "locks" the ribose in the 3'-endo (North) conformation, which is often found in A-form duplexes. LNA nucleotides can be used in both DNA and RNA probes. In some examples, 50% or less (e.g., 40%, 30%, 20%, or 10%) of the nucleotides in the probe are LNAs. In some examples, the oligonucleotide may contain 10, 8, 6, 5, 4, 3, 2, or 1 LNA.

[0061] Oligonucleotides may be designed based on the sequence of the RNA biomarker one wishes to detect and may be prepared by conventional methods (eg, chemical synthesis or in vitro transcription).

[0062] The oligonucleotides described herein may be immobilized on a support member by conventional methods. As used herein, "immobilized" means covalently or non-covalently attached, bound, or fixed to prevent dissociation or loss of the oligonucleotide, but does not require absolute immobility for either the oligonucleotide or the support member. The support member may be a solid or semi-solid member having a surface that can be used to specifically attach, bind, or capture a nucleotide probe (e.g., an oligonucleotide of the present disclosure) so that the nucleotide probe is immobilized on the support member.

[0063] Support members of the present disclosure may be made from one or more suitable materials, such as plastics or synthetic polymers (e.g., polyethylene, polypropylene, polystyrene, polyamide, polyurethane, phenolic polymers, or nitrocellulose), naturally occurring polymers (e.g., latex rubber, polysaccharides, polypeptides), composite materials, ceramics, silica or silica-based materials, carbon, metals or metal compounds (e.g., those containing gold, silver, steel, aluminum, or copper), inorganic glass, silica, and various other suitable materials. Non-limiting examples of potentially suitable shapes include beads (e.g., magnetic beads), tubes (e.g., nanotubes), plates, disks, dipsticks, chips, microchips, cover slips, and the like.

[0064] The surface of the support member of the present disclosure may comprise any molecule, other chemical / biological entity, or solid support modification disposed on the solid support that can be used to specifically attach, bind, or capture nucleic acid molecules (e.g., oligonucleotides complementary to RNA biomarkers). Surface compositions that can be used to immobilize nucleic acid molecules can be readily found in the art. For example, the surface may comprise a complementary nucleic acid or a nucleic acid-binding protein that can be attached to the surface by conventional methods. Thus, the link between the nucleic acid to be immobilized (e.g., an oligonucleotide of the present disclosure) and the surface may comprise one or more chemical or physical bonds (e.g., nonspecific attachment via van der Waals forces, hydrogen bonds, electrostatic interactions, hydrophobic / hydrophilic interactions, etc.) and / or a chemical linker that effects such bond(s). Alternatively, the surface of the support member may comprise reactive functional groups capable of forming covalent bonds with the nucleic acid molecules to be immobilized. In some embodiments, the functional groups are chemical functional groups. That is, the binding surface may be derivatized to present chemical functional groups on the binding surface that can react with chemical functional groups on the nucleic acid to be captured to effect attachment. Examples of potentially useful functional groups for attachment include, but are not limited to, amino groups, carboxyl groups, epoxide groups, maleimide groups, oxo groups, and thiol groups. The functional groups can be attached directly or through the use of linkers, a combination of which is sometimes referred to herein as "crosslinkers." Crosslinkers for attaching nucleic acid molecules to support members are known in the art. For example, homobifunctional crosslinkers or heterobifunctional crosslinkers are well known (see, for example, the technical section on crosslinkers in the 1994 Pierce Chemical Company catalog (pages 155-200) or "Bioconjugate Techniques" by Greg T. Hermanson (Academic Press, 1996)).Non-limiting examples of crosslinkers include alkyl groups (including substituted alkyl groups and alkyl groups containing heteroatom moieties), esters, amides, amines, epoxy groups, and ethylene glycol and derivatives. The linker may also be a sulfone group to form sulfonamides. In some embodiments, the functional group is a photoactivatable functional group. That is, the functional group can be activated with light to attach the capture component to the capture object surface. One example is PhotoLink™ technology available from SurModics, Inc. (Eden Prairie, MN).

[0065] It should be understood that the examples provided herein with respect to support members and surface compositions are not intended to be limiting: any support member known in the art to be suitable for immobilizing nucleic acid molecules may be used in accordance with the methods and kits described herein.

[0066] In some embodiments, a binding agent (e.g., an oligonucleotide complementary to an RNA biomarker) may be conjugated to a label. As used herein, "conjugated" means that the label is covalently or non-covalently attached to the binding agent. The labeling agent may be any molecule, particle, etc. that facilitates detection, directly or indirectly, using a suitable detection technique. For direct detection, the labeling agent may be a molecule or moiety (e.g., a fluorescent label or fluorescent dye) capable of emitting a signal that can be directly interrogated and / or detected. In a non-limiting example of indirect detection, the label may be a molecule or moiety (e.g., an enzyme) capable of converting a substrate into a product capable of emitting a detectable signal. For example, the label may be luciferase, which converts luciferin to oxyluciferin, emitting detectable light. In another non-limiting example of indirect detection, the label is a binding ligand for a molecule or moiety (e.g., an enzyme) capable of converting a substrate, where the converted substrate emits a detectable signal.

[0067] In some embodiments, the label is a fluorescent label. Examples include fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, and fluorescent metal ions. 152 Eu or other metals from the lanthanide series), CYE dyes, and fluorescent proteins (such as eGFP, eYFP, eCFP, mKate2, mCherry, mPlum, mGrape2, mRaspberry, mGrapel, mStrawberry, mTangerine, mBanana, and mHoneydrew).

[0068] Other exemplary labels include biotin, phosphorescent labels, chemiluminescent or bioluminescent labels (such as luminal, isoluminol, theromatic acridinium esters, imidazoles, acridinium salts, oxalate esters and dioxetanes), radioisotopes ( 3 H, 125 I, 32 P, 35 S, 14 C. 51 Cr, 36 Cl, 57 Co, 58 Co, 59 Fe and 75 Se), metals, metal chelates, or metal cations (e.g., 99m Tc, 123 I, 111 In, 131 I, 97 Ru, 67 Cu, 67 Ga and 68 Examples of suitable cations include, but are not limited to, metal cations such as Ga. Other examples include chromophores and enzymes (e.g., malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase).

[0069] In some embodiments, biomarkers are measured using polymerase chain reaction. In some embodiments, RNA extracted from biological samples is subjected to polymerase chain reaction. In some embodiments, cDNA corresponding to RNA of biological samples is used in polymerase chain reaction. Nucleic acid can be double-stranded or single-stranded. Various methods using polymerase chain reaction are known in the art, including reverse transcription RNA, quantitative PCR and multiplex PCR.

[0070] Generally, the polymerase chain reaction relies on a repeated series of steps involving annealing and extension / elongation. A pair of oligonucleotides (e.g., primers) has sufficient complementarity to a desired nucleic acid (e.g., a biomarker) so that, if the desired nucleic acid is present in a sample, the primers will anneal (hybridize) to the desired nucleic acid under appropriate annealing conditions (e.g., annealing temperature). The reaction then proceeds to the extension / elongation step, where a polymerase synthesizes a complementary nucleic acid strand. The double-stranded nucleic acid product is denatured, initiating another reaction cycle.

[0071] In some embodiments, the polymerase chain reaction involves a different temperature setting for each step of the cycle, hi some embodiments, the polymerase chain reaction is carried out at a single temperature (e.g., an isothermal reaction).

[0072] The polymerase chain reaction as described above allows for selective amplification of nucleic acids based on the selection of oligonucleotide primers. In some embodiments, nucleic acids corresponding to a set of RNA biomarkers are selectively amplified and then detected using any method known in the art. In some embodiments, the amplification products may be detected using hybridization methods. Alternatively, or in addition, the amplification products may be detected by one or more modifications introduced during the polymerase chain reaction. In some embodiments, a dye, fluorophore, or other indicator substance is intercalated into the nucleic acid during the polymerase chain reaction. In some embodiments, one or more of the nucleic acid primers may include a tag (e.g., a nucleic acid tag) that can be detected.

[0073] In some embodiments, RNA biomarkers are measured using methods involving hybridization, e.g., with oligonucleotides that are complementary (partially or fully) to the RNA biomarker. In some embodiments, hybridization methods are performed on biological samples to detect RNA biomarkers, or on compositions (e.g., PCR reactions) containing amplification products (e.g., amplified RNA biomarkers or cDNA corresponding to the RNA biomarkers). In some embodiments, hybridization involves contacting the sample with oligonucleotides (e.g., probes) that specifically bind to the biomarkers. Examples of hybridization methods include, but are not limited to, Southern blotting, Northern blotting, and microarrays. As described herein, in some embodiments, the oligonucleotides may be conjugated to labels for detection and / or immobilized on a support member.

[0074] Selection of appropriate buffers, polymerases, and hybridization conditions will be apparent to one of skill in the art, and optimization may involve routine experimentation.

[0075] In some embodiments, biomarkers are measured by nucleic acid sequencing.For example, the abundance of specific nucleic acid sequence (for example, biomarker) in sample can be compared with the abundance of the same nucleic acid sequence in another sequence (for example, whole transcriptome sequencing, RNASeq).This comparison can be carried out, for example, by comparing the read number of specific sequence between samples.

[0076] Generally, nucleic acid sequencing may be performed using any method known in the art, and the selection of an appropriate method will be apparent to one skilled in the art. For example, nucleic acids may be sequenced using Sanger sequencing or high-throughput sequencing methods.

[0077] In some embodiments, a biological sample is subjected to an RNA extraction process to isolate RNA present in the sample (e.g., before measuring biomarkers). Such RNA extraction processes are well known in the art. Examples of RNA extraction processes include commercially available kits and phenol-chloroform extraction. In some embodiments, RNA extracted from a biological sample is subjected to in vitro translation, thereby producing proteins encoded by the RNA, including proteins encoded by RNA biomarkers, if present. In some embodiments, a biological sample or RNA extracted from a biological sample is subjected to reverse transcription, thereby producing cDNA corresponding to the RNA, including RNA biomarkers, if present.

[0078] In some embodiments, biomarkers are measured using immunoassays. In some embodiments, a biological sample is contacted with a binding substance (such as a nucleic acid-binding protein) that binds to an RNA biomarker. An immunoassay can then be performed to detect the binding substance as an indirect measure of the amount of biomarker in the sample. Examples of immunoassays include, but are not limited to, immunoblotting assays (Western blots), enzyme-linked immunosorbent assays (ELISAs) (e.g., sandwich ELISAs), radioimmunoassays, electrochemiluminescence-based detection assays, magnetic immunoassays, lateral flow assays, and related techniques. Additional suitable immunoassays for detecting the biomarkers presented herein, for example, by detecting binding substances that bind to the biomarkers, will be apparent to those skilled in the art. However, it will be apparent to those skilled in the art that the present disclosure is not limited to immunoassays, and that detection assays that rely on chromogenic substrates may also be useful for detecting and / or quantifying contact-based biomarkers as presented herein.

[0079] ELISAs are known in the art (see, e.g., Crowther, John R (2009), "The ELISA Guidebook," 2nd ed., Humana Press and Lequin R (2005), "Enzyme immunoassay (EIA) / enzyme-linked immunosorbent assay (ELISA)," Clin. Chem. 51(12):2415-8), and exemplary ELISAs are described herein. Kits for performing ELISAs are also known in the art and commercially available (see, e.g., ELISA kits from Life Technologies and BD Biosciences).

[0080] The immunoassays described herein may be in the format of a sandwich ELISA, in which a first binding substance that specifically binds to a binding substance (e.g., a nucleic acid-binding protein that binds to an RNA biomarker) is immobilized on a support member. The support member may then be incubated with a biological sample as described herein for a suitable time under conditions that allow the formation of a complex between the binding substance and the biomarker in the sample. Such complexes may then be detected using a detection substance that binds to the biomarker, the binding substance-biomarker complex, or the binding substance. The detection substance may be conjugated to a label as described herein that can directly or indirectly emit a signal. The intensity of the signal represents the level of the RNA biomarker in the sample. In some embodiments, the detection substance is detected, and its level represents the level of the RNA biomarker in the sample.

[0081] Any binding substance that specifically binds to a desired nucleic acid may be used in the methods and kits described herein to measure the level of an RNA biomarker in a biological sample. In some embodiments, the binding substance is an antibody that specifically binds to a desired nucleic acid. In some embodiments, a sample may be contacted simultaneously or sequentially with two or more binding substances that bind to different RNAs (e.g., multiplex analysis; e.g., SOMAScan™ assay (SOMALogic)). The biological sample is contacted with the binding substance under appropriate conditions. Generally, the term "contacting" refers to bringing the binding substance into contact with the biological sample or agent for a suitable time sufficient to form a complex between the binding substance and RNA (if present) in the sample. In some embodiments, contacting is performed by capillary action, whereby the biological sample or agent crosses the surface of a support membrane.

[0082] In some embodiments, immunoassays may be performed on low-throughput platforms, including simple immunoassay formats. For example, low-throughput platforms may be used to measure the presence and amount of nucleic acids in a biological sample (e.g., biological tissue, tissue extract) for diagnostic methods, for monitoring disease and / or treatment progression, and / or for predicting whether a disease or disorder may benefit from a particular treatment.

[0083] In some embodiments, it may be necessary to immobilize the binding substance on a support member. The method for immobilizing the binding substance will depend on factors such as the nature of the binding substance and the material of the support member, and may require a specific buffer. Such methods will be apparent to those skilled in the art. For example, a set of biomarkers in a biological sample as described herein may be measured using any of the kits and / or detection devices also described herein.

[0084] As used herein, the terms "measuring" or "measuring" or "detecting" or "detection" mean assessing the presence, absence, quantity or amount (which may be an effective amount) of a substance in a sample (including deriving a qualitative or quantitative concentration level of such a substance), or assessing a value or classification of interest.

[0085] The assay (e.g., Southern blot or Northern blot assay) may further involve the use of commercially available quantitative imaging systems (e.g., LICOR imaging technology) (see, e.g., LI-COR Biosciences' Odyssey® CLx Infrared Imaging System). In some embodiments, an electrochemiluminescence detection assay, or an assay relying on a combination of electrochemiluminescence and patterned array technology, is used (e.g., Meso Scale Discovery's (MSD) ECL or MULTI-ARRAY technology assay).

[0086] In any of the methods described herein, the level of RNA of the set of biomarkers may be compared to the level of RNA in a control or reference sample.

[0087] The methods and kits described herein (also involving any of the sets of RNA biomarkers described herein) may be applied to the assessment of diseases associated with the contact activation system (such as those described herein).

[0088] (ii) Diagnostic and / or prognostic uses The levels of the RNAs presented in Table 1 detected in samples from subjects can be used as reliable biomarkers for diagnosing diseases associated with the contact activation system (e.g., HAE), monitoring the progression of such diseases, assessing the effectiveness of treatments for the diseases, identifying patients suitable for particular treatments, and / or predicting attacks of the disease in subjects.

[0089] Thus, described herein are diagnostic and prognostic methods for diseases associated with the contact activation system based on the levels of a set of biomarkers in a biological sample obtained from a subject. In some embodiments, the levels of the biomarkers measured using any of the methods described herein can be used to assess whether a subject (e.g., a human patient) from whom the biological sample was obtained has or is at risk for a disease associated with the contact activation system, such as a plasma kallikrein-associated disease (e.g., HAE) or an autoimmune disease (such as RA, UC, and Crohn's disease).

[0090] In some embodiments, the level of the biomarker may then be compared to a reference or control sample to determine a value indicative of the amount of RNA in the sample. In some embodiments, the value for the biomarker is obtained by comparing the level of the RNA in the sample to the level of another RNA (e.g., an internal control or internal standard) in the sample. Such a biomarker value may be normalized to the internal control or internal standard. The value of the biomarker may be compared to a reference value to determine whether a subject has or is at risk for a disease associated with the contact activation system. The reference value may represent the level of the corresponding biomarker in a subject (e.g., a human subject) who does not have the target disease. In some embodiments, if the level or value of the biomarker is higher than the reference level or value, the subject may be identified as having or at risk for a disease associated with the contact activation system. In some embodiments, if the level or value of the biomarker is lower than the reference level or value, the subject may be identified as having or at risk for a disease associated with the contact activation system.

[0091] In some embodiments, the level of the biomarker may be compared to a predetermined threshold for that RNA biomarker, deviation from which may indicate that the subject has a disease associated with the contact system. The predetermined threshold may represent a value of the biomarker that distinguishes between the level of the biomarker in patients with the target disease and the level of the biomarker in patients without the target disease.

[0092] In some embodiments, the set of biomarkers includes two or more RNA biomarkers that are differentially present (elevated and / or decreased) in subjects with or at risk of having a disease compared to healthy subjects. In some examples, the set of biomarkers includes at least one RNA biomarker that is elevated in levels in subjects with or at risk of having a disease and at least one RNA biomarker that is decreased in levels in subjects with or at risk of having a disease. Examples of "elevated" and "decreased" RNA biomarkers are listed in Table 1. In some embodiments, the set of biomarkers includes two or more RNAs, each of which an elevated level indicates that the subject has or is at risk of having a disease. In some embodiments, the set of biomarkers includes two or more RNAs, each of which a decreased level indicates that the subject has or is at risk of having a disease.

[0093] In some embodiments, control sample or reference sample is a biological sample obtained from a healthy individual.In some embodiments, control sample or reference sample comprises a known amount of RNA to be evaluated.In some embodiments, control sample or reference sample is a biological sample obtained from a control subject.

[0094] As used herein, a control subject may be a healthy subject or individual who is known to be free of the target disease (e.g., a disease associated with a contact system) or has no history of the disease at the time the level of RNA(s) is measured. The term "control subject" encompasses an individual subject or a group of subjects with similar characteristics (e.g., a group of healthy subjects with specific characteristics (e.g., age, sex, ethnicity, etc.) matching those of a candidate subject). In some embodiments, the level of a biomarker in a control subject is used to establish a reference value (e.g., the average level of a biomarker in control subjects encompassing a group of subjects) to which the level of the biomarker in a candidate or test subject may be compared.

[0095] Control level refers to the level of a set of RNA biomarkers as described herein in control subjects.Control level can be a predetermined level or threshold.This predetermined level can represent the level of that RNA in a group of subjects that do not have or are not at risk of having target disease (for example, the average level in a group of healthy subjects).It can also represent the level of that RNA in a group of subjects that have target disease.

[0096] The predetermined level can take various forms. For example, it can be a single cutoff value, such as a median or mean value. In some embodiments, such a predetermined level can be established based on comparative groups (such as a comparative group in which one defined group is known to have the target disease and another defined group is known to not have the target disease). Alternatively, the predetermined level can be a range (e.g., a range representing the level of that RNA in a control population).

[0097] The control level as described herein can be determined by conventional techniques.In some cases, the control level can be obtained by carrying out conventional methods (for example, the same assay as that for obtaining the level of the RNA in the test sample as described herein) on the control sample as described herein.In other cases, the level of the RNA can be obtained from the members of a control population, and the result can be analyzed, for example, by a computer program, to obtain the control level (predetermined level) that represents the level of the RNA in the control population.

[0098] By comparing the level of biomarkers in a sample obtained from a candidate subject with a reference value as described herein, it can be determined whether the candidate subject has or is at risk for a disease associated with the contact system (e.g., HAE). For example, if the level of biomarker(s) in the candidate subject's sample deviates from the reference value (e.g., is increased compared to the reference value), the candidate subject may be identified as having or at risk for the disease. If the reference value represents a range of values for the level of biomarkers in a population of subjects with the target disease, a value of biomarker in the candidate sample within that range indicates that the candidate subject has or is at risk for the target disease.

[0099] As used herein, an "elevated level" or "level above a reference value" means that the level of a biomarker is higher than a reference value (e.g., a predetermined threshold for the level of the biomarker in a control sample). Control levels are described in detail herein. Elevated levels of a biomarker include levels of the biomarker that are, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500% or more above the reference value. In some embodiments, the level of the biomarker in the test sample is at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 15 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 50 fold, 100 fold, 150 fold, 200 fold, 300 fold, 400 fold, 500 fold, 1000 fold, or 10,000 fold or more higher than the level of the biomarker in the reference sample.

[0100] As used herein, a "decreased level" or "level below a reference value" means that the level of a biomarker is lower than a reference value (e.g., a predetermined threshold value for the biomarker in a control sample). Control levels are described in detail herein. A reduced level of a biomarker includes a level of the biomarker that is, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500% or more lower than the reference value. In some embodiments, the level of the biomarker in the test sample is at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 15 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 50 fold, 100 fold, 150 fold, 200 fold, 300 fold, 400 fold, 500 fold, 1000 fold, or 10,000 fold or more lower than the level of the biomarker in the reference sample.

[0101] In some embodiments, the candidate subject is a human patient with symptoms of a disease associated with the contact activation system, such as a pKal-mediated disorder (e.g., HAE) or an autoimmune disease (such as RA, UC, and Crohn's disease). For example, the subject has edema; swelling that is entirely or mainly peripheral; hives; redness, pain, and swelling in the absence of evidence of infection; non-histamine-mediated edema; recurrent bouts of swelling; or a combination thereof. In other embodiments, the subject does not have symptoms of a pKal-mediated disorder at the time the sample is collected, or does not have a history of symptoms of a pKal-mediated disorder, or does not have a history of a pKal-mediated disorder, such as HAE. In yet other embodiments, the subject is resistant to antihistamine therapy, corticosteroid therapy, or both.

[0102] Subjects identified by the methods described herein may receive suitable treatment, such as treatment with a pKal inhibitor, as described herein.

[0103] Given the correlation between biomarker levels and such diseases, the assays and kits described herein can also be applied to assess the effectiveness of treatments for diseases associated with the contact system (such as those described herein). For example, multiple biological samples (e.g., blood or plasma samples) may be collected from a subject receiving treatment before and after treatment or during the course of treatment. The level of the biomarker may be measured by any of the assays described herein, and the value (e.g., amount) of the biomarker may be determined accordingly. For example, if an elevated level of a biomarker indicates that the subject has the target disease, and the level of the biomarker decreases after or over the course of treatment (the level of the biomarker in a later sample compared to the level of the biomarker in an earlier sample), it indicates that the treatment is effective. As another example, if a decreased level of a biomarker indicates that the subject has the target disease, and the level of the biomarker increases after or over the course of treatment (the level of the biomarker in a later sample compared to the level of the biomarker in an earlier sample), it indicates that the treatment is effective. In some examples, the treatment involves an effective amount of a therapeutic agent, such as a plasma kallikrein inhibitor, a bradykinin B2 receptor antagonist, or a C1 esterase inhibitor (C1-INH). Examples of therapeutic agents include, but are not limited to, lanadelumab, ecallantide, icatibant, and human plasma-derived C1-INH.

[0104] If a subject is identified as not responding to treatment, a higher dose and / or more frequent administration of the therapeutic agent is administered to the identified subject. In some embodiments, the dosage or administration frequency of the therapeutic agent is maintained, reduced, or discontinued in subjects who are identified as responding to treatment or not requiring further treatment. Alternatively, a different treatment may be administered to subjects who are found not to respond to the first treatment.

[0105] In other embodiments, the value of a biomarker or set of biomarkers can also be used to identify a disorder associated with the contact system or that the disorder may be treatable, for example, by a pKal inhibitor. To carry out this method, the level of a biomarker in a sample (e.g., a blood sample or plasma sample) taken from a subject with a target disease may be measured by a suitable method (e.g., mass spectrometry, chromatography, immunoassay, etc., as described herein). If the level of the biomarker deviates from the reference value (e.g., elevated or decreased), this indicates that a pKal inhibitor may be effective in treating the disease. If the disease is identified as sensitive to a pKal inhibitor (i.e., treatable by a pKal inhibitor), the method may further include administering to the subject with the disease an effective amount of a pKal inhibitor (e.g., an anti-pKal antibody or an inhibitory peptide (e.g., lanadelumab, ecallantide, etc.)), a bradykinin 2 receptor inhibitor (e.g., icatibant), and / or C1-INH (e.g., human plasma-derived C1-INH).

[0106] Also within the scope of the present disclosure are methods for assessing the severity of or pathology of diseases associated with the contact system. For example, as described herein, HAE can be in a quiescent state (basal state) during which the subject does not experience symptoms of the disease. HAE attacks are recurrent episodes that can typically last 2-5 days and during which the subject may experience pain and swelling, for example, in the hands, feet, face, gastrointestinal tract, genitals, and pharynx (throat). In some embodiments, the level of one or more biomarkers indicates whether the subject will experience, is experiencing, or will soon experience an HAE attack. In some embodiments, the method involves comparing the level of a biomarker in a sample obtained from a subject with HAE with the level of that biomarker in a sample obtained from the same subject (e.g., a sample obtained from the same subject in a basal state or a sample obtained from the same subject during an HAE attack).

[0107] Another aspect of the present disclosure provides a method for assessing the risk of a disease attack (e.g., an HAE attack). As described herein, HAE attacks are typically recurring episodes during which a subject may experience symptoms (such as pain and swelling). In some embodiments, the level of one or more biomarkers indicates whether a subject is at risk for an HAE attack. In some embodiments, the method involves comparing the level of a biomarker in a sample obtained from a subject with HAE to the level of that biomarker in a sample obtained from the same subject (e.g., a sample obtained from the same subject in an underlying condition or during an HAE attack). For example, the level of a biomarker in another sample from the subject may indicate that the subject has an elevated (or decreased) level of a biomarker associated with an HAE attack compared to when the subject is at risk for an HAE attack.

[0108] In some embodiments, the method involves comparing the level of a biomarker in a sample obtained from a subject with HAE with the level of a biomarker in a control sample or reference sample. In some embodiments, the level of a biomarker in a control sample or reference sample represents the level of a biomarker that indicates an HAE attack state. For example, a biomarker level in a sample obtained from a subject with HAE that is similar to the level of a biomarker in a reference that indicates an HAE attack state may indicate that the subject is at risk of having an HAE attack. In some embodiments, the level of a biomarker in a control sample or reference sample represents the level of a biomarker that indicates an underlying HAE state. For example, a biomarker level in a sample obtained from a subject with HAE that deviates (is elevated or decreased) from the level of a biomarker in a reference that indicates an underlying HAE state may indicate that the subject is at risk of having an HAE attack.

[0109] (iii) Non-clinical use Furthermore, the levels of any of the sets of biomarkers described herein may be used for research purposes. Although a number of diseases associated with the contact activation system have been identified, it is possible that other diseases are mediated by similar mechanisms or involve similar components. In some embodiments, the methods described herein may be used to identify diseases as being associated with the contact activation system or components of the contact activation system. In some embodiments, the methods described herein may be used to study the mechanisms (e.g., discovering new biological pathways or processes involved in the development of a disease) or progression of a disease.

[0110] In some embodiments, the levels of a set of biomarkers as described herein may be utilized in the development of new treatments for diseases associated with the contact activation system. For example, the levels of the set of biomarkers may be measured in samples obtained from subjects undergoing a new treatment (e.g., a clinical trial). In some embodiments, the levels of the set of biomarkers may indicate the effectiveness of the new treatment or the progression of disease in the subject before, during, or after the new treatment.

[0111] Kits and detection devices for measuring a set of RNA biomarkers The present disclosure also provides kits and detection devices for use in measuring levels of a set of biomarkers as described herein. Such kits or detection devices may include binding substances that specifically bind to RNA biomarkers (such as those listed in Table 1). For example, such kits or detection devices may include at least two binding substances specific for two different RNA biomarkers selected from Table 1. In some examples, the kits or detection devices include binding substances specific for all members of the set of RNA biomarkers described herein.

[0112] In some embodiments, the binding agent is an oligonucleotide as described herein that is complementary to the RNA biomarker. In some embodiments, the binding agent comprises a pair of oligonucleotides (e.g., a pair of primers), each of which binds (hybridizes) to a specific nucleotide sequence in the RNA biomarker. In some embodiments, the pair of oligonucleotides hybridizes to the RNA biomarker when subjected to a method such as polymerase chain reaction, allowing amplification of the RNA biomarker. In some embodiments, the amplification product may be detected directly, for example, by detecting a dye, fluorophore, or other indicator substance intercalated into the nucleic acid during polymerase chain reaction. In some embodiments, at least one of the oligonucleotides is conjugated to a detectable label.

[0113] In some embodiments, RNA biomarkers are measured using nucleic acid hybridization methods. In some embodiments, the binding agent is an oligonucleotide that is complementary to the RNA biomarker and hybridizes to the RNA biomarker if it is present in a sample (e.g., a biological sample or a sample from a polymerase chain reaction). In some embodiments, the oligonucleotide is conjugated to a detectable label (such as any of the labels described herein). Non-limiting examples of hybridization methods as described herein include Northern blotting, Southern blotting, and microarrays.

[0114] In some embodiments, one or more of the binding agents is a nucleic acid binding protein that specifically binds to the RNA of the set of biomarkers. In some embodiments, the binding agent may be directly detected or conjugated to a tag that can be directly or indirectly identified.

[0115] In some embodiments, the kit or device further comprises a support member as described herein. In the kit or detection device, one or more of the binding substances may be immobilized on a support member (e.g., a membrane, a bead, a slide, or a multi-well plate). The selection of an appropriate support member for an immunoassay will depend on various factors, such as the number of samples and the method for detecting the signal emitted from the label conjugated to the second agent.

[0116] The kit may also include one or more buffers as described herein (including but not limited to a coating buffer, a blocking buffer, a washing buffer and / or a stopping buffer).

[0117] In some embodiments, the kit may include instructions for use according to any of the methods described herein, including instructions on how to use the components included in the kit to measure RNA levels of a set of RNA biomarkers in a biological sample taken from a subject, such as a human patient.

[0118] The instructions for using the kit generally include information about the amount of each component and suitable conditions for carrying out the assay methods described herein. The components in the kit may be in unit dose, bulk package (e.g., multi-dose package), or sub-unit dose. The instructions provided in the kits of the present disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0119] The label or package insert indicates that the kit is used for assessing RNA levels of a set of biomarkers. Instructions may be provided for performing any of the methods described herein.

[0120] The kits of the present disclosure are contained in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with specific devices, such as inhalers, intranasal administration devices (e.g., nebulizers), or infusion devices (e.g., minipumps). The kits may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle).

[0121] The kit may optionally provide additional components, such as information for determination (such as a control sample and / or a standard or reference sample). Typically, the kit includes a container and a label or package insert(s) on or associated with the container. In some embodiments, the present disclosure provides an article of manufacture comprising the contents of the above-described kit.

[0122] Treatment of diseases associated with the contact activation system Subjects identified using the methods described herein as being at risk for or suffering from a disease associated with the contact activation system may be treated with any appropriate therapeutic agent. In some embodiments, the methods provided include selecting a treatment for the subject based on the output of the described methods (e.g., measuring the levels of a set of biomarkers).

[0123] In some embodiments, the methods described herein provide a method for identifying a subject as a candidate for preventative (prophylactic) treatment. As used herein, "preventative" treatment encompasses any therapy or treatment regimen aimed at preventing or reducing the occurrence of a disease (e.g., an HAE attack). In some embodiments, the method further includes administering a preventative treatment to the subject. Any of the therapeutic agents described herein (e.g., a pKal inhibitor such as lanadelumab). In some embodiments, the level of a set of RNA biomarkers in a sample obtained from the subject indicates that the patient has or is at risk of having HAE (e.g., by comparing the level of the RNA biomarkers with levels in a reference or control sample). Any subject who has or is at risk of having HAE may be administered a prophylactic treatment. The selection of an appropriate therapeutic agent and administration regimen for preventative treatment will be apparent to one skilled in the art.

[0124] In some embodiments, the method includes one or both of selecting or administering a therapeutic agent (e.g., a kallikrein inhibitor, a bradykinin B2 receptor inhibitor, and / or a C1 esterase inhibitor) for administration to the subject based on the output of the assay (e.g., detection of a biomarker).

[0125] In some embodiments, the therapeutic agent is administered to the subject one or more times. In some embodiments, a plasma kallikrein inhibitor is administered to the subject. In some embodiments, the kallikrein inhibitor is a peptide, a small molecule inhibitor, a kallikrein antibody or a fragment thereof. In some embodiments, a bradykinin B2 receptor antagonist is administered to the subject. In some embodiments, C1-INH is administered to the subject.

[0126] A therapeutic agent (e.g., a kallikrein inhibitor, a bradykinin B2 receptor inhibitor, and / or C1-INH) may be administered together with another therapy as part of a combination therapy for the treatment of a disease or condition involving the contact activation system. The combination therapy (e.g., a combination therapy with one or more of a kallikrein inhibitor, a bradykinin B2 receptor antagonist, or a C1-INH replacement agent, e.g., a combination therapy with one or more of a kallikrein inhibitor, a bradykinin B2 receptor antagonist, or a C1-INH replacement agent and another therapy) may be provided in several different configurations. A first agent may be administered before or after the administration of the other therapy. In some situations, the first agent and the other therapy (e.g., a therapeutic agent) are administered simultaneously or closely in time (e.g., with a short injection interval, such as during the same treatment session). The first agent and the other therapy may also be administered at a longer time interval.

[0127] Treatment drugs Plasma kallikrein binding agents (e.g., binding proteins, e.g., polypeptides, e.g., inhibitory polypeptides, e.g., antibodies, e.g., inhibitory antibodies, or other binding agents (e.g., small molecules)) are useful therapeutic agents for various diseases and conditions (e.g., diseases and conditions associated with plasma kallikrein activity). For example, in some embodiments, the disease or condition associated with plasma kallikrein activity is hereditary angioedema (HAE). In some embodiments, a plasma kallikrein binding agent, such as a plasma kallikrein inhibitor, is administered to a subject at risk for or suffering from a disease associated with the contact activation system.

[0128] Some useful protein-based inhibitors of kallikrein (either tissue kallikrein and / or plasma kallikrein) contain a Kunitz domain. As used herein, a "Kunitz domain" is a polypeptide domain having at least 51 amino acids and containing at least two (preferably three) disulfides. This domain folds so that the first and sixth cysteines, the second and fourth, and the third and fifth cysteines form disulfide bonds (e.g., in a 58-amino acid Kunitz domain, cysteines may be present at positions corresponding to amino acids 5, 14, 30, 38, 51, and 55 according to the numbering of the BPTI homologous sequence presented below, and disulfides may be formed between the cysteines at positions 5 and 55, 14 and 38, and 30 and 51). Alternatively, if two disulfides are present, they may be formed between corresponding subsets of the cysteines. The spacing between each cysteine can be within 7, 5, 4, 3, 2, 1, or 0 amino acids of the spacing between positions corresponding to positions 5-55, 14-38, and 30-51 according to the numbering of the BPTI sequence presented below. The BPTI sequence can be used as a reference to point to specific positions in any general Kunitz domain. Comparison of a Kunitz domain of interest to BPTI can be performed by identifying the best-match alignment that maximizes the number of matching cysteines.

[0129] The 3D structure (at high resolution) of the Kunitz domain of BPTI is known. One X-ray structure has been deposited in the Brookhaven Protein Data Bank as "6PTI." The 3D structures of several BPTI homologs (Eigenbrot et al., Protein Engineering (1990) 3(7):591-598; Hynes et al., Biochemistry (1990) 29:10018-10022) are known. The sequences of at least 81 Kunitz domains are known. Known human homologs include the three Kunitz domains of LACI, also known as tissue factor pathway inhibitor (TFPI) (Wun et al., J.Biol.Chem.(1988) 263(13):6001-6004; Girard et al., Nature(1989) 338:518-20; Novotny et al., J.Biol.Chem.(1989) 264(31):18832-18837), the two Kunitz domains of inter-α-trypsin inhibitor APP-I (Kido et al. J.Biol.Chem.(1988) 263(34):18104-18107), the Kunitz domains of collagen, and the three Kunitz domains of TFPI-2 (Sprecher et al., PNAS USA(1994) 91:3353-3357), the Kunitz domain of hepatocyte growth factor activator inhibitor type 1, the Kunitz domain of hepatocyte growth factor activator inhibitor type 2, and the Kunitz domain described in U.S. Patent Application Publication No. 2004-0152633. LACI is a human serum phosphoglycoprotein with a molecular weight of 39 kDa that contains three Kunitz domains (amino acid sequence in Table 2).

[0130] [Table 2]

[0131] The Kunitz domains are designated LACI-K1 (residues 50-107), LACI-K2 (residues 121-178), and LACI-K3 (residues 213-270). The cDNA sequence of LACI was reported by Wun et al. (J. Biol. Chem. (1988) 263(13):6001-6004). Girard et al. (Nature (1989) 338:518-20) reported mutational studies in which the P1 residues of each of the three Kunitz domains were altered. LACI-K1 inhibits factor VIIa (F.VIIa) when it is complexed with tissue factor, and LACI-K2 inhibits factor Xa.

[0132] Exemplary Kunitz domain-containing proteins include the following (SWISS-PROT accession numbers in parentheses): [ka]

[0133] Kunitz domains can be identified from sequence databases using various methods. For example, known amino acid sequences, consensus sequences, or motifs (e.g., ProSite Motifs) of Kunitz domains can be searched against the GenBank sequence database (National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD) using BLAST (e.g., using default parameters for Pfam searches; against the SMART database; or against the ProDom database) for HMMs (hidden Markov models). For example, Pfam Release 9, Pfam accession number PF00014, provides numerous Kunitz domains and HMMs for identifying Kunitz domains. A description of the Pfam database can be found in Sonhammer et al. Proteins (1997) 28(3):405-420, and a detailed description of HMMs can be found, for example, in Gribskov et al. Meth. Enzymol. (1990) 183:146-159; Gribskov et al. Proc. Natl. Acad. Sci. USA (1987) 84:4355-4358; Krogh et al. J. Mol. Biol. (1994) 235:1501-1531; and Stultz et al. Protein Sci. (1993) 2:305-314. The SMART database of HMMs (Simple Modular Architecture Research Tool, EMBL, Heidelberg, DE) is as described in Schultz et al. Proc. Natl. Acad. Sci. USA (1998) 95:5857 and Schultz et al. Nucl. Acids Res (2000) 28:231.The SMART database contains domains identified by hidden Markov model profiling using the HMMer2 search program (R. Durbin et al. (1998) "Biological sequence analysis: probabilistic models of proteins and nucleic acids," Cambridge University Press). This database is also annotated and monitored. The ProDom protein domain database consists of an automated compilation of homologous domains (Corpet et al. Nucl. Acids Res. (1999) 27:263-267). The current version of ProDom was constructed using recursive PSI-BLAST searches of the SWISS-PROT 38 and TREMBL protein databases (Altschul et al. Nucleic Acids Res. (1997) 25:3389-3402; Gouzy et al. Computers and Chemistry (1999) 23:333-340). This database automatically generates consensus sequences for each domain. Prosite lists Kunitz domains as motifs and identifies proteins containing Kunitz domains (see, e.g., Falquet et al. Nucleic Acids Res. (2002) 30:235-238).

[0134] Kunitz domains interact with target proteases primarily using amino acids in two loop regions ("binding loops"). The first loop region is located approximately between residues corresponding to amino acids 13-20 of BPTI. The second loop region is located approximately between residues corresponding to amino acids 31-39 of BPTI. In an exemplary library of Kunitz domains, one or more amino acid positions within the first and / or second loop regions are varied. When screening for Kunitz domains that interact with kallikrein or selecting variants with improved affinity, particularly useful positions to vary include positions 13, 15, 16, 17, 18, 19, 31, 32, 34, and 39 relative to the sequence of BPTI. At least some of these positions are expected to be in close contact with the target protease. Varying other positions (e.g., positions adjacent to the aforementioned positions in the three-dimensional structure) is also useful.

[0135] The "framework regions" of a Kunitz domain are defined as residues that are part of the Kunitz domain, but specifically exclude residues within the first and second binding loop regions (i.e., around residues corresponding to amino acids 13-20 of BPTI and amino acids 31-39 of BPTI). Conversely, residues not within the binding loops may tolerate a broader range of amino acid substitutions (e.g., conservative and / or non-conservative substitutions).

[0136] In one embodiment, these Kunitz domains are variants of the loop structure comprising Kunitz domain 1 of human lipoprotein-associated coagulation inhibitor (LACI). LACI contains three well-defined internal peptide loop structures that are paradigm Kunitz domains (Girard, T. et al., Nature (1989) 338:518-520). Variants of Kunitz domain 1 of LACI described herein have been screened and isolated, and they bind to kallikrein with improved affinity and specificity (see, e.g., U.S. Patent Nos. 5,795,865 and 6,057,287). These methods can also be applied to the frameworks of other Kunitz domains to obtain other Kunitz domains that interact with kallikrein (e.g., plasma kallikrein). Useful modulators of kallikrein function typically bind to and / or inhibit kallikrein as determined using kallikrein binding and kallikrein inhibition assays.

[0137] In some aspects, the plasma kallikrein inhibitor binds to the active form of plasma kallikrein. In some embodiments, the plasma kallikrein inhibitor binds to and inhibits plasma kallikrein (e.g., human plasma kallikrein and / or mouse kallikrein). Exemplary polypeptide-based plasma kallikrein drugs are disclosed in U.S. Patent No. 5,795,865, U.S. Patent No. 5,994,125, U.S. Patent No. 6,057,287, U.S. Patent No. 6,333,402, U.S. Patent No. 7,628,983, U.S. Patent No. 8,283,321, U.S. Patent No. 7,064,107, U.S. Patent No. 7,276,480, U.S. Patent No. 7,851,442, U.S. Patent No. 8,124,586, U.S. Patent No. 7,811,991 and U.S. Patent Application Publication No. 20110086801, the entire contents of each of which are incorporated herein by reference. In some embodiments, the plasma kallikrein inhibitor is an inhibitory polypeptide or inhibitory peptide. In some embodiments, the inhibitory peptide is ecallantide (also known as DX-88 or KALBITOR®; SEQ ID NO: 3). In some embodiments, the kallikrein inhibitor comprises or consists of a DX-88 polypeptide having a sequence of about 58 amino acids from amino acids 3 to 60 of SEQ ID NO: 3, or the 60 amino acid sequence of SEQ ID NO: 3.

[0138] Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (SEQ ID NO: 3).

[0139] The plasma kallikrein inhibitor may be a full-length antibody (e.g., IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgA1, IgA2), IgD, and IgE) or may comprise only an antigen-binding fragment (e.g., a Fab fragment, a F(ab')2 fragment, or an scFv fragment). The binding protein may comprise two heavy chain immunoglobulins and two light chain immunoglobulins, or may be a single-chain antibody. The plasma kallikrein inhibitor may be a recombinant protein, such as a humanized antibody, a CDR-grafted antibody, a chimeric antibody, a deimmunized antibody, or an in vitro generated antibody, and may optionally comprise a constant region derived from a human germline immunoglobulin sequence. In one embodiment, the plasma kallikrein inhibitor is a monoclonal antibody.

[0140] Exemplary plasma kallikrein binding proteins are disclosed in U.S. Patent Application Publication No. 20120201756, the entire contents of which are incorporated herein by reference. In some embodiments, the kallikrein binding protein is selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-E10, X115-E11, X115-E12, X115-E13, X115-E14, X115-E15, X115-E16, X115-E17, X115-E18, X115-E19, X115-E20, X115-E21, X115-E22, X115-E23, X115-E24, X115-E25, X115-E26, X115-E27, X115-E28, X115-E29, X115-E30, X115-E31, X115-E32, X115-E33, X115-E34, X115-E35, X115-E36, X115-E37, X115-E38, X115-E39, X115-E40, X115-E41, X115-E42, X115-E43, X115-E and an antibody (e.g., a human antibody) having a light chain and / or a heavy chain of an antibody selected from the group consisting of 115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (also referred to herein as DX-2930 or lanadelumab), X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04. In some embodiments, the plasma kallikrein binding protein competes with or binds to the same epitope as M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04. In some embodiments, the plasma kallikrein binding protein is lanadelumab. See U.S. Patent Application Publication Nos. 20110200611 and 20120201756, which are incorporated herein by reference.

[0141] An example of a plasma kallikrein inhibitor antibody is lanadelumab. The amino acid sequences of the heavy and light chain variable regions of lanadelumab are provided below, with the CDR regions identified in bold and underlined.

[0142] Lanadelumab heavy chain variable region sequence (SEQ ID NO: 4) EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR IGVPRRDEFD IWGQGTMVTV SS

[0143] Lanadelumab light chain variable region sequence (SEQ ID NO: 5) DIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPKLLIY KASTLESGVP SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEI

[0144] In some embodiments, a plasma kallikrein inhibitor may have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to a plasma kallikrein inhibitor described herein. In some embodiments, a plasma kallikrein inhibitor may have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity in the framework regions of the HC and / or LC (e.g., FR1, 2, 3, and / or 4 of the HC and / or LC) to a plasma kallikrein inhibitor described herein. In some embodiments, a plasma kallikrein inhibitor may have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity in the CDRs of the HC and / or LC (e.g., CDR1, 2, and / or 3 of the HC and / or LC) to a plasma kallikrein inhibitor described herein. In some embodiments, a plasma kallikrein inhibitor may have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity in the constant region (e.g., CH1, CH2, CH3, and / or CL1) to a plasma kallikrein inhibitor described herein.

[0145] In some aspects, the small molecule binds to and inhibits the active form of plasma kallikrein.

[0146] Bradykinin B2 receptor inhibitor In some embodiments, a bradykinin B2 receptor inhibitor (e.g., antagonist) is administered to the subject. Exemplary bradykinin B2 receptor antagonists include icatibant (Firadyl®), a 10-amino acid peptidomimetic that blocks the binding of native bradykinin to the bradykinin B2 receptor.

[0147] C1-INH replacement In some embodiments, a C1 esterase inhibitor (C1-INH), such as a C1-INH replacement drug, is administered to the subject. Exemplary C1-INH replacement drugs are publicly available and include, for example, human plasma-derived C1-INH (e.g., Verinert® and Sinrise®).

[0148] Without further elaboration, it is believed that, based on the above description, one skilled in the art can utilize the present disclosure to its fullest extent. Accordingly, the following specific embodiments are to be construed as merely illustrative, and not limiting in any way to the remainder of the disclosure. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein. [Example]

[0149] Example Example 1: Identification of RNA molecules that are differentially present in samples from HAE patients compared to healthy individuals To investigate novel RNA biomarkers for hereditary angioedema, we analyzed circulating small RNAs present in plasma samples obtained from patients with HAE compared with samples obtained from healthy individuals. Circulating citrated plasma was collected from healthy individuals (n = 3) as well as patients with HAE during a quiescent state of the disease ("basal state"; N = 19) and during an edematous attack ("attack"; N = 20). RNA was extracted from the plasma samples. Library fragments of 128–158 nucleotides were sequenced using a HiSeq 2500 sequencing system (Illumina). Reads were demultiplexed, adapter sequences were removed, and unique sequences with more than five reads were generated. Using the sequence alignment tool Bowtie2 (bowtie-bio.sourceforge.net / bowtie2 / manual.shtml), non-redundant sequences were aligned to the reference genome (hg19). Sequences with perfect and single-nucleotide mismatches were further aligned to cDNA, non-coding RNA, miRNA, and piRNA databases. RNA-Seq data identified 1,811 detectable human RNA transcripts and 457 human microRNAs (≥10 reads in ≥25% of samples). The abundance of each detected RNA was compared between samples from healthy individuals and those from patients with HAE. RNA transcripts encoding specific mitochondrial proteins were found to be significantly elevated in samples from HAE patients compared to those from healthy individuals (Figure 1). Furthermore, RNA-Seq analysis identified 24 microRNAs that were differentially expressed in samples from HAE patients compared to those from healthy individuals. The identified RNA transcripts and microRNAs were validated using RT-qPCR on separate samples obtained from healthy individuals (N=20), patients with HAE during the quiescent state of the disease (N=34), and patients with HAE during edematous attacks (N=18).

[0150] As shown in Figure 1, in the initial RNASeq as well as in RT-qPCR analysis, RNA transcripts of mitochondrial-encoded NADH:ubiquinone oxidoreductase core subunit 3 (MT-ND3) and mitochondrial-encoded cytochrome C oxidase III were higher in samples from patients with HAE (during attacks or during quiescence) compared to samples from healthy individuals. These results indicated that these RNAs could be used as biomarkers to distinguish samples from patients with HAE from samples from healthy individuals.

[0151] As shown in panels A and B of Figure 2, several microRNAs were found to be differentially present in samples obtained from HAE patients and healthy individuals. For example, hsa-miR-423-3p, hsa-miR-1307-3p, hsa-miR-355-3p, and hsa-miR-485-5p were elevated in samples from HAE patients. In addition, several microRNAs, including hsa-miR-16-5p, hsa-miR-19a-3p, hsa-miR-20a-5p, hsa-miR-17-5p, and hsa-miR-885-5p, were found to be at reduced levels in samples from HAE patients. Finally, this analysis also identified several microRNAs (e.g., has-miR-1307-3p, hsa-miR-335-3p, and hsa-miR-485-5p) that were decreased in samples from HAE patients during attacks compared with those in the basal state (Figure 2, panel C).

[0152] Any of the RNAs identified herein (e.g., RNAs with significant fold changes between HAE patients and healthy individuals) may be used (individually or in combination (in sets of biomarkers)) as biomarkers for diseases associated with the contact activation system in methods, e.g., to identify patients at risk for diseases associated with the contact activation system (e.g., HAE), to select candidates for treatment, to monitor disease progression or pathology, to assess the effectiveness of treatments for diseases, to determine a course of treatment, to identify whether a disease or disorder is associated with the contact activation system, and / or for research purposes (e.g., including studying disease mechanisms that may be exploited for the development of new therapies).

[0153] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each disclosed feature is only one example of a generic series of equivalent or similar features.

[0154] From the foregoing description, those skilled in the art can readily ascertain the essential features of the present disclosure, and can make various changes and modifications to the present disclosure to adapt it to various uses and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are within the scope of the following claims.

[0155] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present disclosure described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0156] In the claims, articles such as "a," "an," and "the" can mean one or more unless indicated otherwise or clear from the context. A claim or description including "or" between one or more members of a group is deemed to stand if one, more than one, or all of the group members are present in, used in, or associated with a given product or process, unless indicated otherwise or clear from the context. The present disclosure includes embodiments in which exactly one member of a group is present in, used in, or associated with a given product or process. The present disclosure includes embodiments in which two or more, or all of the group members are present in, used in, or associated with a given product or process.

[0157] Furthermore, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim that depends on another claim may be modified to include one or more limitations found in any other claim that depends from the same base claim. Where elements are presented as a list (e.g., in Markush group format), each subgroup of elements is also disclosed, and any element(s) may be deleted from the group. In general, when the present disclosure or aspects of the present disclosure are referred to as comprising certain elements and / or features, it should be understood that certain embodiments of the present disclosure or aspects of the present disclosure consist of or consist essentially of such elements and / or features. For the sake of brevity, those embodiments have not been specifically described in those terms herein. It should also be noted that the terms "comprising" and "containing" are intended to be open-ended, permitting the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or apparent from the context and the understanding of one of ordinary skill in the art, values expressed as ranges may, in various embodiments of the present disclosure, assume any specific value or subrange within the stated range, to the nearest tenth of the lower limit of the range, unless the context clearly dictates otherwise.

[0158] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, this specification shall control. Furthermore, any specific embodiments of the present disclosure that fall within the prior art may be expressly excluded from one or more of the claims. Such embodiments may be excluded even if the exclusion is not explicitly set forth herein because they are deemed to be known to those of ordinary skill in the art. Any specific embodiments of the present disclosure may be excluded from any claim for any reason, whether related to the existence of prior art or not.

[0159] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above description, but rather is as set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure as defined in the following claims.

Claims

[Claim 1] 1. A method of analyzing a sample, comprising: (i) providing a biological sample obtained from a subject having, suspected of having, or at risk for having a disease associated with the contact activation system; and (ii) measuring the level of a set of RNA biomarkers comprising at least one RNA biomarker, wherein when the set of biomarkers consists of one RNA biomarker, the RNA biomarker is not any of hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-19a-3p, and hsa-miR-20a-5p.

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