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 on September 16, 2016. The entire content of the said application is incorporated herein by reference.

Background Art

[0002] Background The plasma contact activation system is a pro - inflammatory and pro - coagulant system involving a group of plasma proteases. The plasma contact activation system is activated by factor XIIa when exposed to foreign surfaces or negatively charged surfaces, or by prolyl carboxypeptidase on the endothelial cell surface (Sainz I.M. et al., Thromb. Haemost. (2007) 98, 77 - 83). Inappropriate or disordered 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 airway). Since the symptoms of HAE 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 enable better management of emergencies associated with acute HAE attacks, and early diagnosis would also help manage HAE patients to prevent or attenuate acute HAE episodes (for example, by having HAE patients avoid exposure to stimuli that may trigger HAE episodes).

[0004] Accordingly, it is highly interesting to identify biomarkers for HAE and to develop reliable diagnostic and prognostic methods for identifying subjects having a particular type of HAE or at risk of suffering an acute HAE attack. Such biomarkers would also be useful in studies of the disease mechanism that could facilitate the development of effective new therapies 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 having, suspected of having, or at risk of having a disease 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., attack vs. baseline).

[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 a plasma sample) obtained from a subject (such as a human subject) having, suspected of having, or at risk of having a disease associated with the contact activation system; and (ii) measuring the levels 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 biomarker is messenger RNA encoding a mitochondrial protein which may be cytochrome C oxidase III (MT-CO3) encoded by mitochondria or oxidoreductase core subunit (MT-ND3) encoded by mitochondria. In some embodiments, the RNA biomarker is a microRNA (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 level of the 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 that the subject has a disease associated with the contact system when the level of the set of RNA biomarkers of the subject deviates from the level of the same set of RNA biomarkers of a control subject. In some embodiments, the method further comprises administering to the subject an effective amount of a therapeutic agent (such as a plasma kallikrein (pKal) inhibitor, a bradykinin 2 receptor inhibitor, and / or a C1 esterase inhibitor, etc.) for treating the disease when 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 a human plasma-derived C1 esterase inhibitor.

[0010] In some embodiments, the subject is a human patient undergoing treatment for a disease, where the method further comprises evaluating the effectiveness of the treatment based on the levels of a set of RNA biomarkers, and deviation of the levels of the set of RNA biomarkers of the subject from those 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 of a 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 evaluating the risk of onset of a disease in the subject based on the levels of the set of RNA biomarkers, and deviation of the levels of the set of RNA biomarkers of the subject from those of a control subject indicates the risk of onset of the disease.

[0012] The present disclosure provides RNA biomarkers capable of identifying patients having a disease associated with a contact activation system (e.g., HAE). Measuring the levels of a set of biomarkers may also be useful in the evaluation 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 a disease associated with a contact system, 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 RNA biomarker and the second RNA biomarker are different. In some examples, the first binding substance is an oligonucleotide (fully or partially) complementary to the first RNA biomarker and / or the second binding substance is an oligonucleotide (fully or partially) complementary and specific 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] Details of one or more embodiments of the present disclosure are set forth in the following description. Other features or advantages of the present disclosure will become apparent from the following drawings and detailed description of several embodiments, and still further from the appended claims.

[0015] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present 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 Description of the Drawings

[0016]

Figure 1

Figure 2

Mode for Carrying Out the Invention

[0017] Detailed Description The contact activation system initiates the intrinsic pathway of blood coagulation and promotes inflammation through the release of bradykinin, a pro-inflammatory peptide. Factor XII (FXII), also known as Hageman factor, is a serine protease that plays a role in the activation of the intrinsic pathway of blood coagulation as well as the kallikrein-kinin system. FXII is activated by negatively charged surfaces (e.g., polyanionic surfaces, glass, polyphosphate, ellagic acid) to yield the active form, FXIIa. Activated FXIIa has the ability to cleave prekallikrein, generating active pKal. Subsequently, activated pKal can cleave FXII to FXIIa, resulting in a positive feedback loop where FXIIa generates more pKal, which further activates additional FXII to FXIIa. 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, an increase in bradykinin levels can induce vasodilation and inflammation that lead to edematous HAE attacks. For example, it is desirable to identify novel biomarkers that can be used to identify subjects having or at risk of having such diseases in order to identify such diseases mediated by the contact activation system.

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

[0019] Accordingly, provided herein is a method for analyzing a biological sample from a subject having, suspected of having, or at risk of having 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 methods can be useful, for example, to identify patients at risk of a disease associated with the contact activation system (e.g., HAE), to select candidates for treatment, to monitor disease progression or condition, to evaluate the effectiveness of treatment for a disease, to determine the course of treatment, to assess whether a subject is at risk of an attack of a disease, to identify whether a disease or disorder is associated with the contact activation system, and / or for research purposes (e.g., including studying the mechanism of a disease and / or the biological pathways / processes involved in that disease, which may be utilized for the development of 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 RNA that has been found to be present differently in samples from subjects with HAE than in samples from healthy subjects and / or to be present differently in samples at different stages of such a disease (e.g., baseline 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 contact activation system-related disease (e.g., HAE) vs. healthy subjects (e.g., subjects without a disease), or subjects with a disease in a quiescent stage vs. subjects during an attack of the disease). Such biomarkers / sets of biomarkers may be used for both diagnostic / prognostic uses and non-clinical uses (e.g., for research purposes).

[0021] In some embodiments, an RNA biomarker may be present at elevated levels in samples from subjects with a contact activation system-related disease (e.g., HAE) compared to the levels of the same RNA biomarker in samples from healthy subjects. In some embodiments, an RNA biomarker may be present at reduced levels in samples from subjects with a contact activation system-related disease (e.g., HAE) compared to the levels of the biomarker in samples from healthy subjects. In still other examples, an RNA biomarker may be present at elevated levels in samples obtained from subjects during an attack of a disease as described herein compared to subjects during a quiescent state of the disease. Alternatively, an RNA biomarker may be present at reduced levels in samples obtained from subjects during an attack of a disease as described herein compared to subjects during a quiescent state of the disease.

[0022] In some embodiments, a set of RNA biomarkers comprising one or more biomarkers may be analyzed by the methods described herein. If the set of RNA biomarkers comprises two or more biomarkers, all of those biomarkers may be present at elevated or reduced levels in a subject having a disease compared to a healthy subject. Alternatively, the set of RNA biomarkers may comprise at least one biomarker that is elevated in a subject having a disease compared to a healthy subject and at least one biomarker that is reduced in a subject having a disease compared to a healthy subject.

[0023] Similarly, a set of RNA biomarkers for distinguishing a subject during an episode of a disease from a subject in a quiescent state of the disease may comprise a plurality of biomarkers all of which are elevated or reduced in a first disease stage (e.g., episode) compared to a second disease stage (e.g., quiescent state). Alternatively, the set of biomarkers may comprise at least one biomarker that is elevated in a first disease stage compared to a second disease stage and at least one biomarker that is reduced in a first disease stage compared to a second disease stage.

[0024] Table 1 below presents RNA biomarkers that can be evaluated by the methods described herein for evaluating a subject or a biological sample from a subject for diseases associated with contact activation systems.

[0025]

Table 1

[0026] In some embodiments, a set of biomarkers measured and analyzed by any of the methods described herein comprises at least one (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more than eleven) RNA selected from Table 1. If the set of biomarkers comprises a single RNA biomarker, that RNA biomarker may not be any of hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-19a-3p, and hsa-miR-20a-5p. In some examples, a set of RNA biomarkers measured and analyzed by the methods described herein does not include any combination of any one of hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-19a-3p, and hsa-miR-20a-5p.

[0027] As described in Example 1, it was unexpectedly found that several RNAs encoding proteins involved in similar cell processes and pathways are 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 terms "involved in" or "associated with" are applied to RNAs that contribute to a pathway. For example, an RNA (such as an RNA encoding a protein or a microRNA) involved in or associated with a pathway may be capable of functioning within that pathway (e.g., regulating the expression of another nucleic acid or the activity of a protein), or may encode a molecule (such as a protein) that functions within that pathway or within a cell process.

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

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

[0031] As also described in Example 1, it was also found that some RNAs are present differently 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 comprises one or more microRNAs (e.g., one or more microRNAs selected from Table 1).

[0032] Usefulness 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 of a disease associated with a contact activation system by measuring the level of a set of biomarkers as described herein in the sample. Results obtained from such an assay would 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 preparing a biological sample obtained from a subject. As used herein, "biological sample" refers to a composition comprising tissue (e.g., blood, plasma, or protein) from a subject. Samples include both the initially untreated sample taken from the subject as well as subsequently processed (e.g., partially purified or preserved) forms thereof. Exemplary samples include blood, plasma, tears, or mucus. In some embodiments, the sample is a body fluid sample such as a serum sample or a plasma sample. In some embodiments, multiple (e.g., at least 2, 3, 4, 5, or 6 or more) biological samples may be taken from the subject over time or at specific time intervals, for example, to assess disease progression or the effectiveness of treatment.

[0034] A biological sample can be obtained from a subject using any means known in the art. In some embodiments, the sample is obtained from the subject by collecting the sample (e.g., a blood sample) into a vacuum collection tube (e.g., a vacuum blood collection tube). In some embodiments, the vacuum collection tube contains one or more protease inhibitors, for example, to reduce or prevent ex vivo activation of the contact system during sample collection. Such protease inhibitors may be included in a liquid formulation. In some embodiments, the protease inhibitor includes at least one serine protease inhibitor and at least one cysteine protease inhibitor. Such vacuum collection tubes are known in the art. See, for example, PCT Application No. US2016 / 046681. Optionally, the vacuum blood 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 the 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 having 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., genetic factors associated with the disease (e.g., genetic defects in C1-INH)).

[0036] Alternatively, the subject in need of the analysis described herein may be a patient with the disease. Such a subject may currently be suffering from an episode of the disease or may have suffered from the disease in the past (e.g., currently in a quiescent state of the disease). In some examples, the subject is a human patient who may be undergoing treatment for the disease (e.g., treatment involving a C1 esterase inhibitor (C1-INH), a plasma kallikrein inhibitor, or a bradykinin inhibitor). In other examples, 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, such as bradykinin-mediated disorders (e.g., 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 a ventricular assist device or stent, heparin-induced thrombocytopenia with thrombosis, thromboembolic disease, and coronary heart disease with unstable angina, edema, eye diseases, gout, intestinal bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis - degenerative spinal disorders, postoperative ileus, aortic aneurysm, osteoarthritis, hereditary angioedema, pulmonary embolism, stroke, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic events (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 nephropathy and diabetic nephropathy, allergic diseases 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 injury).

[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) that can affect, for example, the limbs, 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 that may be accompanied by swelling of the larynx and sudden hoarseness; repeated episodes of abdominal cramps without an obvious cause; and / or swelling of the intestine that can be 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] Swelling of the airway can be life-threatening and, in some patients, can be fatal. The mortality rate is estimated to be 15 - 33%. HAE results in approximately 15,000 - 30,000 emergency hospital visits per year. Trauma or stress (e.g., dental procedures, illness (e.g., viral diseases such as colds and influenza), menstruation, and surgery) can trigger an attack of angioedema. To prevent acute attacks of HAE, patients can try to avoid certain stimuli that have previously triggered an attack. However, in many cases, attacks occur without a known trigger. Typically, the symptoms of HAE first appear in childhood and worsen during puberty. On average, untreated individuals have an attack every 1 - 2 weeks, and most episodes last about 3 - 4 days (ghr.nlm.nih.gov / condition / hereditary-angioedema). The frequency and duration of attacks vary widely among people with hereditary angioedema, even among people in the same family.

[0040] There are three types of HAE known as type I, type II, and type III. One in 50,000 people has HAE. Type I accounts for approximately 85% of cases, type II accounts for approximately 15% of cases, and type III is thought to be very rare. Type III is the most recently described form and was initially thought to occur only in women, but families with affected males have been identified.

[0041] HAE is inherited in an autosomal dominant pattern, so an affected person may inherit the mutation from one affected parent. Also, new mutations in the gene can occur, and thus HAE can occur in people with no family history of this disorder. 20 - 25% of cases are estimated to result from new spontaneous mutations.

[0042] Mutations in the SERPING1 gene cause hereditary angioedema types I and II. The SERPING1 gene gives instructions to produce C1 inhibitor protein, which is important for the suppression of inflammation. C1 inhibitor blocks the activity of certain proteins that promote inflammation. Mutations that cause type I hereditary angioedema reduce the level of C1 inhibitor in the blood. In contrast, mutations that cause type II result in the production of abnormally functioning C1 inhibitor. When the level of functional C1 inhibitor is not appropriate, an excessive amount of bradykinin is produced. Bradykinin promotes inflammation by increasing the leakage of body fluids into body tissues through the blood vessel walls. Excessive accumulation of body fluids in tissues causes the swelling episodes seen in individuals with types I and 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 coagulation factor XII. In addition to playing a decisive role in blood coagulation (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 factor XII with increased activity. As a result, more bradykinin is generated, increasing the leakiness of blood vessel walls, which leads to episodes of swelling. The causes of other cases of type III hereditary angioedema remain unknown. In these cases, mutations in one or more genes that have not yet been identified may be responsible for this disorder.

[0044] HAE can present similarly to other forms of angioedema resulting from allergy or other medical conditions, but 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 laparotomies in patients with abdominal swelling, and in some HAE patients, abdominal pain has been incorrectly diagnosed as being psychogenic.

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

[0046] The acute treatment of HAE attacks is carried out to stop the progression of swelling as quickly as possible. Intravenous administration of C1 inhibitor concentrate derived from donor blood is one of the acute treatments. However, this treatment is not available in many countries. In emergencies where C1 inhibitor concentrate is not available, fresh frozen plasma (FFP) can be used as an alternative because fresh frozen plasma (FFP) also contains C1 inhibitor.

[0047] Purified C1 inhibitor derived from human blood has been used in Europe since 1979. Several C1 inhibitor therapies are currently available in the United States, and two C1 inhibitor products are currently available in Canada. CSL Behring's pasteurized Berinert was approved by the FDA in 2009 for acute attacks. Shire's nanofiltrated Cinryze (registered trademark) was approved by the FDA in 2008 for prophylaxis. Ruconest (Pharming) is a recombinant C1 inhibitor in development that does not carry the risk of transmission of infections caused by human blood-derived pathogens.

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

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

[0050] Helicobacter pylori can cause abdominal attacks. Antibiotics for treating H. pylori will reduce abdominal attacks.

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

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

[0053] The symptoms of HAE can be evaluated, for example, using a questionnaire (e.g., a questionnaire answered by the patient, clinician, or family). Such questionnaires are known in the art and include, for example, visual analog scales. See, for example, McMillan, C.V. et al. Patient. 2012;5(2):113-26.

[0054] The biological samples described herein may be subjected to analysis by measuring the levels of a set of RNA biomarkers as described herein in the biological samples. The levels (e.g., amounts) of the biomarkers disclosed herein or changes in the levels of the biomarkers may be evaluated using the assays described herein and / or assays 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 evaluated or measured by directly detecting the RNA biomarker in the biological sample. In some embodiments, the RNA biomarker may be amplified prior to detection, e.g., by PCR.

[0055] The type of detection assay used for the detection and / or quantification of biomarkers of the contact activation system (such as those presented herein) will depend on several parameters, including the specific situation (e.g., clinical or research use) in which the assay will be used, and the type and number of biomarkers to be detected, and the type and number of patient samples to be run in parallel.

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

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

[0058] As used herein, "complementary" is applied to the complementarity of nucleobases generally 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 "nucleic acid sequences being complementary to each other" 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 to each other, and that the two nucleic acid molecules can hybridize to form a double strand under appropriate conditions (e.g., hybridization temperature). As is known in the art, for two nucleic acid molecules to hybridize and form a double strand, 100% sequence complementarity is not required. The sequence complementarity between an oligonucleotide and an RNA biomarker (or cDNA corresponding to the RNA biomarker) may be at least 80% complementary to the corresponding region in the RNA biomarker. In some embodiments, the oligonucleotide comprises a fragment that is 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 examples, the oligonucleotide comprises a fragment that is completely (100%) complementary to a portion of the RNA biomarker (or cDNA corresponding to the RNA biomarker). Such oligonucleotides can be used to distinguish an RNA biomarker from a nucleic acid that is substantially similar (e.g., a nucleic acid having 1, 2 or 3 base differences from the target nucleic acid).

[0059] The oligonucleotide may contain 100 nucleotides or less (e.g., 80 nt, 60 nt, 50 nt, or 30 nt or less). In some embodiments, the 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, the 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 molecule of the oligonucleotide is complementary to a portion of the RNA biomarker. In other examples, a fragment of the oligonucleotide is complementary to a portion of the RNA biomarker. For example, the oligonucleotide may contain a linker (e.g., a polyA linker or a polyT linker) for attachment to a support member. Alternatively, or additionally, the detection probe may contain 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 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 comprises one or more modified nucleotides, including nucleotides modified with, for example, 2'-O-methoxyl groups, 2'-O-methoxyethyl groups, and / or phosphorothioate groups. In some examples, the oligonucleotide comprises one or more locked nucleic acids (LNAs). LNAs, often referred to as inaccessible RNA, are modified RNA nucleotides in which the ribose moiety is modified with an extra bridge linking the 2'-oxygen and 4'-carbon. This bridge "locks" the ribose in the 3'-endo (North) conformation often found in A-form duplexes. LNA nucleotides can be used in both DNA probes 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 comprise 10, 8, 6, 5, 4, 3, 2, or 1 LNA.

[0061] The oligonucleotide may be designed based on the sequence of the RNA biomarker whose detection is desired and may be prepared by conventional methods (e.g., chemical synthesis or in vitro transcription).

[0062] The oligonucleotides as described herein may be immobilized on a support member by conventional methods. As used herein, "immobilized" means attached, bound, or fixed covalently or non-covalently to prevent dissociation or loss of the oligonucleotide, but does not require absolute immobility with respect to 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) such that the nucleotide probe is immobilized to the support member.

[0063] The support member of the present disclosure may be made from one or more suitable materials (e.g., plastics or synthetic polymers (e.g., polyethylene, polypropylene, polystyrene, polyamide, polyurethane, phenolic polymers, or nitrocellulose), naturally derived 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, etc.

[0064] The surface of the support member of the present disclosure may include any molecule, other chemical entity / biological entity, or solid support modification disposed on a solid support that can be used to specifically adhere, bind, or capture nucleic acid molecules (e.g., oligonucleotides complementary to RNA biomarkers). Surface compositions that may be used to immobilize nucleic acid molecules can be readily found in the art. For example, the surface may include a complementary nucleic acid or nucleic acid-binding protein that can be attached to the surface by conventional methods. Thus, the linkage between the nucleic acid to be immobilized (e.g., the oligonucleotide of the present disclosure) and the surface may include one or more chemical or physical bonds (e.g., non-specific attachment by van der Waals forces, hydrogen bonding, electrostatic interactions, hydrophobic / hydrophilic interactions, etc.), and / or a chemical linker that effects such bond(s). Alternatively, the surface of the support member may include reactive functional groups that can form a covalent bond with the nucleic acid molecule to be immobilized. In some embodiments, the functional groups are chemical functional groups. That is, the binding surface may be derivatized such that a chemical functional group capable of reacting with a chemical functional group on the captured nucleic acid to effect attachment is presented on the binding surface. Examples of functional groups for attachment that may be useful 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 a linker, and combinations of these may be 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, e.g., 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 that forms a sulfonamide. In some embodiments, the functional group is a functional group that is activated by light. That is, the functional group can be activated by light to attach a capture component to a capture object surface. One example is the PhotoLink™ technology available from SurModics, Inc. (Eden Prairie, MN).

[0065] It should be understood that the examples presented herein with respect to the support member and surface composition are not intended to be limiting. Any support member known in the art to be suitable for the immobilization of nucleic acid molecules may be used in accordance with the methods and kits described herein.

[0066] In some embodiments, the binding substance (e.g., an oligonucleotide complementary to an RNA biomarker) may be conjugated to a label. As used herein, "conjugated" means that the label is attached to the binding substance either covalently or non-covalently. The labeling substance may be any molecule, particle, etc. that facilitates direct or indirect detection using suitable detection techniques. In the case of direct detection, the labeling substance may be a molecule or moiety (e.g., a fluorescent label or a fluorescent dye) that is capable of emitting a signal that can be directly investigated and / or detected. In a non-limiting example of indirect detection, the label may be a molecule or moiety (e.g., an enzyme) that is capable of converting a substrate into a product that can emit a detectable signal. For example, the label may be luciferase that converts luciferin to oxyluciferin and emits 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) that is 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, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, and fluorescent metals ( 152 such as Eu or other metals from the lanthanide series), CYE dyes, and fluorescent proteins (such as eGFP, eYFP, eCFP, mKate2, mCherry, mPlum, mGrape2, mRaspberry, mGrape1, mStrawberry, mTangerine, mBanana, and mHoneydrew).

[0068] Other exemplary labels include biotin, a phosphorescent label, a chemiluminescent label or a bioluminescent label (such as luminal, isoluminol, aromatic acridinium ester, imidazole, acridinium salt, oxalate ester and dioxetane), a radioisotope ( 3 H, 125 I, 32 P, 35 S, 14 C, 51 Cr, 36 Cl, 57 Co, 58 Co, 59 Fe and 75 Se, etc.), a metal, a metal chelate or a metal cation (e.g., 99m Tc, 123 I, 111 In, 131 I, 97 Ru, 67 Cu, 67 Ga and 68 Ga, etc. metal cations), but are not limited thereto. 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, the biomarker is measured using polymerase chain reaction. In some embodiments, RNA extracted from a biological sample is subjected to polymerase chain reaction. In some embodiments, cDNA corresponding to the RNA of the biological sample is used in the polymerase chain reaction. The nucleic acid may 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, polymerase chain reaction relies on the repetition of a series of steps including annealing and extension. A pair of oligonucleotides (e.g., primers) has sufficient complementarity to the desired nucleic acid (e.g., biomarker), such that if the desired nucleic acid is present in the sample, the primers anneal (hybridize) to the desired nucleic acid under appropriate annealing conditions (e.g., annealing temperature). The reaction proceeds to an extension step where the polymerase synthesizes a complementary strand of the nucleic acid. The double-stranded nucleic acid product is denatured and another reaction cycle is initiated.

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

[0072] The polymerase chain reaction as described above enables the 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, dyes, fluorophores or other indicator substances are intercalated into the nucleic acids 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 with oligonucleotides that are (partially or fully) complementary to the RNA biomarkers, for example. In some embodiments, the hybridization method is performed on a biological sample for detecting an RNA biomarker or on a composition (e.g., a PCR reaction) containing an amplification product (e.g., an amplified RNA biomarker or cDNA corresponding to the RNA biomarker). In some embodiments, hybridization involves contacting the sample with an oligonucleotide (e.g., a probe) that specifically binds to the biomarker. Examples of hybridization methods include, but are not limited to, Southern blotting, Northern blotting and microarray methods. As described herein, in some embodiments, the oligonucleotide may be conjugated to a label for detection and / or immobilized on a support member.

[0074] The selection of appropriate buffers, polymerases, hybridization conditions will be apparent to those skilled in the art and optimization may involve routine experimentation.

[0075] In some embodiments, biomarkers are measured using nucleic acid sequencing. For example, the abundance of a particular nucleic acid sequence in a sample (e.g., a biomarker) can be compared to the abundance of the same nucleic acid sequence in another sequence (e.g., whole transcriptome sequencing, RNASeq). Such comparisons can be performed, for example, by comparing the read counts of a particular sequence between samples.

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

[0077] In some embodiments, a biological sample is subjected to an RNA extraction process to isolate the RNA present in the sample (e.g., before measuring a biomarker). 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, the RNA extracted from a biological sample is subjected to in vitro translation, thereby generating the proteins encoded by the RNA, including the proteins encoded by RNA biomarkers if present. In some embodiments, a biological sample or the RNA extracted from a biological sample is subjected to reverse transcription, thereby generating cDNA corresponding to the RNA, including the RNA biomarker if present.

[0078] In some embodiments, the biomarker is measured using an immunoassay. In some embodiments, the biological sample is contacted with a binding substance (such as a nucleic acid binding protein) that binds to the RNA biomarker. An immunoassay may 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 blot), enzyme-linked immunosorbent assay (ELISA) (e.g., sandwich ELISA), radioimmunoassay, detection assays utilizing electrochemiluminescence, magnetic immunoassay, lateral flow assay, and related techniques. For example, by detecting a binding substance that binds to a biomarker, additional suitable immunoassays for detecting the biomarkers presented herein 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] ELISA is known in the art (see, e.g., Crowther, John R (2009), "The ELISA Guidebook", Second Edition, Humana Press and Lequin R (2005), "Enzyme immunoassay (EIA) / enzyme-linked immunosorbent assay (ELISA)", Clin.Chem. 51(12):2415-8), and an exemplary ELISA is described herein. Kits for performing ELISA are also known in the art and are 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 where 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 for the formation of a complex between the binding substance and the biomarker in the sample. Such a complex 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 emit a signal directly or indirectly. 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, the sample may be contacted with two or more binding substances that bind to different RNAs, simultaneously or sequentially (e.g., multiplex analysis; e.g., SOMAScan™ assay (SOMALogic)). The biological sample is contacted with the binding substance under appropriate conditions. Generally, the term "contact" refers to bringing the binding substance into contact with the biological sample or agent for a suitable time sufficient for the formation of a complex between the substance and the RNA (if present) in the sample. In some embodiments, the contact is effected by capillary action across the surface of a support membrane by the biological sample or agent.

[0082] In some embodiments, the immunoassay may be performed on a low-throughput platform that includes a single immunoassay format. For example, using a low-throughput platform, the presence and amount of nucleic acids in a biological sample (e.g., a biological tissue, a tissue extract) may be measured for diagnostic methods, for monitoring the progression of a disease and / or treatment, 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 a 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 also be measured using any of the kits and / or detection devices described herein.

[0084] As used herein, the terms “measuring” or “measurement” or “detecting” or “detection” mean evaluating the presence, absence, quantity or amount (which may be an effective amount) of a substance in a sample (including deriving qualitative or quantitative concentration levels of such substance), or evaluating a value or classification of a subject.

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

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

[0087] The methods and kits described herein (also with 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 RNA levels presented in Table 1 detected in a sample from a subject can be used as reliable biomarkers for diagnosing a disease associated with the contact activation system (e.g., HAE), monitoring the progression of such a disease, evaluating the effectiveness of treatment for the disease, identifying patients suitable for a particular treatment, and / or predicting the onset of the disease in a subject.

[0089] Accordingly, methods for diagnosis and prognosis 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 are described herein. In some embodiments, the biomarker levels measured using any of the methods described herein can be utilized to assess whether a subject (e.g., a human patient) from whom the biological sample was obtained has or is at risk of having a disease associated with the contact activation system, such as a disease associated with plasma kallikrein (e.g., HAE) or an autoimmune disease (such as RA, UC, and Crohn's disease).

[0090] In some embodiments, the biomarker level may then be compared to a reference sample or control sample to determine a value indicative of the amount of RNA in the sample. In some embodiments, the value for a biomarker is obtained by comparing the level of RNA in the sample to the level of another RNA in the sample (e.g., an internal control or internal standard). Such a biomarker value may be a value normalized relative to an internal control or internal standard. The biomarker value may be compared to a reference value to determine whether the subject has or is at risk of having a disease associated with the exposure activation system. The reference value may represent the level of the corresponding biomarker in a subject (e.g., a human subject) without the target disease. In some embodiments, when the biomarker level or value is higher than the reference level or reference value, the subject may be identified as having or being at risk of having a disease associated with the exposure activation system. In some embodiments, when the biomarker level or value is lower than the reference level or reference value, the subject may be identified as having or being at risk of having a disease associated with the exposure activation system.

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

[0092] In some embodiments, the set of biomarkers comprises two or more RNA biomarkers that are present differently (elevated and / or decreased) in a subject having or at risk of having a disease compared to a healthy subject. In some examples, the set of biomarkers comprises at least one RNA biomarker that is elevated in a subject having or at risk of having a disease and at least one RNA biomarker that is decreased in a subject having 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 comprises two or more RNAs, and for each of them, an elevated level indicates that the subject has or is at risk of having a disease. In some embodiments, the set of biomarkers comprises two or more RNAs, and for each of them, a decreased level indicates that the subject has or is at risk of having a disease.

[0093] In some embodiments, the control sample or reference sample is a biological sample obtained from a healthy individual. In some embodiments, the control sample or reference sample comprises a known amount of the RNA being evaluated. In some embodiments, the 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 healthy individual who is clearly free of the target disease (e.g., a disease related to an exposure system) at the time when the level of RNA(s) is measured, or a healthy subject or healthy individual without a medical history of the disease. The term "control subject" encompasses an individual subject, or a group of subjects having similar characteristics (e.g., a group of healthy subjects having specific characteristics (such as age, gender, ethnicity, etc.) that match those of the candidate subject). In some embodiments, the level of a biomarker in a control subject may be used to establish a reference value (e.g., the average level of the biomarker in control subjects encompassing a group of subjects) for comparison with the level of the biomarker in a candidate subject or test subject.

[0095] A control level refers to the level of a set of RNA biomarkers as described herein in a control subject. The control level may be a predetermined level or threshold. Such a predetermined level may represent the level of that RNA in a population of subjects free of or at risk of the target disease (e.g., the average level in a population of healthy subjects). It may also represent the level of that RNA in a population of subjects having the target disease.

[0096] The predetermined level can take various forms. For example, it may be a single cut-off value such as a median or an average value. In some embodiments, such a predetermined level may be established based on a comparison group (such as a comparison group where one defined group is known to have the target disease and another defined group is known to be free of the target disease). Alternatively, the predetermined level may be a range (e.g., a range representing the level of that RNA in a control population).

[0097] The control levels as described herein can be determined by conventional techniques. In some examples, the control levels can be obtained by performing a conventional method (e.g., the same assay as obtaining the level of that RNA in a test sample as described herein) on a control sample also as described herein. In other examples, the level of that RNA can be obtained from members of a control population and the results analyzed, for example, by a computational program, to obtain a control level (a predetermined level) representing the level of that RNA in the control population.

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

[0099] As used herein, "elevated level" or "level above a reference value" means that the level of a biomarker is higher than a reference value (such as a predetermined threshold of the level of the biomarker in a control sample). The control level is described in detail herein. Elevated levels of a biomarker include levels of the biomarker that exceed the reference value by, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400% or 500% or more. In some embodiments, the level of a biomarker in a test sample is at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-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 10000-fold or more higher than the level of the biomarker in a reference sample.

[0100] As used herein, "decreased level" or "level below a reference value" means that the level of a biomarker is lower than a reference value (such as a predetermined threshold of the biomarker in a control sample). The control level is described in detail herein. Decreased levels of a biomarker include levels of the biomarker that are lower than the reference value by, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400% or 500% or more. In some embodiments, the level of a biomarker in a test sample is at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-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 10000-fold or more lower than the level of the biomarker in a reference sample.

[0101] In some embodiments, the candidate subject is a human patient having symptoms of a disease associated with a 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 complete or mainly peripheral; urticaria; erythema, pain, and swelling in the absence of evidence of infection; non-histamine-mediated edema; recurrent episodes of swelling; or combinations thereof. In other embodiments, the subject does not have symptoms of a pKal-mediated disorder, 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 at the time the sample is taken. In still other embodiments, the subject is resistant to antihistamine therapy, corticosteroid therapy, or both.

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

[0103] Considering the correlation between the level of a biomarker and such a disease, the assay methods and kits described herein can also be applied to evaluate the effectiveness of treatment for diseases related to the contact system (such as those described herein). For example, a plurality of biological samples (e.g., blood samples or plasma samples) may be collected from a subject to be treated before and after treatment, or during the course of treatment. The level of the biomarker may be measured by any of the assay methods as described herein, and accordingly, the value (e.g., amount) of the biomarker may be determined. For example, if an elevated level of the biomarker indicates that the subject has the target disease and the level of the biomarker decreases after treatment or over the course of treatment (compared to the level of the biomarker in a previously collected sample, the level of the biomarker in a subsequently collected sample), this indicates that the treatment is effective. As another example, if a decreased level of the biomarker indicates that the subject has the target disease and the level of the biomarker increases after treatment or over the course of treatment (compared to the level of the biomarker in a previously collected sample, the level of the biomarker in a subsequently collected sample), this 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 frequency of the therapeutic agent is administered to the identified subject. In some embodiments, the dosage or frequency of administration of the therapeutic agent is maintained, reduced, or discontinued in a subject identified as responding to treatment or not in need of further treatment. Alternatively, a different treatment may be applied to a subject found not to respond to the initial treatment.

[0105] In other embodiments, the value of a biomarker or set of biomarkers can also be used to identify that the disorder is 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 having the target disease may be measured by a suitable method (e.g., those described herein such as mass spectrometry, chromatography, immunoassay, etc.). If the level of the biomarker deviates from a reference value (e.g., is elevated or decreased), this indicates that a pKal inhibitor may be effective in treating the disease. If the disease is identified as being sensitive to a pKal inhibitor (treatable by a pKal inhibitor), the method may further comprise administering to the subject having the disease an effective amount of a pKal inhibitor (such as 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 is a method of assessing the severity or the condition of a disorder associated with the contact system. For example, as described herein, HAE may be in a quiescent state (baseline state) during which the subject may not experience symptoms of the disease. HAE attacks are typically recurrent episodes that can last from 2 to 5 days during which the subject may experience pain and swelling, for example, in the hands, feet, face, gastrointestinal tract, genitalia and pharynx (throat). In some embodiments, the level of one or more biomarkers indicates whether a subject is going to experience an HAE attack, is experiencing an HAE attack, or is about to experience an HAE attack soon. In some embodiments, the method involves comparing the level of a biomarker in a sample obtained from a subject having 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 baseline state or a sample obtained from the same subject during an HAE attack).

[0107] Other aspects of the disclosure provide methods for assessing the risk of a disease attack (e.g., an HAE attack). As described herein, an HAE attack is typically a recurrence episode 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 of having an HAE attack. In some embodiments, the method involves comparing the level of a biomarker in a sample obtained from a subject having 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 a basal state or a sample obtained from the same subject during an HAE attack). For example, the level of a biomarker in another sample from the subject may indicate that the level of the biomarker associated with an HAE attack is elevated (or decreased) compared to when the subject is at risk of having an HAE attack.

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

[0109] (iii) Non-clinical uses Furthermore, any level of the set of biomarkers described herein may be used for research purposes. Although a number of diseases associated with the contact activation system have been identified, there is a possibility that other diseases are mediated by similar mechanisms or involve similar components. In some embodiments, the methods described herein may be used to identify a disease as being associated with the contact activation system or a component of the contact activation system. In some embodiments, the methods described herein may be used to study the mechanism (e.g., discovery of a novel biological pathway or process involved in the development of the disease) or progression of a disease.

[0110] In some embodiments, the levels of the set of biomarkers as described herein may be utilized in the development of new therapies 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 a new therapy or the progression of a disease in a subject before, during, or after a new treatment.

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

[0112] In some embodiments, the binding substance is an oligonucleotide as described herein that is complementary to an RNA biomarker. In some embodiments, the binding substance 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 and enables amplification of the RNA biomarker when subjected to a method such as polymerase chain reaction. In some embodiments, the amplification product may be detected directly, for example, by detecting a dye, fluorophore, or other indicator substance that intercalates into nucleic acids during polymerase chain reaction. In some embodiments, at least one of the oligonucleotides is conjugated to a detectable label.

[0113] In some embodiments, the RNA biomarker is measured using a nucleic acid hybridization method. In some embodiments, the binding substance is an oligonucleotide that is complementary to the RNA biomarker and that hybridizes to the RNA biomarker in a sample (e.g., a biological sample or a sample from a polymerase chain reaction) if the RNA biomarker is present therein. 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 microarray.

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

[0115] In some embodiments, the kit or apparatus further includes 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, beads, slide, or 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 of 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 coating buffer, blocking buffer, washing buffer and / or stopping buffer).

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

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

[0119] The label or insert indicates that the kit is used to evaluate the 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 within a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (e.g., sealed Mylar or plastic bags), etc. Also contemplated are packages for use in combination with certain devices such as inhalers, nasal delivery devices (e.g., atomizers) or infusion devices (such as mini-pumps). The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper penetrable 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 having a stopper penetrable by a hypodermic needle).

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

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

[0123] In some embodiments, the methods described herein provide a method of identifying a subject as a candidate for prophylactic treatment. As used herein, "prophylactic" 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 comprises administering prophylactic treatment to the subject. Any of the therapeutic agents described herein (e.g., a pKal inhibitor such as lanadelumab). In some embodiments, the levels of a set of RNA biomarkers in a sample obtained from a subject indicate that the patient has or is at risk of having HAE (e.g., by comparing the levels of the RNA biomarkers to the levels in a reference or control sample). Any subject who has or is at risk of having HAE may be administered prophylactic treatment. The selection of an appropriate therapeutic agent and dosing regimen for prophylactic treatment will be apparent to those skilled in the art.

[0124] In some embodiments, the method comprises either or both of the selection or administration of 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 an assay (e.g., the 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, an antagonist of the bradykinin B2 receptor is administered to the subject. In some embodiments, C1-INH is administered to the subject.

[0126] Therapeutic agents (e.g., kallikrein inhibitors, bradykinin B2 receptor inhibitors and / or C1-INH) may be given together with another therapy as part of a combination therapy for the treatment of diseases or conditions involving the contact activation system. The combination therapy (e.g., combination therapy with one or more of a kallikrein inhibitor, bradykinin B2 receptor antagonist or C1-INH replacement agent, e.g., combination therapy with one or more of a kallikrein inhibitor, bradykinin B2 receptor antagonist or C1-INH replacement agent and another therapy) may be provided in a plurality of different configurations. The first agent may be administered before or after the implementation of the other therapy. In some situations, the first agent and another therapy (e.g., a therapeutic agent) are given simultaneously or in close temporal proximity (e.g., during the same treatment session, at short injection intervals). The first agent and the other therapy may also be given at longer time intervals.

[0127] Therapeutic agent Plasma kallikrein-binding substances (e.g., binding proteins, e.g., polypeptides, e.g., inhibitory polypeptides, e.g., antibodies, e.g., inhibitory antibodies, or other binding substances (e.g., small molecules)) are useful therapeutic agents for various diseases and conditions (e.g., diseases and conditions involving plasma kallikrein activity). For example, in some embodiments, the diseases and conditions involving plasma kallikrein activity are hereditary angioedema (HAE). In some embodiments, plasma kallikrein-binding substances such as plasma kallikrein inhibitors are administered to subjects at risk of or suffering from diseases associated with the contact activation system.

[0128] Some useful proteinaceous 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 is folded such that the first and sixth cysteines, the second and fourth, and the third and fifth cysteines form disulfide bonds (e.g., in a Kunitz domain having 58 amino acids, the cysteines may be at positions corresponding to amino acids 5, 14, 30, 38, 51 and 55 of the BPTI homology sequence presented below, and the disulfides may be formed between cysteines at positions 5 and 55, 14 and 38, and 30 and 51), or, if two disulfides are present, they may be formed between the corresponding subsets of those cysteines. The spacing between each pair of cysteines can be within 7, 5, 4, 3, 2, 1 or 0 amino acids of the spacing between the positions corresponding to 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 for indicating a particular position in any common Kunitz domain. Comparison of a Kunitz domain of interest with BPTI can be performed by identifying the best aligned alignment that maximizes the number of matching cysteines.

[0129] The (high-resolution) 3D structure of the knotted domain of BPTI is known. One of the X-ray structures 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 knotted domains are known. Known human homologs include the three knotted 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 knotted domains of inter-α-trypsin inhibitor APP-I (Kido et al. J. Biol. Chem. (1988) 263(34):18104-18107), the knotted domain of collagen, the three knotted domains of TFPI-2 (Sprecher et al., PNAS USA (1994) 91:3353-3357), the knotted domain of hepatocyte growth factor activator inhibitor type 1, the knotted domain of hepatocyte growth factor activator inhibitor type 2, and the knotted domain described in US Patent Application Publication No. 2004-0152633. LACI is a human serum phosphoglycoprotein with a molecular weight of 39 kDa that contains three knotted domains (amino acid sequence in Table 2).

[0130]

Table 2

[0131] The above-mentioned knotted domains are designated as LACI-K1 (residues 50-107), LACI-K2 (residues 121-178), and LACI-K3 (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 a mutagenesis study in which the P1 residue of each of the three knotted domains was altered. LACI-K1 inhibits factor VIIa (F.VIIa) when F.VIIa forms a complex with tissue factor, and LACI-K2 inhibits factor Xa.

[0132] Proteins containing exemplary knotted domains include the following (the numbers in parentheses are SWISS-PROT accession numbers):

Chemical formula

[0133] Knitted domains can be identified from an array database using various methods. For example, a known amino acid sequence, consensus sequence, or motif of a knitted domain (e.g., ProSite Motif) can be searched against, for example, the Pfam database of HMMs (Hidden Markov Models) using BLAST (e.g., using default parameters for Pfam searches; against the SMART database; or against the ProDom database), the GenBank sequence database (National Center for Biotechnology Information, National Institutes of Health, Bethesda MD). For example, the Pfam accession number PF00014 of Pfam Release 9 provides numerous knitted domains and the HMMs for identifying knitted domains. The 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 profiling with the Hidden Markov Model of 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 the automatic 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 the consensus sequence of each domain. Prosite lists knotted domains as motifs and identifies proteins containing knotted domains. See, for example, Falquet et al. Nucleic Acids Res. (2002) 30:235-238.

[0134] The knotted domain interacts with the target protease mainly using the amino acids within two loop regions ("binding loops"). The first loop region is around the residues corresponding to amino acids 13 - 20 of BPTI. The second loop region is around the residues corresponding to amino acids 31 - 39 of BPTI. In an exemplary library of the knotted domain, one or more amino acid positions within the first and / or second loop regions are altered. When screening for a knotted domain that interacts with kallikrein, or when selecting variants with improved affinity, particularly useful altered positions include positions 13, 15, 16, 17, 18, 19, 31, 32, 34, and 39 with respect to the sequence of BPTI. At least some of these positions are expected to be in close contact with the target protease. Also, it is useful to alter other positions (e.g., positions adjacent to the aforementioned positions in the three-dimensional structure).

[0135] The "framework region" of the knotted domain is defined as the residues that are part of the knotted domain, specifically excluding the residues within the first and second binding loop regions (i.e., around the residues corresponding to amino acids 13 - 20 of BPTI and amino acids 31 - 39 of BPTI). Conversely, residues outside the binding loops may allow a wider range of amino acid substitutions (e.g., conservative substitutions and / or non-conservative substitutions).

[0136] In one embodiment, these knotted domains are variant forms of a loop structure that includes knotted domain 1 of human lipoprotein-associated coagulation inhibitor (LACI). LACI contains three clearly defined internal peptide loop structures that are paradigmatic knotted domains (Girard, T. et al., Nature (1989) 338:518-520). Variants of LACI's knotted domain 1 as described herein have been screened and isolated, and these 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 knotted domains to obtain other knotted domains that interact with kallikrein (e.g., plasma kallikrein). A useful modulator of kallikrein function, as determined using kallikrein binding assays and kallikrein inhibition assays, typically binds to and / or inhibits kallikrein.

[0137] In some embodiments, 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 agents are disclosed in U.S. Patent Nos. 5,795,865, 5,994,125, 6,057,287, 6,333,402, 7,628,983, 8,283,321, 7,064,107, 7,276,480, 7,851,442, 8,124,586, 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 an inhibitory peptide. In some embodiments, the inhibitory peptide is ecallantide (also referred to as DX-88 or KALBITOR®; SEQ ID NO: 3). In some embodiments, the kallikrein inhibitor comprises, or consists of, a sequence of about 58 amino acids of amino acids 3-60 of SEQ ID NO: 3, or the DX-88 polypeptide having 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 only contain an antigen-binding fragment (e.g., Fab fragment, F(ab’)2 fragment or scFv fragment). The binding protein may contain 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, CDR-grafted antibody, chimeric antibody, deimmunized antibody or in vitro generated antibody, and optionally may contain a constant region derived from the sequence of a human germline immunoglobulin. 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 content of which is incorporated herein by reference. In some embodiments, the kallikrein-binding protein is 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 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-H06, X115-A03, X115-D01, X115-F02, X124-G01 (also referred to herein as DX-2930 or ranalizumab), 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 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 ranalizumab. See U.S. Patent Application Publication No. 20110200611 and U.S. Patent Application Publication No. 20120201756, which are incorporated herein by reference.

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

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

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

[0144] In some embodiments, the plasma kallikrein inhibitor may have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the plasma kallikrein inhibitors described herein. In some embodiments, the plasma kallikrein inhibitor may have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% 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 the plasma kallikrein inhibitors described herein. In some embodiments, the plasma kallikrein inhibitor may have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% 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 the plasma kallikrein inhibitors described herein. In some embodiments, the plasma kallikrein inhibitor may have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity in the constant regions (e.g., CH1, CH2, CH3 and / or CL1) to the plasma kallikrein inhibitors described herein.

[0145] In one aspect, a 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., an antagonist) is administered to a subject. Exemplary bradykinin B2 receptor antagonists include icatibant (Firazyr®), a peptidomimetic that contains 10 amino acids that block the binding of native bradykinin to the bradykinin B2 receptor.

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

[0148] Without further elaboration, based on the above description, those skilled in the art should be able to make the fullest use of the present disclosure. Accordingly, the following specific embodiments should be construed as merely illustrative and not as limiting the other parts of the present disclosure in any way. All publications cited herein are hereby incorporated by reference for the purposes or subject matter referred to herein.

Examples

[0149] Examples 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, circulating small RNAs present in plasma samples obtained from patients with HAE were analyzed in comparison with samples obtained from healthy individuals. Circulating citrated plasma was collected from healthy individuals (n = 3) as well as patients with HAE during the disease quiescent state (“baseline”; N = 19) and patients with HAE during an edematous attack (“attack”; N = 20). RNA was extracted from the plasma samples. Library fragments of 128 - 158 nucleotides were sequenced using the HiSeq 2500 sequencing system (Illumina). Reads were demultiplexed, adapter sequences were removed, and unique sequences with read counts exceeding 5 were created. Non-redundant sequences were sequence-compared with the reference genome (hg19) using the sequence alignment tool Bowtie2 (bowtie-bio.sourceforge.net / bowtie2 / manual.shtml), and sequences with perfect alignments and alignments with 1 nucleotide mismatch were further sequence-compared with databases of cDNA, non-coding RNA, miRNA, and piRNA. RNASeq data identified 1,811 detectable human RNA transcripts and 457 human microRNAs (≥10 reads in ≥25% of the samples). The abundance of each of the detected RNAs was compared between samples from healthy individuals and samples from patients with HAE. RNA transcripts encoding specific mitochondrial proteins were found to be significantly elevated in samples from HAE patients compared to samples from healthy individuals (Figure 1). Furthermore, RNASeq analysis identified 24 microRNAs that were differentially expressed in samples from HAE patients compared to samples from healthy individuals. The identified RNA transcripts and microRNAs were verified using RT-qPCR on separate samples obtained from healthy individuals (N = 20), patients with HAE during the disease quiescent state (N = 34), and patients with HAE during an edematous attack (N = 18).

[0150] As shown in Figure 1, in the initial RNASeq as well as in the RT-qPCR analysis, the RNA transcripts of the mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 3 (MT-ND3) and the mitochondrially encoded cytochrome c oxidase III were higher in samples from patients with HAE (during an attack or during a quiescent state) 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. Also, 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 reduced in samples from HAE patients during an attack compared to HAE patients in a basal state (Panel C of Figure 2).

[0152] Any of the RNAs identified herein (e.g., RNAs having a significant fold change between HAE patients and healthy individuals) may be used as a biomarker for diseases associated with the contact activation system (e.g., HAE) in methods for identifying patients at risk of diseases associated with the contact activation system (e.g., HAE), for selecting candidates for treatment, for monitoring disease progression or condition, for assessing the effectiveness of treatment for a disease, for determining the course of treatment, for determining whether a disease or disorder is associated with the contact activation system, and / or for research purposes (including research on the mechanisms of diseases that may be utilized for the development of new therapies), either individually or in combination (as a set of biomarkers).

[0153] Other embodiments All of the features disclosed herein may be combined in any combination. Each of the features disclosed herein may be replaced by alternative features that serve the same purpose, equivalent purpose, or similar purpose. Accordingly, unless otherwise specified, each of the disclosed features is merely an example of a general series of equivalent or similar features.

[0154] From the above description, those skilled in the art can easily identify the essential features of the present disclosure and can make various changes and modifications to the present disclosure to adapt it to various applications and conditions without departing from its spirit and scope. Accordingly, other embodiments also fall within the scope of the claims.

[0155] Equivalents and scope Those skilled in the art will recognize many equivalents to the particular embodiments of the present disclosure described herein or will be able to confirm them using only routine experimentation. 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" may mean one or more unless otherwise indicated or unless the context clearly dictates otherwise. A claim or description that includes "or" between one or more members of a group is considered to be satisfied if, unless otherwise indicated or unless the context clearly dictates otherwise, one, two or more, or all of the members of the group are present in, used in, or relevant to a given product or process. The present disclosure encompasses embodiments where exactly one member of the group is present in, used in, or relevant to a given product or process. The present disclosure encompasses embodiments where two or more or all of the members of the group are present in, used in, or relevant to a given product or process.

[0157] Furthermore, the present disclosure encompasses all variations, combinations, and substitutions in which one or more limitations, elements, clauses, and descriptive terms from one or more of the recited 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 on the same base claim. When elements are presented as a list (e.g., in Markush group form), each subgroup of the elements is also disclosed, and any element(s) may be deleted from the group. Generally, when the present disclosure or an aspect of the present disclosure is referred to as including a particular element and / or feature, it should be understood that a particular embodiment of the present disclosure or aspect of the present disclosure consists of or consists essentially of such element and / or feature. For the sake of brevity, those embodiments are not specifically described herein by those words. It should also be noted that the terms "comprising" and "containing" are intended to be open and allow the inclusion of additional elements or steps. When a range is given, the endpoints are included. Furthermore, unless otherwise indicated or not apparent from the context and the understanding of one of ordinary skill in the art, values expressed as a range may be assumed to be any specific value or sub-range within the recited range, to the tenth of a unit of the lower limit of the range, in various embodiments of the present disclosure, unless the context clearly indicates otherwise.

[0158] This application references various issued patents, published patent applications, academic papers, and other publications, all of which are hereby incorporated by reference into this specification. In the event of any conflict between any of the incorporated references and this specification, this specification shall prevail. Furthermore, any particular embodiment of the present disclosure that is included in the prior art may be explicitly excluded from one or more of the claims. Such embodiments may be considered known to one of ordinary skill in the art, and thus may be excluded even if such exclusion is not explicitly stated in this specification. Any particular embodiment of the present disclosure may be excluded from any claim for any reason, regardless of whether it is related to the existence of the prior art.

[0159] One of ordinary skill in the art will recognize many equivalents to the specific embodiments described herein or will be able to ascertain them using only routine experimentation. 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. One of ordinary skill in the art will understand that various changes and modifications may be made to this description without departing from the spirit or scope of the disclosure as defined by the following claims.

Claims

1. (i) preparing a biological sample obtained from a subject having, suspected of having, or at risk of having an acute attack of hereditary angioedema (HAE); (ii) measuring the levels of a set of RNA biomarkers comprising RPL23 and at least one RNA biomarker selected from the group consisting of hsa-miR-16-5p, hsa-miR-19a-3p, hsa-miR-20a-5p, hsa-miR-17-5p, hsa-miR-28-5p, hsa-miR-423-3p, hsa-miR-26a-5p, hsa-miR-1307-3p, hsa-miR-335-3p, hsa-miR-139-5p, hsa-miR-485-5p, hsa-miR-26b-5p, hsa-miR-885-5p, hsa-miR-361-3p, hsa-miR-769-5p, hsa-miR-140-5p, hsa-miR-485-3p, hsa-miR-889-3p, hsa-miR-941, hsa-miR-328-3p, and hsa-miR-484; A method for analyzing a sample, comprising:

2. The method according to claim 1, wherein the set of RNA biomarkers consists of 2 to 10 RNA biomarkers.

3. The method according to claim 1 or 2, wherein the biological sample is a serum sample or a plasma sample.

4. The method according to any one of claims 1 to 3, wherein the HAE is type I HAE or type II HAE.

5. The method according to any one of claims 1 to 4, wherein the set of RNA biomarkers further comprises messenger RNA encoding cytochrome c oxidase III (MT-CO3) encoded by mitochondria and / or messenger RNA encoding NADH:ubiquinone oxidoreductase core subunit 3 (MT-ND3) encoded by mitochondria.

6. The method according to any one of claims 1 to 5, wherein step (ii) involves polymerase chain reaction and / or nucleic acid hybridization.

7. The method according to any one of claims 1 to 6, wherein the subject is a human patient.

8. The method according to any one of claims 1 to 7, wherein the level of the set of RNA biomarkers of the subject deviates from the level of the same set of RNA biomarkers of a control subject, indicating that the subject has an acute attack of HAE.

9. The method according to claim 8, wherein the deviation of the level of the set of RNA biomarkers of the subject includes an elevated level of RPL23 when compared to the level of the same set of RNA biomarkers of a control subject.

10. The method according to claim 9, wherein the deviation of the level of the set of RNA biomarkers of the subject further includes the level of one or more of hsa-miR-16-5p, hsa-miR-19a-3p, hsa-miR-20a-5p, hsa-miR-17-5p, hsa-miR-885-5p, hsa-miR-140-5p, hsa-889-3p, hsa-miR-941, hsa-miR-484, or any combination thereof that is elevated when compared to the level of the same set of RNA biomarkers of a control subject.

11. The method according to claim 9, wherein the deviation of the level of the set of RNA biomarkers of the subject further includes the level of one or more of hsa-miR-1307-3p, hsa-miR-335-3p, hsa-miR-485-5p, hsa-miR-28-5p, hsa-miR-423-3p, hsa-miR-26a-5p, hsa-miR-139-5p, hsa-miR-26b-5p, hsa-miR-361-3p, hsa-miR-769-5p, hsa-miR-485-3p, hsa-miR-328-3p, or any combination thereof that is decreased when compared to the level of the same set of RNA biomarkers of a control subject.

12. The subject is a human patient undergoing treatment for HAE, and the method further includes evaluating the effectiveness of the treatment based on the level of the set of RNA biomarkers, wherein the deviation of the level of the set of RNA biomarkers of the subject from that of a control subject indicates the effectiveness of the treatment. The method according to any one of claims 1 to 11.

13. The method according to any one of claims 1 to 12, wherein the level of the set of RNA biomarkers of the subject deviates from the level of the same set of RNA biomarkers of a control subject, indicating that the subject is a candidate for the treatment of HAE.

14. The method according to any one of claims 1 to 13, wherein the at least one RNA biomarker 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.

15. A kit for analyzing a sample of a subject having, suspected of having, or at risk of having an acute attack of hereditary angioedema (HAE), comprising: (i) a first binding substance specific for a first RNA biomarker that is RPL23; (ii) a second binding substance specific for a second RNA biomarker selected from the group consisting of hsa-miR-16-5p, hsa-miR-19a-3p, hsa-miR-20a-5p, hsa-miR-17-5p, hsa-miR-28-5p, hsa-miR-423-3p, hsa-miR-26a-5p, hsa-miR-1307-3p, hsa-miR-335-3p, hsa-miR-139-5p, hsa-miR-485-5p, hsa-miR-26b-5p, hsa-miR-885-5p, hsa-miR-361-3p, hsa-miR-769-5p, hsa-miR-140-5p, hsa-miR-485-3p, hsa-miR-889-3p, hsa-miR-941, hsa-miR-328-3p, and hsa-miR-484; comprising the first binding substance comprises an oligonucleotide capable of specifically hybridizing to the first RNA biomarker or cDNA corresponding to the first RNA biomarker, and the second binding substance comprises an oligonucleotide capable of specifically hybridizing to the second RNA biomarker or cDNA corresponding to the second RNA biomarker; kit.

16. The kit according to claim 15, wherein the first binding substance is an oligonucleotide that can specifically hybridize to the first RNA biomarker or cDNA corresponding to the first RNA biomarker, and / or the second binding substance is an oligonucleotide that can specifically hybridize to the second RNA biomarker or cDNA corresponding to the second RNA biomarker.

17. The kit according to claim 15 or 16, wherein the first binding substance, the second binding substance, or both are conjugated to a label, and / or the first binding substance and the second binding substance are immobilized on a support member.

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