MICRORNAS AS PREDICTORS OF RESPONSE TO ANTI-IGE THERAPIES IN CHRONIC SPONTANEOUS URTICARIA

CROSS-REFERENCE TO REUATED APPUICATIONS [0001] This application claims benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63/128,515 filed December 21, 2020 and U.S. Provisional Application No. 63/036,663 filed June 9, 2020, the contents of each of which are incorporated herein by reference in their entirety.

TECHNICAU FIEUD

[0002] The technology described herein relates to the treatment of chronic spontaneous urticaria.

BACKGROUND

[0003] Chronic spontaneous urticaria (CSU) is a disease characterized by itching and hives on the skin of a subject that can last for more than 6 weeks. The treatment of choice for CSU is antihistamines although other treatments are available, such as those targeting IgE/FceRI-mediated mast cell and basophil activation by anti-IgE antibodies. Anti-IgE treatment has emerged as a novel treatment strategy for patients with Hi-antihistamine-resistant (refractory) CSU. However, approximately one-third of patients fail to respond to anti-IgE antibody treatments. Thus, there is an unmet clinical need for biomarkers predictive of response to anti-IgE therapies for the treatment of CSU and related skin diseases.

SUMMARY

[0004] The technology described herein relates to the discovery of biomarkers indicating whether a subject’s CSU will respond to anti-IgE therapy. It was discovered that certain miRNAs are differentially expressed in plasma exosomes, and basophils of subjects who do and do not respond to anti-IgE therapy, and it was also discovered that baseline number of circulating basophils can also be indicative of treatment response. Described herein are applications of these discoveries, which include diagnostic and prognostic methods and kits, and methods of treatment for CSU, including CSU that is refractory to antihistamine treatment.

[0005] In one aspect, described herein is a method of predicting a subject’s response to an anti- IgE therapeutic agent for the treatment of spontaneous urticaria (CSU), the method comprising: receiving the results of an assay performed on a sample of plasma exosomes from the subject to detect the level of one or more miRNAs relative to a reference level, wherein detection of an increase in the level of one or more of the miRNAs miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR- 6877-3p, or miR-3976 or a decrease in the level of miR-141-3p relative to the reference level indicates the subject’s CSU is likely to respond to an anti-IgE therapeutic agent.

[0006] In another aspect, described herein is a method of predicting a subject’s response to an anti-IgE therapeutic agent for the treatment of spontaneous urticaria (CSU), the method comprising: receiving the results of an assay performed on a sample of plasma extracellular vesicles from the subject to detect the level of one or more miRNAs relative to a reference level, wherein detection of an increase in the level of one or more of the miRNAs miR-6499-5p, miR-7848-3p, miR-4494, miR- 450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in the level of miR-141-3p relative to the reference level indicates the subject’s CSU is likely to respond to an anti-IgE therapeutic agent.

[0007] In another aspect, described herein is a method of predicting a subject’s response to an anti-IgE therapeutic agent for the treatment of chronic spontaneous urticaria (CSU), the method comprising: receiving the results of an assay performed on a sample of basophils from the subject to detect the level of one or more miRNAs relative to a reference level, wherein detection of an increase in the level of one or more of the miRNAs miR-1200, miR-1236-3p and miR-4664-3p relative to the reference level indicates the subject’s CSU is likely to respond to an anti-IgE therapeutic agent.

[0008] In another aspect, described herein is a method of predicting a subject’s response to an anti-IgE therapeutic agent for the treatment of chronic spontaneous urticaria (CSU), the method comprising: performing an assay to detect the level of one or more miRNAs selected from 6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, miR-3976 and miR-141-3p in a sample of plasma exosomes from the subject; wherein detection of an increase in the level of one or more of miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in the level of miR-141-3p relative to a reference level indicates the subject’s CSU is likely to respond to an anti-IgE therapeutic agent.

[0009] In another aspect, described herein is a method of predicting a subject’s response to an anti-IgE therapeutic agent for the treatment of chronic spontaneous urticaria (CSU), the method comprising: performing an assay to detect the level of one or more miRNA species selected from the group consisting of miR-1200, miR-1236-3p and miR-4664-3p in a sample of basophils from the subject; wherein detection of an increase in the level of one or more of the miRNAs relative to a reference level indicates the subject’s CSU is likely to respond to an anti-IgE therapeutic agent.

[0010] In another aspect, described herein is a method of treating chronic spontaneous urticaria in a subject in need thereof, the method comprising: (a) receiving results of an assay to detect the level of one or more miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, miR-3976 and miR-141-3p in an exosome sample from the subject; and (b) when the level of one or more of miR-6499-5p, miR-7848-3p, miR-4494, miR-450a- 2-3p, miR-6877-3p, and miR-3976 is above a reference level, and/or when the level of miR-141-3p is below the reference level, administering an anti-IgE therapeutic agent; and (c) when the level of one or more of miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 is at or below the reference level, and/or when the level of miR-141-3p is at or above the reference level, administering a non-steroidal immunosuppressant.

[0011] In another aspect, described herein is a method of reducing at least one symptom of chronic spontaneous urticaria in a subject in need thereof, the method comprising: (a) receiving results of an assay to detect the level of one or more miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, miR-3976 and miR-141-3p in an exosome sample from the subject; and (b) when the level of one or more of miR-6499-5p, miR- 7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 is above a reference level, and/or when the level of miR-141-3p is below the reference level, administering an anti-IgE therapeutic agent; and (c) when the level of one or more of miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2- 3p, miR-6877-3p, and miR-3976 is at or below the reference level, and/or when the level of miR-141- 3p is at or above the reference level, administering a non-steroidal immunosuppressant, thereby reducing the at least one symptom of chronic spontaneous urticaria. In one embodiment of this aspect and all other aspects provided herein, the at least one symptom of CSU comprises itching, redness, swelling, wheal and flare rash, hives, or burning pain.

[0012] In another aspect, described herein is a method of treating chronic spontaneous urticaria or reducing at least one symptom of CSU in a subject in need thereof, the method comprising: (a) performing an assay to detect the level of one or more miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, miR-3976 and miR-141-3p in an exosome sample from the subject; and (b) when the level of one or more of miR- 6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 is above a reference level, and/or when the level of miR-141-3p is below the reference level, administering an anti-IgE therapeutic agent; and (c) when the level of one or more of miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 is at or below the reference level, and/or when the level of miR-141-3p is at or above the reference level, administering a non-steroidal immunosuppressant. [0013] In another aspect, described herein is a method of treating chronic spontaneous urticaria or reducing at least one symptom of CSU in a subject in need thereof, the method comprising: (a) performing an assay to detect the level of one or more miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, miR-3976 and miR-141-3p in an extracellular vesicle sample from the subject; and (b) when the level of one or more of miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 is above a reference level, and/or when the level of miR-141-3p is below the reference level, administering an anti-IgE therapeutic agent; and (c) when the level of one or more of miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 is at or below the reference level, and/or when the level of miR-141-3p is at or above the reference level, administering a non steroidal immunosuppressant.

[0014] In another aspect, described herein is a method of treating chronic spontaneous urticaria (CSU) or reducing at least one symptom of CSU, the method comprising: (a) receiving the results of an assay performed on a sample of basophils from the subject to detect the level of one or more miRNAs selected from miR-1200, miR-1236-3p and miR-4664-3p, and (b) when the level of one or more of miR-1200, miR-1236-3p and miR-4664-3p is above a reference level, administering an anti- IgE therapeutic agent; and (c) when the level of one or more of miR-1200, miR-1236-3p and miR- 4664-3p is at or below the reference level, administering a non-steroidal immunosuppressant.

[0015] In another aspect, described herein is a method of predicting a subject’s response to an anti-IgE therapeutic agent for the treatment of chronic spontaneous urticaria (CSU), the method comprising: (a) performing an assay to detect the level of one or more miRNA species selected from the group consisting of miR-1200, miR-1236-3p and miR-4664-3p in a sample of basophils from the subject; and (b) when the level of one or more of miR-1200, miR-1236-3p and miR-4664-3p is above a reference level, administering an anti-IgE therapeutic agent; and (c) when the level of one or more of miR-1200, miR-1236-3p and miR-4664-3p is at or below the reference level, administering a non steroidal immunosuppressant.

[0016] Another aspect provided herein relates to a method of treating CSU or reducing at least one symptom of CSU, the method comprising administering an anti-IgE therapeutic agent to a subject with CSU and comprising an increased level of one or more miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 and/or a reduced level of miR-141-3p.

[0017] Another aspect provided herein relates to a method of treating CSU or reducing at least one symptom of CSU, the method comprising administering a non-steroidal immunosuppressant to a subject with CSU and comprising a decreased level of one or more of miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 is at or below the reference level, and/or an increased level of miR-141-3p.

[0018] In one embodiment of any of the aspects, two or more of an increase in miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the reference level are detected.

[0019] In another embodiment of any of the aspects, three or more of an increase in miR-6499- 5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141- 3p relative to the reference level are detected.

[0020] In another embodiment of any of the aspects, four or more of an increase in miR-6499- 5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141- 3p relative to the reference level are detected. [0021] In another embodiment of any of the aspects, five or more of an increase in miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the reference level are detected.

[0022] In another embodiment of any of the aspects, six or more of an increase in miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the reference level are detected.

[0023] In another embodiment of any of the aspects, an increase in each of miR-6499-5p, miR- 7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 and a decrease in miR-141-3p relative to the reference level are detected.

[0024] In another embodiment of any of the aspects, the reference is the level of the one or more miRNAs in a plasma exosome sample from one or more subjects with CSU whose disease did not respond to an anti-IgE therapeutic agent. In another embodiment of any of the aspects, the reference is the level of the one or more miRNAs in an extracellular vesicle sample from one or more subjects with CSU whose disease did not respond to an anti-IgE therapeutic agent. In another embodiment of any of the aspects, the reference is the level of the one or more miRNAs in a plasma sample from one or more subjects with CSU whose disease did not respond to an anti-IgE therapeutic agent. It is contemplated that one can also use an individual whose CSU does respond to anti-IgE therapy as a reference. In such situation, a level of miRNA marker at or above the level of miR-6499-5p, miR- 7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 in the responder’s plasma exosomes, or a level of miR-141-3p at or below the level in the responder’s plasma exosomes is likely to be predictive of a positive response to anti-IgE therapy

[0025] In another embodiment of any of the aspects, the subject’s CSU is resistant to Hi- antihistamine therapy.

[0026] In another embodiment of any of the aspects, the anti-IgE therapeutic agent is selected from omalizumab, ligelizumab, UB-221, quilizumab, and MEDI-4212, among others.

[0027] In another embodiment of any of the aspects, the method further comprises detecting the level of basophils in a biological sample from the subject compared to a reference level. In another embodiment of any of the aspects, the method further comprises detecting the level of basophils in a blood sample from the subject. In another embodiment of any of the aspects, detecting > 21 basophils/mΐ of blood is indicative of a likely response to an anti-IgE therapeutic agent.

[0028] In another embodiment of any of the aspects, the subject has two or more miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR- 6877-3p, and miR-3976 above the reference level; and/or miR-141-3p below the reference level.

[0029] In another embodiment of any of the aspects, the subject has three or more of the miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2- 3p, miR-6877-3p, and miR-3976 above the reference level; and/or miR-141-3p below the reference level.

[0030] In another embodiment of any of the aspects, the subject has four or more of the miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2- 3p, miR-6877-3p, and miR-3976 above the reference level; and/or miR-141-3p below the reference level.

[0031] In another embodiment of any of the aspects, the subject has five or more of the miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2- 3p, miR-6877-3p, and miR-3976 above the reference level; and/or miR-141-3p below the reference level.

[0032] In another embodiment of any of the aspects, the subject has six or more of the miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2- 3p, miR-6877-3p, and miR-3976 above a reference level; and/or miR-141-3p below the reference level.

[0033] In another embodiment of any of the aspects, the subject has each of the miRNA species selected from miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 above the reference level; and miR-141-3p below the reference level.

[0034] In another aspect, described herein are methods of predicting whether a subject’s CSU will respond to or be successfully treated with an anti-IgE therapy, as well as methods of treatment based upon detection of miRNAs described herein that are differentially expressed or found in plasma exosomes and detection of miRNAs described herein that are differentially expressed or found in basophils, as well as the combination of detection of basophil number and either or both of plasma exosomal miRNA markers or basophil miRNA markers of anti-IgE response as described herein. [0035] In another aspect, described herein are methods of predicting whether a subject’s CSU will respond to or be successfully treated with an anti-IgE therapy, as well as methods of treatment based upon detection of miRNAs described herein that are differentially expressed or found in plasma extracellular vesicles and detection of miRNAs described herein that are differentially expressed or found in basophils, as well as the combination of detection of basophil number and either or both of plasma extracellular vesicle miRNA markers or basophil miRNA markers of anti-IgE response as described herein.

[0036] In another aspect, described herein is a method of predicting a subject’s response to an anti-IgE therapeutic agent for the treatment of chronic spontaneous urticaria (CSU), the method comprising: receiving the results of an assay performed on a sample of plasma exosomes from the subject to detect the level of one or more miRNAs relative to a reference, and receiving the results of an assay performed on a sample of basophils from the subject to detect the level of one or more miRNAs relative to a reference, and/or receiving the results of a blood count indicating the number of basophils in a blood sample from the subject, wherein detection of an increase in the level of one or more of the miRNAs miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 or a decrease in the level of miR-141-3p in plasma exosomes relative to the respective reference, and/or wherein the detection of an increase in the level of one or more of the miRNAs miR-1200, miR-1236- 3p and miR-4664-3p in basophils relative to the respective reference, and/or wherein the detection of a number of basophils greater than or equal to 21 basophils per microliter of blood indicates the subject’s CSU is likely to respond to an anti-IgE therapeutic agent.

[0037] In another aspect, described herein is a method of predicting a subject’s response to an anti-IgE therapeutic agent for the treatment of chronic spontaneous urticaria (CSU), the method comprising: performing an assay on a sample of plasma exosomes from the subject to detect the level of one or more miRNAs relative to a reference, and performing an assay performed on a sample of basophils from the subject to detect the level of one or more miRNAs relative to a reference, and/or performing a blood count to detect the number of basophils in a blood sample from the subject, wherein detection of an increase in the level of one or more of the miRNAs miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 or a decrease in the level of miR-141-3p in plasma exosomes relative to the respective reference, and/or wherein the detection of an increase in the level of one or more of the miRNAs miR-1200, miR-1236-3p and miR-4664-3p in basophils relative to the respective reference, and/or wherein the detection of a number of basophils greater than or equal to 21 basophils per microliter of blood indicates the subject’s CSU is likely to respond to an anti-IgE therapeutic agent.

[0038] In another aspect, described herein is a method of treating chronic spontaneous urticaria (CSU) or reducing at least one symptom of CSU in a subject, the method comprising: receiving the results of an assay performed on a sample of plasma exosomes from the subject to detect the level of one or more miRNAs relative to a reference, and receiving the results of an assay performed on a sample of basophils from the subject to detect the level of one or more miRNAs relative to a reference, and/or receiving the results of a blood count indicating the number of basophils in a blood sample from the subject, and administering to the subject an anti-IgE therapeutic agent when the level of one or more of the miRNAs miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877- 3p, and miR-3976 in plasma exosomes is increased, or the level of miR-141-3p in plasma exosomes is decreased relative to the respective reference, and/or when the level of one or more of the miRNAs miR-1200, miR-1236-3p and miR-4664-3p in basophils is increased relative to the respective reference, and/or wherein the number of basophils is greater than or equal to 21 basophils per microliter of blood, or administering to the subject a non-steroidal immunosuppressant when the level of one or more of the miRNAs miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877- 3p, and miR-3976 in plasma exosomes is not increased, or the level of miR-141-3p in plasma exosomes is not decreased relative to the respective reference, and/or when the level of one or more of the miRNAs miR-1200, miR-1236-3p and miR-4664-3p in basophils is not increased relative to the respective reference, and/or wherein the number of basophils is not greater than or equal to 21 basophils per microliter of blood. [0039] In another aspect, described herein is a kit comprising reagents permitting the quantitative detection of target miRNAs including miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR- 6877-3p, miR-3976, and miR-141-3p.

[0040] In another aspect, described herein is a kit comprising reagents permitting the quantitative detection of target miRNAs including miR-1200, miR-1236-3p and miR-4664-3p.

[0041] In another aspect, described herein is a kit comprising reagents permitting the quantitative detection of target miRNAs including miR-6499-5p, miR-7848-3p, miR-4494, miR-450a- 2-3p, miR-6877-3p, miR-3976, miR-141-3p, miR-1200, miR-1236-3p and miR-4664-3p.

[0042] In one embodiment of any of the kits, the kit further comprises reagents permitting the isolation or quantitation of basophils in a biological sample.

[0043] In another embodiment of any of the kits, the kit further comprises reagents permitting the quantitative detection of miR-1200, miR-1236-3p and miR-4664-3p target miRNAs.

[0044] In another embodiment of any of the kits, the kit further comprises reagents permitting the isolation of miRNAs.

[0045] In another embodiment of any of the kits, the kit further comprises PCR primers permitting amplification of the target miRNAs.

[0046] In another embodiment of any of the kits, the kit further comprises a reference composition comprising a known amount of target miRNA.

[0047] In another embodiment of any of the kits, the kit further comprises a reference sample, wherein the reference sample comprises miRNAs isolated from exosomes isolated from a subject with refractory CSU that did not respond to anti-IgE therapy.

[0048] In another embodiment of any of the kits, the kit further comprises one or more of the reagents arrayed on a solid support.

[0049] In another embodiment of any of the kits, the solid support comprises a microscope slide, a microwell plate, or a capillary electrophoresis plate.

[0050] In another embodiment of any of the kits, the kit further comprises a detectable label. [0051] Another aspect provided herein relates to the use of an anti-IgE therapeutic agent in the treatment of chronic spontaneous urticaria (CSU) or at least one symptom thereof in a subject in need thereof, wherein the subject is determined to be in need thereof by (i) receiving the results of an assay performed on a sample of plasma exosomes from the subject to detect the level of one or more miRNAs relative to a reference, (ii) receiving the results of an assay performed on a sample of basophils from the subject to detect the level of one or more miRNAs relative to a reference, and/or (iii) receiving the results of a blood count indicating the number of basophils in a blood sample from the subject, and when the level of one or more of the miRNAs miR-6499-5p, miR-7848-3p, miR- 4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 in plasma exosomes is increased, or the level of miR-141-3p in plasma exosomes is decreased relative to the respective reference, and/or when the level of one or more of the miRNAs miR-1200, miR-1236-3p and miR-4664-3p in basophils is increased relative to the respective reference, and/or wherein the number of basophils is greater than or equal to 21 basophils per microliter of blood.

[0052] Another aspect provided herein relates to the use of a non-steroidal immunosuppressant in the treatment of chronic spontaneous urticaria (CSU) or at least one symptom thereof in a subject in need thereof, wherein the subject is determined to be in need thereof by (i) receiving the results of an assay performed on a sample of plasma exosomes from the subject to detect the level of one or more miRNAs relative to a reference, (ii) receiving the results of an assay performed on a sample of basophils from the subject to detect the level of one or more miRNAs relative to a reference, and/or (iii) receiving the results of a blood count indicating the number of basophils in a blood sample from the subject, and when the level of one or more of the miRNAs miR-6499-5p, miR-7848-3p, miR- 4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 in plasma exosomes is not increased, or the level of miR-141-3p in plasma exosomes is not decreased relative to the respective reference, and/or when the level of one or more of the miRNAs miR-1200, miR-1236-3p and miR-4664-3p in basophils is not increased relative to the respective reference, and/or wherein the number of basophils is not greater than or equal to 21 basophils per microliter of blood.

[0053] Another aspect provided herein relates to the use of an anti-IgE therapeutic agent for the treatment of chronic spontaneous urticaria or reducing at least one symptom of CSU in a subject, wherein the subject comprises an increased level of one or more miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR- 3976 and/or a reduced level of miR-141-3p.

[0054] Another aspect provided herein relates to the use of a non-steroidal immunosuppressant for the treatment of CSU or reduction of at least one symptom of CSU in a subject, wherein the subject comprises a decreased level of one or more of miR-6499-5p, miR-7848-3p, miR-4494, miR- 450a-2-3p, miR-6877-3p, and miR-3976 is at or below the reference level, and/or an increased level of miR-141-3p.

Definitions

[0055] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Wemer Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-14664- 3p59055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties. [0056] An “agent” can be any chemical, entity or moiety, including without limitation synthetic and naturally-occurring proteinaceous and non-proteinaceous entities. In some embodiments, an agent is a nucleic acid, nucleic acid analog, protein, antibody, peptide, aptamer, oligomer of nucleic acids, amino acids, or carbohydrates including without limitation a protein, oligonucleotide, ribozyme, DNAzyme, glycoprotein, siRNAs, lipoprotein and/or a modification or combinations thereof etc. In certain embodiments, agents are small molecule chemical moieties. For example, chemical moieties include unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof. Compounds can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds.

[0057] An agent can be a molecule from one or more chemical classes, e.g., organic molecules, which may include organometallic molecules, inorganic molecules, genetic sequences, etc. Agents may also be fusion proteins from one or more proteins, chimeric proteins (for example domain switching or homologous recombination of functionally significant regions of related or different molecules), synthetic proteins or other protein variations including substitutions, deletions, insertions and other variants.

[0058] As used herein, “antibodies” or “antigen-binding fragments thereof’ include monoclonal, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, and/or antigen-binding fragments of any of the above. Antibodies can also refer to immunoglobulin molecules and immunologically active portions that contain antigen or target binding sites or “antigen-binding fragments.” The immunoglobulin molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule, as is understood by one of skill in the art. IgE primes the IgE-mediated allergic response by binding to Fc receptors found on the surface of mast cells and basophils. The structure and function of IgE are known in the art, e.g., Wu and Zarin, Nature Reviews Immunology 14, 247-259 (2014), which is incorporated herein by reference in its entirety.

[0059] As used herein, an “anti-IgE therapeutic agent” or “anti-IgE immunosuppressant” refers to any agent, antibody, or small molecule that reduces the level or activity of IgE. An anti-IgE immunosuppressant therapy can be used to neutralize free IgE, inhibit IgE production by B cells, and/or downregulate high-affinity IgE Fc receptors (FceRI) on, e.g., basophils or mast cells. Non limiting examples of anti-IgE immunosuppressants include but are not limited to: Omalizumab (E25), a glycosylated IgGl monoclonal antibody; talizumab (TNX-901), a humanized monoclonal antibody; ligelizumab (QGE031), an anti-IgE monoclonal antibody; E26; CGP56901; CGP51901; UB-221, an anti-IgE monoclonal antibody developed by United BioPharma, Inc.; MEDI-4212, quilizumab; and antigen-binding fragments and derivatives thereof. For non-limiting examples of anti-IgE therapeutics and derivatives, see, e.g., U.S. Patent Nos. 5,994,511 A, 6,172,213 Bl; US Pub. 2019/0144565 Al; WO 2019/186369A1, Kocaturk et al. Clin Transl Allergy (2017) 7:1; and Nyborg et al. Cellular & Molecular Immunology (2016) 13, 391-400; the contents of each of which are incorporated herein by reference in their entireties. Other anti-IgE immunosuppressants (see e.g., P Guntem, A Eggel. Allergy. (2020) 75(10):2491-2502) include but are not limited to: 1) anti-IgE antibody XmAb7195, a humanized, affinity maturated version of the murine parental antibody of omalizumab (MaEl 1) with a mutated Fc-part for increased binding to the inhibitory receptor FcyRIIb, developed by Xencor and in clinical trials (NCT03468790), 2) monoclonal anti-IgE antibody, called 8D6, which binds to the Cs3 domain of IgE with high affinity, 3) Omalizumab biosimilars in development such as monoclonal anti-IgE antibody CMAB007, STI-004, FB317, GBR310, CTP-39, BP001, 4) anti-IgE binders based on designed ankyrin repeat protein (DARPin) scaffolds such as DARPin bi53 _ 79 and other DARPin- based drug candidates in clinical trials (NCT02462486), 5) IgG-Fc-fusion proteins that aggregate FceRI with FcyRIIb such as GE2 (developed by Tunitas Therapeutics), DE53-Fc, and DE53-Fc mut+), and 6) anti-IgE nanobodies or single-domain antibody (sdab) to generate specific binders against IgE such as anti-IgE nanobody IgE026 or 026 sdab (Ablynx, Ghent, Belgium).

[0060] As used herein, a “non-steroidal immunosuppressant” refers to an agent that suppresses an inflammatory immune response. Non-limiting examples of non-steroidal immunosuppressants include calcineurin inhibitors, such as cyclosporine, tacrolimus, FK1012; leukotriene antagonists, such as zafirlukast, montelukast, zileuton; hydroxychloroquine, and sulfasalazine. As used herein, a non-steroidal immunosuppressant is distinct from an anti-IgE therapeutic agent.

[0061] As used herein, “chronic spontaneous urticaria,” or “CSU” refers to a disease or skin condition that causes red, swollen, itchy, and sometimes painful hives on the skin. Urticaria symptoms can spontaneously present or reoccur daily or almost daily for a period of at least 6 weeks. Complications of CSU include sleep deprivation, depression, social isolation, lack of energy, anxiety, and emotional upset. Current treatments for CSU include, but are not limited to, antihistamines, non steroidal immunosuppressants, and anti-IgE immunosuppressants.

[0062] The term "therapeutically effective amount" refers to an amount of a therapeutic as described herein, that is effective to treat a disease or disorder as the terms “treat” or “treatment” are defined herein. Amounts will vary depending on the specific disease or disorder, its state of progression, age, weight and gender of a subject, among other variables. Thus, it is not possible to specify an exact “effective amount" . However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0063] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with, a disease or disorder. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a disease or disorder. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of at least slowing of progress or worsening of symptoms that would be expected in absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e.. not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total). The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0064] The terms “decrease”, “reduce”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction", “decrease" or “inhibit” means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “reduction” or

“inhibition” does not encompass complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level.

[0065] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0066] As used herein, a "subject" is a human or a non-human animal. Usually the non-human animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.

[0067] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of diseases including diseases and disorders involving inappropriate immunosuppression. A subject can be male or female. [0068] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors. [0069] As used herein, a “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition. In one embodiment, a subject in need has or is diagnosed as having the condition.

[0070] As used herein, the terms “extracellular vesicle” or “extracellular microvesicle” can be used interchangeably with the term “exosome.”

[0071] As used herein, a “reference level” refers to the level of a given, e.g., biomarker or parameter useful as a gauge for an experimental or diagnostic measurement. In one embodiment, a reference level is the level of such marker or parameter in a normal, otherwise unaffected cell population or tissue (e.g., a biological sample obtained from a healthy subject, or a level of the marker or parameter from a sample obtained from the subject at a prior time point, e.g., a biological sample obtained from a patient prior to being diagnosed with a disease or disorder, or a biological sample from an individual that has not been contacted with a therapeutic composition. It is contemplated that one can also use a level of a biomarker or parameter from an individual whose disease or disorder, such as CSU, does not respond to anti-IgE therapy as a reference. It is also contemplated that one can alternatively use a level of a biomarker or parameter from an individual whose disease or disorder, such as CSU, does respond to anti-IgE therapy as a reference.

[0072] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectable either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified or presented as a liposome composition. The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient. The therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of an active agent used with the methods described herein that will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.

[0073] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0074] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

[0075] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0076] The term "consisting of' refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0077] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

BRIEF DESCRIPTION OF THE DRAWINGS

[0078] FIG. 1A-1E demonstrates the study plan and clinical response to omalizumab. The study plan is shown in FIG. 1A. Omalizumab injections were on Days 0, 30, and 60 with UAS7 scores and blood obtained for microRNA analysis as indicated. The area under the curve (AUC) for weekly UAS7 over the last 6 weeks of omalizumab therapy for each subject in the complete response (CR), partial response (PR), and non-response (NR) groups is shown in FIG. IB. The weekly UAS7 scores over the 12-week treatment period for the CR, PR, and NR groups are shown in FIG. 1C-1E respectively with arrows indicating omalizumab injection day. Asterix in FIG. ID indicates Subject 26 was treated with cyclosporine A for urticaria exacerbation between Day 60 and Day 90 visits.

[0079] FIG. 2A-2F shows characteristics of study participants including eosinophil and basophil levels. FIG. 2A shows the baseline characteristics of the study participants. Values are presented as N (%) for categorical variables and mean ± SD for continuous variables. Statistical differences between responders and non-responders were analyzed using Fisher exact test for categorical variables or unpaired Welch’s t-test for continuous variables. FIG. 2B shows Welch’s ANOVA comparing the baseline eosinophil levels between the three response groups: complete response (CR), partial response (PR), and non-response (NR). Welch’s ANOVA comparing the mean baseline basophil levels between the CR, PR, and NR groups is shown in FIG. 2C. FIG. 2D shows the response to omalizumab as measured by the area under curve (AUC) for the weekly urticaria activity scores (UAS7) over the 12-week treatment period (weeks 0-12) as a function of baseline basophil levels assessed by linear regression model. Baseline basophil levels were negatively correlated with the UAS7 AUC. The receiver operating characteristic (ROC) curve for baseline basophil levels obtained from all study participants is shown in FIG. 2E. The area under the ROC curve was 0.81 (95% Cl 0.61 - 1, P = 0.044). FIG. 2F compares the mean weekly urticaria activity score (UAS7) over the 12-week treatment period between basophil -high subjects (defined as those with a baseline basophil level >21 cells/pL) and basophil-low subjects (<21 cells/pL). Error bars represent standard error of the mean. Arrows indicate omalizumab injection day. There was a statistically significant difference in the mean UAS7 between the two groups (P <0.001).

[0080] FIG. 3A-3H demonstrates the differential expression of exosomal microRNAs between complete responder and non-responder groups at baseline. FIG. 3A shows a heatmap of the exosomal microRNAs differentially expressed between the complete response (CR) and non-response (NR) groups on Day 0. Expression values (CPM) were converted to z-score. The upper sections of FIG. 3B-3H show comparisons of the geometric mean expression levels for the seven differentially expressed exosomal microRNAs at baseline between the CR and NR groups. Error bars represent the standard deviation. The lower sections of FIG. 3B-3H compare the mean weekly urticaria activity score (UAS7) over the 12-week treatment period between study participants with high expression (defined as those with expression level above the geometric mean expression level for that microRNA; †) and those with low expression (defined as those with expression level below the geometric mean expression level for that microRNA; ††).

[0081] FIG. 4A-4B demonstrates the differential expression of basophil microRNAs between complete responder and non-responder groups at baseline. FIG. 4A shows a heatmap of the basophil microRNAs differentially expressed between the complete response (CR) and non-response (NR) groups on Day 0. Expression values (CPM) were converted to z-scores. The upper panels show geometric mean expression levels (±SD) between CR and NR at baseline. Lower panels show UAS7 scores (±SE) over 12-week treatment period of subjects with high (†) versus low (††) expression. Statistical significance was determined by LRT after fitting a linear mixed model with a random intercept for each subject and fixed effects defined by an interaction between a 2 degrees of freedom spline on the observational week and the expression level (high or low).

[0082] FIG. 5 shows modulation of Tec Kinase signaling pathway by exosomal microRNAs differentially expressed between complete responders and non-responders. Ingenuity Pathway Analysis (IPA) was used to identify mRNA targets for the seven differentially expressed microRNAs (i.e., miR-450a-2-3p, miR-6877-3p, miR-4494, miR-6499-5p, miR-7848-3p-3p, miR-3976, and miR- 141-3p) between the complete responder and non-responder groups on Day 0. IPA identified Tec Kinase Signaling pathway with significant modulation by the seven differentially expressed microRNAs (FDR = 0.003). The predicted effects of the differential microRNA expression on this pathway are shown.

[0083] FIG. 6 shows modulation of Oncostatin M signaling pathway by exosomal microRNAs differentially expressed between complete responders and non-responders. Ingenuity Pathway Analysis (IPA) was used to identify mRNA targets for the seven differentially expressed microRNAs (i.e., miR-450a-2-3p, miR-6877-3p, miR-4494, miR-6499-5p, miR-7848-3p-3p, miR-3976, and miR- 141-3p) between the complete responder and non-responder groups on Day 0. IPA showed modulation of the Oncostatin M Signaling pathway by the seven differentially expressed microRNAs (FDR = 0.062). The predicted effects of the differential microRNA expression on this pathway are shown.

[0084] FIG. 7 shows modulation of IL-17a homodimer signaling pathway by exosomal microRNAs differentially expressed between complete responders and non-responders.

Ingenuity Pathway Analysis (IPA) was used to identify mRNA targets for the seven differentially expressed microRNAs (i.e., miR-450a-2-3p, miR-6877-3p, miR-4494, miR-6499-5p, miR-7848-3p- 3p, miR-3976, and miR-141-3p) between the complete responder and non-responder groups on Day 0. IPA showed modulation of the IL-17a homodimer signaling pathway by six of the seven differentially expressed microRNAs (FDR = 0.089). The predicted effects of the differential microRNA expression on this pathway are shown.

[0085] FIG. 8 shows modulation of Thl7 activation pathway by exosomal microRNAs differentially expressed between complete responders and non-responders. Ingenuity Pathway Analysis (IPA) was used to identify mRNA targets for the seven differentially expressed microRNAs (i.e., miR-450a-2-3p, miR-6877-3p, miR-4494, miR-6499-5p, miR-7848-3p-3p, miR-3976, and miR- 141-3p) between the complete responder and non-responder groups on Day 0. IPA showed modulation of the Thl7 activation pathway by the seven differentially expressed microRNAs (FDR = 0.085). The predicted effects of the differential microRNA expression on this pathway are shown. [0086] FIG. 9 shows modulation of the role of NFAT in the regulation of the immune response pathway by exosomal microRNAs differentially expressed between complete responders and non-responders. Ingenuity Pathway Analysis (IP A) was used to identify mRNA targets for the seven differentially expressed microRNAs (i.e., miR-450a-2-3p, miR-6877-3p, miR-4494, miR-6499-5p, miR-7848-3p-3p, miR-3976, and miR-141-3p) between the complete responder and non-responder groups on Day 0. IPA showed modulation of the role of NFAT in the regulation of the immune response pathway by the seven differentially expressed microRNAs (FDR = 0.085). The predicted effects of the differential microRNA expression on this pathway are shown.

[0087] FIGs. 10A-10E shows a study plan and clinical response to omalizumab. The study plan is shown in FIG. 10A. Omalizumab injections were on Days 0, 30, and 60 (arrows) with UAS7 scores and blood obtained for microRNA (miRNA) analysis as indicated. Twenty-seven subjects were screened (Day -14 visit) and 6 either failed screening or elected not to enter the treatment phase; 21 subjects received omalizumab on Day 0 with first follow-up on Day 14. Subject 18 withdrew on Day 30 because of worsening urticaria symptoms and did not receive omalizumab then. Twenty subjects received omalizumab on Days 30 and 60. Subject 26 received cyclosporine A for urticaria exacerbation between Days 60 and 90 with no UAS7 scores recorded after Day 60. The area under the curve (AUC) for weekly UAS7 over the last 6 weeks of omalizumab therapy for each subject in the complete responders (CR), partial responders (PR), and non-responders (NR) is shown in FIG. 10B. Weekly UAS7 scores over the 12-week study period for CR, PR, and NR are shown in FIGs. IOC, 10D, and 10E, respectively.

[0088] FIGs. 11A-11C show the relationship between eosinophil and basophil levels and response to omalizumab. FIG. 11A shows Welch’s ANOVA comparing the baseline eosinophil levels between the three response groups: complete responders (CR) (n=6), partial responders (PR) (n=9), and non-responders (NR) (n = 4). Welch’s ANOVA comparing the mean and 95% confidence intervals for basophil levels between CR (n = 6), PR (n=9), and NR (n=4) (FIG. 11B). FIG. 11C shows the response to omalizumab measured by the area under curve (AUC) for the weekly urticaria activity scores (UAS7) over the 12-week study period as a function of baseline basophil and eosinophil levels assessed by linear regression model. Baseline basophil levels were negatively correlated with the UAS7 AUC. Shown are regression lines and 95% confidence intervals.

[0089] FIGs. 12A-12H shows baseline levels of EV microRNAs between CR and NR. (FIG. 12A) Heatmap of EV microRNAs differentially expressed between CR and NR at baseline. Upper sections of FIGs. 12B-12H show geometric mean expression levels of these microRNAs ± standard deviation for CR versus NR. Statistical significance was assessed by empirical Bayes adjusted contrasts. Uower panel sections compare the mean UAS7 score over 12-week treatment period between those with high expression (†) and those with low expression (††) ± standard error of the mean. Statistical significance was assessed by the likelihood ratio test. [0090] FIG. 13 shows modulation of Tec Kinase Signaling pathway by EV microRNAs differentially expressed between CR and NR at baseline. IPA identified mRNA targets for differentially expressed EV microRNAs, miR-450a-2-3p, miR-6877-3p, miR-4494, miR-6499-5p, miR-7848-3p-3p, miR-3976, and miR-141-3p for CR versus NR and found Tec Kinase Signaling pathway with significant modulation (FDR = 0.003). Predicted microRNA effects on this pathway are shown (including IPA predictions inconsistent with literature).

DETAILED DESCRIPTION

[0091] The methods described herein are based, in part, on the discovery that patients with chronic spontaneous urticaria (CSU) that are responsive to anti-IgE therapeutics (e.g., omalizumab) have high basophil levels (e.g., >21 cells/pL), unique or characteristic expression profiles of exosomal and basophil microRNAs, and altered Tec kinase-dependent canonical pathways compared with non responders. The methods described herein allow for predicting a subject’s response to an anti-IgE therapeutic agent for the treatment of CSU. Also provided herein are treatments for chronic spontaneous urticaria, which at a minimum can reduce at least one symptom of CSU in a subject (e.g., pain, swelling, itchiness, redness, rash, hives etc).

[0092] It is to be understood that the foregoing description and the following examples are illustrative only and are not to be taken as limitations upon the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those of skill in the art, may be made without departing from the spirit and scope of the present invention. Further, all patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.

[0093] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that could be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents. The following examples are provided by way of illustration not limitation.

Chronic Spontaneous Urticaria (CSU)

[0094] Urticaria is a heterogeneous group of diseases characterized by itchy hives and/or angioedema. As used herein, the term “chronic urticaria” refers to urticaria that has been continuously or intermittently present for at least 6 weeks. Chronic urticaria can be further divided into two subgroups: Chronic Spontaneous Urticaria (CSU) and Inducible Urticaria (IU) the latter including physical urticaria such as heat-, cold-, or pressure-urticaria, and special variants such as cholinergic urticaria. As used herein, the term “chronic spontaneous urticaria (CSU)” refers to the spontaneous appearance of itchy wheals, angioedema, or both due to known or unknown causes that lasts at least 6 weeks.

[0095] The lifetime prevalence of CSU is approximately 1.8%, and 20% of CSU patients still have the disease after 20 years. Affected patients can experience frequent pruritic hives with associated erythema and/or episodes of angioedema. Typically, urticaria presents with skin lesion, such as a “wheal and flare” with a pale elevated lesion and surrounding erythema, ranging in size from a few millimeters to a few centimeters across, usually occurring in groups and often coalescing to form large confluent lesions. CSU is associated with intense itching and has a major impact on patient well-being and quality of life, including adversely affecting daily activities and sleep. Therefore, when managing patients with urticaria, patient-related outcomes (e.g., DLQI) are important measures of treatment (Kaplan A, Ledford D, Ashby M, et al (2013). J Allergy Clin Immunol; 132(1): 101-9; Maurer M, Magerl M, Metz M, et al (2013) J Dtsch Dermatol Ges; Zuberbier T, Aberer W, Asero R, et al (2014) The EAACI/GA(2) LEN/EDF/WAO Allergy; 69(7):868-87).

[0096] The pathogenesis of CSU is not fully clear. Up to 50% of CSU cases are associated with histamine-releasing autoantibodies against multiple antigens including the high-affinity IgE receptor (FceRI) or IgE antibodies; the clinical significance of these autoantibodies is unclear, though there are suggestions that they may be involved in disease pathogenesis (Kaplan A P (2002) Curr Allergy Asthma Rep; 2(4):263-4; Sabroe R A, Greaves M W (2006) Br J Dermatol; 154(5):813-9). It has also been suggested that CSU patients' basophils may have distinct alterations in FceRIa-mediated degranulation, independent of any potential role of autoantibodies (Eckman J A, Hamilton R G, Gober L M, et al (2008) Basophil phenotypes in chronic idiopathic urticaria in relation to disease activity and autoantibodies. J Invest Dermatol; 128(8): 1956-63).

[0097] Treatment of CSU is a challenge, and non-sedating (second generation) Hl- antihistamines (Hl-AH) are the mainstay of symptomatic therapy of CSU. Conventional treatments for CSU can include non-sedating anti-histamines (e.g., loratidine, cetirizine, fexofenadine, desloratadine, levocetirizine), sedating anti-histamines (e.g., diphenhydramine, hydroxyzine, chlorpheniramine, doxepin), Histamine-2 receptor antagonists (e.g., famotidine, ranitidine, cimetidine), leukotriene receptor antagonists (e.g., montelukast, zafirlukast), and anti-inflammatory agents (e.g., dapsone, hydroxychloroquine, sulfasalazine, cyclosporin A, tacrolimus, sirolimus, methotrexate, omalizumab).

[0098] The level of evidence for the efficacy of leukotriene receptor antagonists (LTRA) in urticaria is low but best for montelukast that consequently led to only a weak recommendation from experts for this off-label treatment. Short courses (max. 10 days) of systemic corticosteroids can be added to the 3rd level treatment regimens, if exacerbations demand this. Due to the adverse effects associated with chronic systemic corticosteroid exposure, a longer duration of treatment is not advisable. Other treatment options that were previously used such as intravenous immunoglobulin G, dapsone, hydroxychloroquine, H2-antihistamines (H2-AH), methotrexate, and cyclophosphamide, have an unfavorable benefit risk profile or significant side-effect profile and are no longer recommended for therapy of CSU, unless the subject is refractory to other treatments.

[0099] Omalizumab is an approved therapy for treatment of CSU refractory to standard of care treatment, and exhibits a favorable benefit-risk profile. It is a recombinant humanized IgGl monoclonal antibody that binds to IgE-specific epitopes within the C3 (FceRI binding) region of the IgE molecule and is indicated in many countries for the treatment of poorly controlled moderate or severe asthma and CSU refractory to standard therapy. The completed Phase 2 and Phase 3 studies demonstrate that omalizumab improves the signs and symptoms of urticaria (e.g., itch, hives) in patients with CSU who have failed treatment with HI -AH as well as those who have failed treatment with a combination of HI and H2-AH and a leukotriene receptor antagonist. Published data from the omalizumab Phase 3 study (Q4882g) demonstrated that omalizumab safely improved the clinical manifestations of CSU (e.g., itch, hives), compared to placebo, with monthly doses of 150 mg or 300 mg in a dose -dependent way, but not a 75 mg dose.

[00100] Treatment efficacy of CSU can be determined or monitored by assessing the change from baseline using an itch-severity score (e.g., 0=none, l=mild, 2=moderate, 3=severe) standard at a given time point (e.g., prior to and/or following onset of a given treatment). Other standards or indices for assessing the degree of CSU or treatment efficacy thereof include, for example, (i) Dermatology Life Quality Index (DLQI)-Assesses 10 items under 6 headings: Symptoms and feelings, daily activities, leisure, work/school, personal relationships, and treatment. Each is graded on a scale of 0-3 with a score ranging from 0-30. A higher score indicates a greater impact on quality of life; (ii) Skindex-29- Consists of 29 items separated into 3 domains (physical symptoms, social functioning, emotional state) with scores ranging from 0-100. A higher score indicates a greater impact on quality of life; (iii) Chronic Urticaria Quality of Life Questionnaire (Cu-Q2oL)-Specifically designed for patients with chronic urticaria, consisting of sections pertaining to physical, emotional, social and practical effects of the disease. Biological Samples

[00101] Responsiveness of CSU to anti-IgE therapy can be predicted using, for example, methods described herein or known in the art to detect the levels of one or more miRNAs disclosed herein in a given biological sample. Preferred biological samples can include isolated extracellular vesicles (e.g., exosomes), a blood sample, isolated basophils, isolated eosinophils, however essentially any sample can be tested using the methods and systems described herein, provided that the sample comprises at least one extracellular vesicle (e.g., an exosome), basophil or eosinophil. The term “biological sample” can refer to any sample containing an extracellular vesicle, basophil or eosinophil, such as, for example, blood, plasma, serum, urine, gastrointestinal secretions, homogenates of tissues or tumors, circulating cells and cell particles (e.g., circulating tumor cells), synovial fluid, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid, tears, prostate fluid, cell culture media, or cellular lysates.

[00102] In one embodiment, the method comprises a step of isolating extracellular vesicles (e.g, exosomes) from the biological sample. In another embodiment, the method can comprise a step of enriching extracellular vesicles (e.g., exosomes) in a biological sample.

Exosomes

[00103] Exosomes are cell-derived nanovesicles of 30-200 nm diameters that are released from most living cells. Exosomes are present in virtually all biological fluids of the body, including blood and urine. Exosomes are formed by membrane invagination of late endosomes, resulting in the vesicles containing some cytosolic components and extracellular domains of plasma membrane receptors of cells. Exosomes are released into the extracellular environment from cells following the fusion of late endosomal multivesicular bodies (MVBs) with the plasma membrane, or they may be released from the plasma membrane directly. Because of their intracellular origin, in addition to other intracellular components including proteins and nucleic acids such as, but not limited to miRNAs, exosomes can harbor specific protein markers of the endosomal pathway, such as tetraspanins (CD63, CD9 and CD81) and heat shock proteins (HSP70), which are not found in other types of nanovesicles of similar size. miRNA

[00104] The terms “microRNA” and “miRNA” as used herein refer to short, single-stranded RNA molecules approximately 21-23 nucleotides in length which are partially complementary to one or more mRNA molecules (target mRNAs). miRNAs can functionally down-regulate gene expression by inhibiting translation or by targeting the mRNA for degradation or deadenylation. MiRNAs base-pair with miRNA recognition elements (MREs) located on their mRNA targets, usually on the 3'-UTR, through a region called the ‘seed region’ which includes nucleotides 2-8 from the 5'-end of the miRNA. Matches between a miRNA and its target are generally asymmetrical. The complementarity of seven or more bases to the 5 '-end miRNA has been found to be sufficient for regulation.

[00105] MicroRNAs play important roles in the regulation of target genes by binding to complementary regions of messenger transcripts to repress their translation or regulate degradation (Griffiths-Jones Nucleic Acids Research. 2006; 34, Database issue: D140-D144). Frequently, one miRNA can target multiple mRNAs and one mRNA can be regulated by multiple miRNAs targeting different regions of, for example, the 3' UTR. Once bound to an mRNA, miRNA can modulate gene expression and protein production by affecting, e.g., mRNA translation and stability (Baek et al. Nature. 2008; 455:64; Selbach et al. Nature. 2008; 455:58; Ambros. Nature. 2004; 431: 350-355; Bartel. Cell. 2004; 116: 281-297; Cullen. Virus Research. 2004; 102: 3-9; He et al. Nat. Rev. Genet. 2004; 5: 522-531; and Ying et al. Gene. 2004; 342: 25-28).

[00106] The methods described herein relate, in part, to the diagnosis and/or treatment of chronic spontaneous urticaria based on the expression level of one or more miRNAs selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, miR-3976, and miR-141-3p. The sequences of the miRNAs are compiled in the following Table.

Table: miRNA Sequences [00107] The level or amount of miRNAs in a biological sample can be determined or measured by any suitable method known to those of skill in the art. For example, miRNA (e.g., exosomal miRNA) can be isolated by various known methods, including the use of commercially available kits such as Norgen Urine Exosome RNA Isolation Kit and RNA Clean-up and Concentration Micro Kit (Norgen biotek, Thorold, Canada), and Qiagen exoRNeasy™ Serum/Plasma Midi Kit (Qiagen, Hilden, Germany). miRNAs, whether exosomal or from other sources, can be detected and quantified by amplification-based methods (e.g., Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Quantitative Polymerase Chain Reaction (qPCR), rolling circle amplification, etc.), hybridization-based methods (e.g., hybridization arrays such as microarrays), NanoString™ analysis, Northern Blot analysis, branched DNA (bDNA) signal amplification, and in situ hybridization), and sequencing -based methods (e.g. next-generation sequencing methods, for example, using the Illumina™ or IonTorrent™ platforms). Other exemplary techniques include ribonuclease protection assay (RPA) and mass spectroscopy.

[00108] In certain embodiments, detection of one or more miRNAs as can be performed using the highly specific and sensitive Droplet Digital PCR (ddPCR) method known in the art. Digital PCR takes advantage of nucleic acid amplification on a single molecule level, and offers a highly sensitive method for quantifying low copy number nucleic acid. Fluidigm® Corporation, BioRad's Digital PCR and Raindance technologies all offer systems for the digital analysis of nucleic acids. ddPCR technology has high sensitivity in addition to the ability for absolute quantification.

[00109] In addition, other methods that are similarly sensitive, accurate and highly reproducible, such as digital molecular barcoding (e.g. NanoString's nCounter technology) and next-generation sequencing (i.e. High-throughput sequencing), are also useful in measuring and determining levels or amounts of miRNAs (e.g., exosomal miRNAs) in a biological sample. In one embodiment, the determining or measuring of exosomal miRNA levels involves using ddPCR, digital molecular barcoding, or next-generation sequencing.

[00110] Other methods of detecting miRNA expression can also include extraction of cellular RNA and Northern blotting using labeled probes that hybridize to a given miRNA; amplification of mRNA using marker-specific primers, followed by quantitative detection of the product by any of a variety of means; extraction of total RNA from the cells, which is then labeled and used to probe miRNAs; or in situ hybridization.

[00111] In some embodiments, a probe or primer can be used to detect or measure a given miRNA; such probes or primers can typically be used to identify a target nucleic acid sequence in a sample by hybridizing to such target nucleic acid sequence under stringent hybridization conditions. As used herein, stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al., ibid., is incorporated by reference herein in its entirety (see specifically, pages 9.31-9.62). In addition, formulae to calculate the appropriate hybridization and wash conditions to achieve hybridization permitting varying degrees of mismatch of nucleotides are disclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid., is incorporated by reference herein in its entirety.

[00112] Agents or probes useful for detecting expression of an miRNA can be conjugated to a detectable tag or detectable label. Such a tag can be any suitable tag which allows for detection of the marker using the selected technique and includes, but is not limited to, any composition or label detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads™), fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3H, 1251, 35S, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

Reference Levels

[00113] The expression of one or more miRNAs determined in a given biological sample can be compared to the expression of the same miRNAs in a control biological sample tested using substantially the same methods. Comparing the expression level or amount of one or more given miRNAs in a biological sample from a subject having or suspected of having chronic spontaneous urticaria with the expression level or amount of the one or more miRNAs in a control biological sample can permit diagnosis, prediction of responsiveness to therapy, or can be used to monitor efficacy of a given treatment. A control biological sample can be a sample from a subject who responded to a given therapy, e.g., anti-IgE therapy; a control or reference level of one or more miRNAs can be a level of the one or more miRNAs in a sample from a subject who responded to such therapy. Comparison of the amount of one or more miRNAs or other markers in a patient or subject sample to the level of the one or more miRNAs or other markers in a control sample or to a predetermined reference level based upon the amount in such a control sample can permit diagnosis, prediction of responsiveness, or monitoring of therapy as described herein. Control biological samples can alternatively be a reference sample taken from the subject at an earlier time point (e.g., during initial diagnosis of CSU) to permit monitoring of disease progress or therapeutic efficacy in the subject. For example, the control biological sample can be a sample taken from the subject one month, two months, three months, six months, or one year prior to the sample to be tested. Typically, a control biological sample will be obtained from a subject that is not experiencing an episode of chronic spontaneous urticaria. When monitoring efficacy of a given therapeutic, a control biological sample can be obtained from the subject having CSU prior to the onset of treatment with the therapeutic. In other embodiments, the control biological sample or reference sample is obtained from the subject during, or following the administration of a given therapy for CSU.

[00114] In other aspects, a reference sample can be a sample from a patient or a population of patients having no detectable urticaria or allergic disease or disorder (i.e., a negative control). Alternatively, a reference sample can be a sample from a patient or population of patients with a known degree or stage of CSU, for example, mild CSU, moderate CSU, advanced CSU, or treatment refractory CSU. Such controls can serve as a positive control to ensure that the assay is working and/or to determine/monitor treatment refractoriness for a given intervention. In other embodiments, one or more controls can comprise a known concentration (or range of concentrations) of each of the miRNAs in order to quantitatively detect the level of a given miRNA in the subject being tested. In certain embodiments, a positive or negative control sample is a sample that is obtained or derived from a corresponding tissue or biological fluid as the sample to be analyzed in accordance with the methods as described herein. This sample will typically be from the same patient at the same or different time points.

[00115] In another embodiment the reference levels for one or more miRNAs are established based on the expression levels of the miRNAs in a sample taken from an individual at an earlier point in time, for example, prior to onset of treatment with a therapeutic agent. Such methods permit one of skill in the art to monitor the efficacy of a given therapeutic. The individual is determined to be responding to treatment for CSU if the relative amounts of the miRNAs in the biological sample have altered favorably from the miRNA levels in a biological sample taken at an earlier first time point from the same individual; i.e. trend towards normal biomarker levels. Similarly, the disease state of the individual may be progressing if the levels of miRNA in a biological fluid sample are changing relative to the levels in the individual taken at an earlier time point or in reference to the control levels.

[00116] The reference level or value can be derived from the level of miRNA expression in a biological sample or population of biological samples.

[00117] As used herein, the term “control level” of a given miRNA expression is a baseline level, and in some embodiments, a normal or negative level, of miRNA expression against which a test level of expression (i.e., in the test sample) can be compared. Therefore, it can be determined, based on the control or baseline level of expression, whether a sample to be evaluated has a higher or lower level of a given miRNA relative to that control or baseline. In one aspect, the baseline level is a negative or normal control level that is indicative of the expression expected in a normal (i.e., healthy, negative control) biological sample. Therefore, the term “negative control” used in reference to a baseline level of miRNA expression typically refers to a baseline level established from a sample from a population of individuals which is believed to be normal (i.e., healthy). Pharmaceutical compositions, Administration and Efficacy

[00118] Pharmaceutical or therapeutic compositions comprising a therapeutic agent for the treatment of CSU can contain a physiologically tolerable carrier, wherein the therapeutic agent is dissolved or dispersed therein as an active ingredient(s). In a preferred embodiment, the pharmaceutical composition is not immunogenic when administered to a mammal or human patient for therapeutic purposes. As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset and the like. A pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired. The preparation of a pharmacological or pharmaceutical composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectable either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified or presented as a liposome composition. The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like which enhance the effectiveness of the active ingredient. The therapeutic composition comprising a therapeutic agent for treatment of CSU can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

[00119] Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of an active agent used in the methods described herein that will be effective in the treatment of CSU or a symptom thereof will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.

[00120] A pharmaceutical composition as described herein can be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multidose containers with, optionally, an added preservative. The compositions can be suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing, and/or dispersing agents. [00121] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients can be prepared as appropriate oily or water-based injection suspensions.

[00122] Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

[00123] Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the active ingredients, to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., a sterile, pyrogen-free, water-based solution, before use.

[00124] In some embodiments, a therapeutic agent can be delivered in an immediate release form. In other embodiments, the therapeutic agent can be delivered in a controlled-release system or sustained-release system. Controlled- or sustained-release pharmaceutical compositions can have a common goal of improving drug therapy over the results achieved by their non-controlled or non- sustained-release counterparts. Advantages of controlled- or sustained-release compositions include extended activity of the therapeutic agents, reduced dosage frequency, and increased compliance. In addition, controlled- or sustained-release compositions can favorably affect the time of onset of action or other characteristics, such as blood levels of the therapeutic agent, and can thus reduce the occurrence of adverse side effects. Controlled- or sustained-release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.

[00125] In one embodiment, a pump can be used (Langer, Science 249:1527-1533 (1990); Sefton, CRC Crit. Ref Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); and Saudek et al., N. Engl. J. Med 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release (Langer and Wise eds., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., 1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); Levy et al., Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); and Howard et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment, a controlled- or sustained-release system can be placed in proximity of a target of infection, e.g., site of urticaria, thus requiring only a fraction of the systemic dose.

[00126] While anti-IgE therapeutics such as antibodies or derivatives thereof tend to be administered parenterally, e.g., intravenously, other modes of administration as suited for a given agent are specifically contemplated. When in tablet or pill form, a pharmaceutical composition as described herein can be coated (e.g., enterically coated) to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotically active driving compound are also suitable for orally administered compositions. In these latter platforms, fluid from the environment surrounding the capsule is imbibed by the driving compound, which swells to displace the agent or agent composition through an aperture. These delivery platforms can provide an essentially zero order delivery profde as opposed to the spiked profdes of immediate release formulations. A time-delay material such as glycerol monostearate or glycerol stearate can also be used. Oral compositions can include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In one embodiment, the excipients are of pharmaceutical grade. [00127] The pharmaceutical composition as described herein can also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, conventional suppository bases such as cocoa butter or other glycerides.

[00128] The appropriate dosage range for a given therapeutic agent depends upon the potency, and includes amounts large enough to produce the desired effect, e.g., reduction in at least one symptom of CSU. The dosage of the therapeutic agent should not be so large as to cause unacceptable or life-threatening adverse side effects or should be used under close supervision by a medical professional. Generally, the dosage will vary with the type of therapeutic, and with the age, condition, and sex of the patient. The dosage can be determined by one of skill in the art and can also be adjusted by the individual physician in the event of any complication.

[00129] Typically, the dosage of a given therapeutic can range from O.OOlmg/kg body weight to 5 g/kg body weight. In some embodiments, the dosage range is from 0.001 mg/kg body weight to lg/kg body weight, from 0.001 mg/kg body weight to 0.5 g/kg body weight, from 0.001 mg/kg body weight to 0.1 g/kg body weight, from 0.001 mg/kg body weight to 50 mg/kg body weight, from 0.001 mg/kg body weight to 25 mg/kg body weight, from 0.001 mg/kg body weight to 10 mg/kg body weight, from 0.001 mg/kg body weight to 5 mg/kg body weight, from 0.001 mg/kg body weight to 1 mg/kg body weight, from 0.001 mg/kg body weight to 0.1 mg/kg body weight, from 0.001 mg/kg body weight to 0.005 mg/kg body weight. Alternatively, in some embodiments the dosage range is from 0.1 g/kg body weight to 5 g/kg body weight, from 0.5 g/kg body weight to 5 g/kg body weight, from 1 g/kg body weight to 5 g/kg body weight, from 1.5 g/kg body weight to 5 g/kg body weight, from 2 g/kg body weight to 5 g/kg body weight, from 2.5 g/kg body weight to 5 g/kg body weight, from 3 g/kg body weight to 5 g/kg body weight, from 3.5 g/kg body weight to 5 g/kg body weight, from 4 g/kg body weight to 5 g/kg body weight, from 4.5 g/kg body weight to 5 g/kg body weight, from 4.8 g/kg body weight to 5 g/kg body weight. In one embodiment, the dose range is from 5mg/kg body weight to 30pg/kg body weight. Alternatively, the dose range will be titrated to maintain serum levels between 5mg/mL and 30pg/mU.

[00130] Currently available therapies, including experimental therapies, for CSU or a symptom thereof and their dosages, routes of administration and recommended usage are known in the art and/or have been described in such literature as the Physician's Desk Reference (60th ed., 2017). With respect to experimental therapies, an appropriate dosage can be estimated based on dose-response modeling in animal models or in silico modeling of drug effects.

[00131] Administration of the doses recited above or as employed by a skilled clinician can be repeated for a limited and defined period of time. In some embodiments, the doses are given once a day, or multiple times a day, for example, but not limited to three times a day. Typically, the dosage regimen is informed by the half-life of the agent as well as the minimum therapeutic concentration of the agent in blood, serum or localized in a given biological tissue. In a preferred embodiment, the doses recited above are administered daily for several weeks or months. The duration of treatment depends upon the subject’s clinical progress and continued responsiveness to therapy. Continuous, relatively low maintenance doses are contemplated after an initial higher therapeutic dose.

[00132] A therapeutically effective amount is an amount of an agent that is sufficient to produce a statistically significant, measurable change of a given symptom of CSU (efficacy measurement is discussed further below). Such effective amounts can be gauged in clinical trials as well as animal studies for a given agent. For example, reduction of a given symptom of CSU can be indicative of adequate therapeutic efficacy of an agent(s).

[00133] Agents useful in the methods and compositions described herein can be administered topically, intravenously (by bolus or continuous infusion), orally, by inhalation, intraperitoneally, intramuscularly, subcutaneously, intracavity, and can be delivered by peristaltic means, if desired, or by other means known by those skilled in the art. The agent can be administered systemically, if so desired.

[00134] Therapeutic compositions containing at least one therapeutic agent can be conventionally administered in a unit dose. The term "unit dose" when used in reference to a therapeutic composition refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of a therapeutic agent calculated to produce the desired therapeutic effect in association with the required physiologically acceptable diluent, i.e., carrier, or vehicle. [00135] The compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered and timing depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and degree of therapeutic effect desired. An agent can be targeted by means of a targeting moiety, e.g., using an antibody or targeted liposome technology, among others.

[00136] Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are particular to each individual. However, suitable dosage ranges for systemic application are disclosed herein and depend on the route of administration. Suitable regimes for administration are also variable, but are typified by an initial administration followed by repeated doses at one or more intervals by a subsequent injection or other administration. Alternatively, continuous intravenous infusion sufficient to maintain concentrations in the blood in the ranges specified for in vivo therapies are contemplated.

[00137] In some embodiments, a therapeutically effective agent is administered to a subject concurrently with a combination therapy. As used herein, the term “concurrently” is not limited to the administration of the two or more agents at exactly the same time, but rather, it is meant that they are administered to a subject in a sequence and within a time interval such that they can act together (e.g., synergistically to provide an increased benefit than if they were administered otherwise). For example, the combination of therapeutics can be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they should be administered sufficiently close in time so as to provide the desired therapeutic effect, preferably in a synergistic fashion. The agents can be administered separately, in any appropriate form and by any suitable route. When each of the therapeutic agents in a combination are not administered in the same pharmaceutical composition, it is understood that they can be administered in any order to a subject in need thereof. For example, the first therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of the second therapeutic agent, to a subject in need thereof (or vice versa). In other embodiments, the delivery of either therapeutic agent ends before the delivery of the other agent/treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the therapeutic agents used in combination are more effective than would be seen with either agent alone. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with either therapeutic agent alone. The effect of such a combination can be partially additive, wholly additive, or greater than additive. The agent and/or other therapeutic agents, procedures or modalities can be administered during periods of active disease, or during a period of persistence or less active disease.

[00138] When administered in combination, one or more of the therapeutic agents can be administered in an amount or dose that is higher, lower or the same as the amount or dosage of the given agent used individually, e.g., as a monotherapy. In certain embodiments, the administered amount or dosage of a first therapeutic agent when administered in combination with a second therapeutic agent is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of the first agent when used individually. In other embodiments, the amount or dosage of a first therapeutic agent, when administered in combination with a second therapeutic agent, results in a desired effect (e.g., improved cognitive functioning) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dosage of the first (or second) agent required to achieve the same therapeutic effect when administered alone.

[00139] The efficacy of a given treatment for chronic spontaneous urticaria can be determined by the skilled clinician. However, a treatment is considered “effective treatment," as the term is used herein, if any one or all of the signs or symptoms of CSU is/are altered in a beneficial manner, or other clinically accepted symptoms or markers of disease are improved, or ameliorated, e.g., by at least 10% following treatment with a therapeutic agent for CSU. Efficacy can also be indicated, for example, by an improvement of at least one unit on a standard scale or index of disease status or severity. Efficacy can also be measured by failure of an individual to worsen as assessed by stabilization of the disease, or the need for medical interventions (i.e., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing progression of the disease; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of the disease, or preventing secondary diseases/disorders associated with CSU (e.g., itching, redness, pain etc).

[00140] An effective amount for the treatment of a disease means that amount which, when administered to a mammal in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of the disease, such as e.g., itching, redness, pain, swelling etc.

Kits

[00141] In one aspect, provided herein are kits containing any one or more of the elements disclosed in or used for measurement or detection in the methods and compositions described herein. In one embodiment, the kit comprises, consists of, or consists essentially reagents and instructions for measuring the expression of one or more miRNAs selected from the group consisting of: miR-6499- 5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, miR-3976, or miR-141-3p (e.g., probes, antibodies, primers etc).

[0001] The kit can comprise the components necessary to assess the level of such miRNAs including PCR primers, nucleic acid probes, PCR enzymes, restriction enzymes, and DNA, RNA or exosome purification means or reagents. The kit can contain at least one pair of primers, or probes.

[0002] Other components include labeling means, and buffers for the reactions. In addition, a control nucleic acid or miRNA sample can be included. The kit will usually also comprise instructions for carrying out the methods as described herein and a key detailing the correlation between the results and the stratification of a subject to a given treatment (e.g., non-steroidal immunosuppressant or anti- IgE therapeutic).

[0003] One or more of the reagents in the kit can be arrayed on a solid support, such as a microscope slide, a microwell plate, or a capillary electrophoresis plate.

[00142] In some embodiments, the kit comprises reagents permitting the quantitative detection of one or more target miRNAs including miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR- 6877-3p, miR-3976, and/or miR-141-3p.

[00143] The kit can also comprise reagents permitting the isolation or quantitation of basophils and/or exosomes in a biological sample.

[00144] Also contemplated herein are kits that comprise reagents permitting the isolation of miRNAs, PCR primers permitting amplification of the target miRNAs or a reference composition comprising a known amount of target miRNA.

[00145] Such kits can also include a reference sample comprising miRNAs isolated from exosomes isolated from a subject with refractory CSU that did (or alternatively, did not) not respond to anti-IgE therapy or a reference table summarizing the data from such a population.

[0004] In some embodiments, a kit comprises one or more reagents for use in a process utilizing one or more of the elements described herein. Reagents can be provided in any suitable container. For example, a kit can provide one or more reaction or storage buffers. Reagents can be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form). A buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH from about 7 to about 10.

[00146] The invention may be as defined in any one of the following numbered paragraphs: [00147] 1. A method of predicting a subject’s response to an anti-IgE therapeutic agent for the treatment of chronic spontaneous urticaria (CSU), the method comprising: receiving the results of an assay performed on a sample of plasma exosomes from the subject to detect the level of one or more miRNAs relative to a reference, wherein detection of an increase in the level of one or more of the miRNAs miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in the level of miR-141-3p relative to the reference indicates the subject’s CSU is likely to respond to an anti-IgE therapeutic agent.

[00148] 2. The method of paragraph 1, wherein two or more of an increase in miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the reference are detected.

[00149] 3. The method of paragraph 1, wherein three or more of an increase in miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the reference are detected.

[00150] 4. The method of paragraph 1, wherein four or more of an increase in miR-6499-5p, miR-

7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the reference are detected.

[00151] 5. The method of paragraph 1, wherein five or more of an increase in miR-6499-5p, miR-

7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the reference are detected.

[00152] 6. The method of paragraph 1, wherein six or more of an increase in miR-6499-5p, miR-

7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the reference are detected.

[00153] 7. The method of paragraph 1, wherein an increase in each of miR-6499-5p, miR-7848-

3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 and a decrease in miR-141-3p relative to the reference are detected.

[00154] 8. The method of any one of paragraphs 1-7, wherein the reference is the level of the one or more miRNAs in a plasma exosome sample from one or more subjects with CSU whose disease did not respond to an anti-IgE therapeutic agent.

[00155] 9. The method of any one of paragraphs 1-8, wherein the subject’s CSU is resistant to Hi- antihistamine therapy.

[00156] 10. The method of any one of paragraphs 1-9, wherein the anti-IgE therapeutic agent comprises omalizumab, ligelizumab, UB-221, quilizumab, and/or MEDI-4212.

[00157] 11. The method of any one of paragraphs 1-10, further comprising detecting the level of basophils in a blood sample from the subject. [00158] 12. The method of paragraph 11, wherein detecting > 21 basophils/mΐ of blood is indicative of a likely response to an anti-IgE therapeutic agent.

[00159] 13. A method of predicting a subject’s response to an anti-IgE therapeutic agent for the treatment of chronic spontaneous urticaria (CSU), the method comprising: performing an assay to detect the level of one or more miRNAs selected from 6499-5p, miR- 7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, miR-3976 and miR-141-3p in a sample of plasma exosomes from the subject; wherein detection of an increase in the level of one or more of miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in the level of miR-141-3p relative to a reference indicates the subject’s CSU is likely to respond to an anti-IgE therapeutic agent. [00160] 14. The method of paragraph 13, wherein two or more of an increase in miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the reference are detected.

[00161] 15. The method of paragraph 13, wherein three or more of an increase in miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the reference are detected.

[00162] 16. The method of paragraph 13, wherein four or more of an increase in miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the reference are detected.

[00163] 17. The method of paragraph 13, wherein five or more of an increase in miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the reference are detected.

[00164] 18. The method of paragraph 13, wherein six or more of an increase in miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the reference are detected.

[00165] 19. The method of paragraph 13, wherein an increase in each of miR-6499-5p, miR-7848-

3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 and a decrease in miR-141-3p relative to the reference are detected.

[00166] 20. The method of any one of paragraphs 13-19, wherein the reference is the level of the one or more miRNAs in an exosome sample from one or more subjects with CSU whose disease did not respond to an anti-IgE therapeutic agent.

[00167] 21. The method of any one of paragraphs 13-20, wherein the subject’s CSU is resistant to

Hi-antihistamine therapy.

[00168] 22. The method of any one of paragraphs 13-21, wherein the anti-IgE therapeutic agent comprises omalizumab, ligelizumab, UB-221, quilizumab, and/or MEDI-4212. [00169] 23. The method of any one of paragraphs 13-22, further comprising detecting the level of basophils in a blood sample from the subject.

[00170] 24. The method of paragraph 23, wherein detecting > 21 basophils/mΐ of blood is indicative of a likely response to an anti-IgE therapeutic agent.

[00171] 25. A method of predicting a subject’s response to an anti-IgE therapeutic agent for the treatment of chronic spontaneous urticaria (CSU), the method comprising: receiving the results of an assay performed on a sample of basophils from the subject to detect the level of one or more miRNAs relative to a reference, wherein detection of an increase in the level of one or more of the miRNAs miR-1200, miR-1236-3p and miR-4664-3p relative to the reference indicates the subject’s CSU is likely to respond to an anti-IgE therapeutic agent.

[00172] 26. The method of paragraph 25, wherein an increase in two or more of miRNAs miR-

1200, miR-1236-3p and miR-4664-3p is detected relative to the reference.

[00173] 27. The method of paragraph 25, wherein an increase in each of miRNAs miR-1200, miR-1236-3p and miR-4664-3p is detected relative to the reference.

[00174] 28. The method of any one of paragraphs 25-27, wherein the reference is the level of the one or more miRNAs in a basophil sample from one or more subjects with CSU whose disease did not respond to an anti-IgE therapeutic agent.

[00175] 29. The method of any one of paragraphs 25-28, wherein the subject’s CSU is resistant to

Hi-antihistamine therapy.

[00176] 30. The method of any one of paragraphs 25-29, wherein the anti-IgE therapeutic agent comprises omalizumab, ligelizumab, UB-221, quilizumab, and/or MEDI-4212.

[00177] 31. The method of any one of paragraphs 25-30, further comprising detecting the level of basophils in a blood sample from the subject.

[00178] 32. The method of paragraph 31, wherein detecting > 21 basophils/mΐ of blood is indicative of a likely response to an anti-IgE therapeutic agent.

[00179] 33. A method of predicting a subject’s response to an anti-IgE therapeutic agent for the treatment of chronic spontaneous urticaria (CSU), the method comprising: performing an assay to detect the level of one or more miRNA species selected from the group consisting of miR-1200, miR-1236-3p and miR-4664-3p in a sample of basophils from the subject; wherein detection of an increase in the level of one or more of the miRNAs relative to a reference indicates the subject’s CSU is likely to respond to an anti-IgE therapeutic agent.

[00180] 34. The method of paragraph 33, wherein an increase in two or more of miRNAs miR-

1200, miR-1236-3p and miR-4664-3p is detected relative to the reference.

[00181] 35. The method of paragraph 33, wherein an increase in each of miRNAs miR-1200, miR-1236-3p and miR-4664-3p is detected relative to the reference. [00182] 36. The method of any one of paragraphs 33-35, wherein the reference is the level of the one or more miRNAs in a basophil sample from one or more subjects with CSU whose disease did not respond to an anti-IgE therapeutic agent.

[00183] 37. The method of any one of paragraphs 33-36, wherein the subject’s CSU is resistant to

Hi-antihistamine therapy.

[00184] 38. The method of any one of paragraphs 33-37, wherein the anti-IgE therapeutic agent comprises omalizumab, ligelizumab, UB-221, quilizumab, and/or MEDI-4212.

[00185] 39. The method of any one of paragraphs 33-38, further comprising detecting the level of basophils in a blood sample from the subject.

[00186] 40. The method of paragraph 39, wherein detecting > 21 basophils/mΐ of blood is indicative of a likely response to an anti-IgE therapeutic agent.

[00187] 41. A method of treating chronic spontaneous urticaria in a subject in need thereof, the method comprising:

(a) receiving results of an assay to detect the level of one or more miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR- 6877-3p, miR-3976 and miR-141-3p in an exosome sample from the subject; and

(b) when the level of one or more of miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 is above a reference level, and/or when the level of miR-141-3p is below the reference level, administering an anti-IgE therapeutic agent; and

(c) when the level of one or more of miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 is at or below the reference level, and/or when the level of miR- 14 l-3p is at or above the reference level, administering anon-steroidal immunosuppressant.

[00188] 42. The method of paragraph 41, wherein the subject has two or more miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR- 6877-3p, and miR-3976 above the reference level; and/or miR-141-3p below the reference level. [00189] 43. The method of paragraph 41, wherein the subject has three or more of the miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2- 3p, miR-6877-3p, and miR-3976 above the reference level; and/or miR-141-3p below the reference level.

[00190] 44. The method of any one of paragraphs 41-43, wherein the subject has four or more of the miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 above the reference level; and/or miR- 141 -3p below the reference level.

[00191] 45. The method of any one of paragraphs 41-44, wherein the subject has five or more of the miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 above the reference level; and/or miR- 141 -3p below the reference level.

[00192] 46. The method of any one of paragraphs 41-45, wherein the subject has six or more of the miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 above a reference level; and/or miR-141-3p below the reference level.

[00193] 47. The method of any one of paragraphs 41-46, wherein the subject has each of the miRNA species selected from miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 above the reference level; and miR-141-3p below the reference level.

[00194] 48. The method of any one of paragraphs 41-47, wherein the method further comprises detecting the level of basophils in a biological sample from the subject compared to a reference level. [00195] 49. A method of treating chronic spontaneous urticaria in a subject in need thereof, the method comprising:

(a) performing an assay to detect the level of one or more miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, miR-3976 and miR-141-3p in an exosome sample from the subject; and

(b) when the level of one or more of miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 is above a reference level, and/or when the level of miR-141-3p is below the reference level, administering an anti-IgE therapeutic agent; and

(c) when the level of one or more of miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 is at or below the reference level, and/or when the level of miR- 14 l-3p is at or above the reference level, administering anon-steroidal immunosuppressant.

[00196] 50. The method of paragraph 49, wherein the subject has two or more miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR- 6877-3p, and miR-3976 above the reference level; and/or miR-141-3p below the reference level. [00197] 51. The method of paragraph 49, wherein the subject has three or more of the miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2- 3p, miR-6877-3p, and miR-3976 above the reference level; and/or miR-141-3p below the reference level.

[00198] 52. The method of paragraph 49, wherein the subject has four or more of the miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2- 3p, miR-6877-3p, and miR-3976 above the reference level; and/or miR-141-3p below the reference level.

[00199] 53. The method of paragraph 49, wherein the subject has five or more of the miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2- 3p, miR-6877-3p, and miR-3976 above the reference level; and/or miR-141-3p below the reference level.

[00200] 54. The method of paragraph 49, wherein the subject has six or more of the miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2- 3p, miR-6877-3p, and miR-3976 above a reference level; and/or miR-141-3p below the reference level.

[00201] 55. The method of paragraph 49, wherein the subject has each of the miRNA species selected from miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 above the reference level; and miR-141-3p below the reference level.

[00202] 56. The method of any one of paragraphs 49-55, wherein the method further comprises detecting the level of basophils in a blood sample from the subject.

[00203] 57. A method of treating chronic spontaneous urticaria (CSU), the method comprising:

(a) receiving the results of an assay performed on a sample of basophils from the subject to detect the level of one or more miRNAs selected from miR-1200, miR-1236-3p and miR- 4664-3p, and

(b) when the level of one or more of miR-1200, miR-1236-3p and miR-4664-3p is above a reference level, administering an anti-IgE therapeutic agent; and

(c) when the level of one or more of miR-1200, miR-1236-3p and miR-4664-3p is at or below the reference level, administering a non-steroidal immunosuppressant.

[00204] 58. The method of paragraph 57, wherein an increase in two or more of miRNAs miR-

1200, miR-1236-3p and miR-4664-3p is detected relative to the reference.

[00205] 59. The method of paragraph 57, wherein an increase in each of miRNAs miR-1200, miR-1236-3p and miR-4664-3p is detected relative to the reference.

[00206] 60. The method of any one of paragraphs 57-59, wherein the reference is the level of the one or more miRNAs in a basophil sample from one or more subjects with CSU whose disease did not respond to an anti-IgE therapeutic agent.

[00207] 61. The method of any one of paragraphs 57-60, wherein the subject’s CSU is resistant to

Hi-antihistamine therapy.

[00208] 62. The method of any one of paragraphs 57-61, wherein the anti-IgE therapeutic agent comprises omalizumab, ligelizumab, UB-221, quilizumab, and/or MEDI-4212.

[00209] 63. The method of any one of paragraphs 57-62, further comprising detecting the level of basophils in a blood sample from the subject.

[00210] 64. The method of any one of paragraphs 57-63, wherein detecting > 21 basophils/mΐ of blood is indicative of a likely response to an anti-IgE therapeutic agent.

[00211] 65. A method of predicting a subject’s response to an anti-IgE therapeutic agent for the treatment of chronic spontaneous urticaria (CSU), the method comprising: (a) performing an assay to detect the level of one or more miRNA species selected from the group consisting of miR-1200, miR-1236-3p and miR-4664-3p in a sample of basophils from the subject; and

(b) when the level of one or more of miR-1200, miR-1236-3p and miR-4664-3p is above a reference level, administering an anti-IgE therapeutic agent; and

(c) when the level of one or more of miR-1200, miR-1236-3p and miR-4664-3p is at or below the reference level, administering a non-steroidal immunosuppressant.

[00212] 66. The method of paragraph 65, wherein an increase in two or more of miRNAs miR-

1200, miR-1236-3p and miR-4664-3p is detected relative to the reference.

[00213] 67. The method of paragraph 65, wherein an increase in each of miRNAs miR-1200, miR-1236-3p and miR-4664-3p is detected relative to the reference.

[00214] 68. The method of paragraph 65, wherein the reference is the level of the one or more miRNAs in a basophil sample from one or more subjects with CSU whose disease did not respond to an anti-IgE therapeutic agent.

[00215] 69. The method of any one of paragraphs 65-68, wherein the subject’s CSU is resistant to

Hi-antihistamine therapy.

[00216] 70. The method of any one of paragraphs 65-69, wherein the anti-IgE therapeutic agent comprises omalizumab or ligelizumab.

[00217] 71. The method of any one of paragraphs 65-70, further comprising detecting the level of basophils in a blood sample from the subject.

[00218] 72. A method of predicting a subject’s response to an anti-IgE therapeutic agent for the treatment of chronic spontaneous urticaria (CSU), the method comprising: receiving the results of an assay performed on a sample of plasma exosomes from the subject to detect the level of one or more miRNAs relative to a reference, and receiving the results of an assay performed on a sample of basophils from the subject to detect the level of one or more miRNAs relative to a reference, and/or receiving the results of a blood count indicating the number of basophils in a blood sample from the subject wherein detection of an increase in the level of one or more of the miRNAs miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 or a decrease in the level of miR-141-3p in plasma exosomes relative to the respective reference, and/or wherein the detection of an increase in the level of one or more of the miRNAs miR-1200, miR-1236-3p and miR-4664-3p in basophils relative to the respective reference, and/or wherein the detection of a number of basophils greater than or equal to 21 basophils per microliter of blood indicates the subject’s CSU is likely to respond to an anti-IgE therapeutic agent. [00219] 73. A method of predicting a subject’s response to an anti-IgE therapeutic agent for the treatment of chronic spontaneous urticaria (CSU), the method comprising: performing an assay on a sample of plasma exosomes from the subject to detect the level of one or more miRNAs relative to a reference, and performing an assay performed on a sample of basophils from the subject to detect the level of one or more miRNAs relative to a reference, and/or performing a blood count to detect the number of basophils in a blood sample from the subject, wherein detection of an increase in the level of one or more of the miRNAs miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 or a decrease in the level of miR-141-3p in plasma exosomes relative to the respective reference, and/or wherein the detection of an increase in the level of one or more of the miRNAs miR-1200, miR-1236-3p and miR-4664-3p in basophils relative to the respective reference, and/or wherein the detection of a number of basophils greater than or equal to 21 basophils per microliter of blood indicates the subject’s CSU is likely to respond to an anti-IgE therapeutic agent.

[00220] 74. A method of treating chronic spontaneous urticaria (CSU), the method comprising: receiving the results of an assay performed on a sample of plasma exosomes from the subject to detect the level of one or more miRNAs relative to a reference, and receiving the results of an assay performed on a sample of basophils from the subject to detect the level of one or more miRNAs relative to a reference, and/or receiving the results of a blood count indicating the number of basophils in a blood sample from the subject, and administering to the subject an anti-IgE therapeutic agent when the level of one or more of the miRNAs miR-6499-5p, miR-7848-3p, miR-4494, miR- 450a-2-3p, miR-6877-3p, and miR-3976 in plasma exosomes is increased, or the level of miR-141-3p in plasma exosomes is decreased relative to the respective reference, and/or when the level of one or more of the miRNAs miR-1200, miR-1236-3p and miR-4664-3p in basophils is increased relative to the respective reference, and/or wherein the number of basophils is greater than or equal to 21 basophils per microliter of blood, or administering to the subject a non-steroidal immunosuppressant when the level of one or more of the miRNAs miR-6499-5p, miR-7848-3p, miR-4494, miR- 450a-2-3p, miR-6877-3p, and miR-3976 in plasma exosomes is not increased, or the level of miR- 141-3p in plasma exosomes is not decreased relative to the respective reference, and/or when the level of one or more of the miRNAs miR-1200, miR-1236-3p and miR-4664-3p in basophils is not increased relative to the respective reference, and/or wherein the number of basophils is not greater than or equal to 21 basophils per microliter of blood.

[00221] 75. The method of any one of paragraphs 72-74, wherein two or more of an increase in miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the plasma exosome reference are detected in plasma exosomes.

[00222] 76. The method of any one of paragraphs 72-74, wherein three or more of an increase in miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the plasma exosome reference are detected in plasma exosomes.

[00223] 77. The method of any one of paragraphs 72-74, wherein four or more of an increase in miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the plasma exosome reference are detected in plasma exosomes.

[00224] 78. The method of any one of paragraphs 72-74, wherein five or more of an increase in miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the plasma exosome reference are detected in plasma exosomes.

[00225] 79. The method of any one of paragraphs 72-74, wherein six or more of an increase in miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, or miR-3976 or a decrease in miR-141-3p relative to the plasma exosome reference are detected in plasma exosomes.

[00226] 80. The method of any one of paragraphs 72-74, wherein an increase in each of miR-

6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 and a decrease in miR-141-3p relative to the plasma exosome reference are detected in plasma exosomes.

[00227] 81. The method of any one of paragraphs 72-80, wherein an increase in two or more of miR-1200, miR-1236-3p and miR-4664-3p relative to the basophil reference are detected in basophils. [00228] 82. The method of any one of paragraphs 72-80, wherein an increase in each of miR-

1200, miR-1236-3p and miR-4664-3p relative to the basophil reference are detected in basophils. [00229] 83. The method of any one of paragraphs 72-82, wherein the respective reference is the level of the one or more miRNAs in a plasma exosome, basophil and/or blood sample from one or more subjects with CSU whose disease did not respond to an anti-IgE therapeutic agent.

[00230] 84. The method of any one of paragraphs 72-83, wherein the subject’s CSU is resistant to

Hi-antihistamine therapy.

[00231] 85. The method of any one of paragraphs 72-84, wherein the anti-IgE therapeutic agent comprises omalizumab, ligelizumab, UB-221, quilizumab, and/or MEDI-4212.

[00232] 86. A kit comprising reagents permitting the quantitative detection of target miRNAs including miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, miR-3976, and miR-141-3p. [00233] 87. A kit comprising reagents permitting the quantitative detection of target miRNAs including miR-1200, miR-1236-3p and miR-4664-3p.

[00234] 88. A kit comprising reagents permitting the quantitative detection of target miRNAs including miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, miR-3976, miR- 141 -3p, miR-1200, miR-1236-3p and miR-4664-3p.

[00235] 89. The kit of any one of paragraphs 86-88, further comprising reagents permitting the isolation or quantitation of basophils in a biological sample.

[00236] 90. The kit of any one of paragraphs 86-89, further comprising reagents permitting the isolation of miRNAs.

[00237] 91. The kit of any one of paragraphs 88-90, further comprising PCR primers permitting amplification of the target miRNAs.

[00238] 92. The kit of any one of paragraphs 88-91, further comprising a reference composition comprising a known amount of target miRNA.

[00239] 93. The kit of any one of paragraphs 88-92, further comprising a reference sample, wherein the reference sample comprises miRNAs isolated from exosomes isolated from a subject with refractory CSU that did not respond to anti-IgE therapy.

[00240] 94. The kit of any one of paragraphs 88-93, further comprising one or more of the reagents arrayed on a solid support.

[00241] 95. The kit of paragraph 94, wherein the solid support comprises a microscope slide, a microwell plate, or a capillary electrophoresis plate.

[00242] 96. The kit of any one of paragraphs 88-95, further comprising a detectable label.

[00243] 97. Use of an anti-IgE therapeutic agent in the treatment of chronic spontaneous urticaria in a subject in need thereof, wherein the subject in need thereof is determined to have a level of one or more of miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 above a reference level, and/or a level of miR-141-3p below the reference level in an exosome sample obtained from the subject.

[00244] 98. Use of a non-steroidal immunosuppressant in the treatment of chronic spontaneous urticaria in a subject in need thereof, wherein the subject in need thereof is determined to have a level of one or more of miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR- 3976 at or below a reference level, and/or when the level of miR-141-3p is at or above a reference level in an exosome sample obtained from the subject.

[00245] 99. The use of paragraph 97 or 98, wherein the subject has two or more miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR- 6877-3p, and miR-3976 above the reference level; and/or miR-141-3p below the reference level. [00246] 100. The use of paragraph 97 or 98, wherein the subject has three or more of the miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2- 3p, miR-6877-3p, and miR-3976 above the reference level; and/or miR-141-3p below the reference level.

[00247] 101. The use of any one of paragraphs 97-100, wherein the subject has four or more of the miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 above the reference level; and/or miR- 141 -3p below the reference level.

[00248] 102. The use of any one of paragraphs 97-101, wherein the subject has five or more of the miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR- 450a-2-3p, miR-6877-3p, and miR-3976 above the reference level; and/or miR-141-3p below the reference level.

[00249] 103. The use of any one of paragraphs 97-102, wherein the subject has six or more of the miRNA species selected from the group consisting of: miR-6499-5p, miR-7848-3p, miR-4494, miR- 450a-2-3p, miR-6877-3p, and miR-3976 above a reference level; and/or miR-141-3p below the reference level.

[00250] 104. The use of any one of paragraphs 97-103, wherein the subject has each of the miRNA species selected from miR-6499-5p, miR-7848-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 above the reference level; and miR-141-3p below the reference level.

[00251] 105. The use of any one of paragraphs 97-104, wherein the method further comprises detecting the level of basophils in a biological sample from the subject compared to a reference level. [00252] 106. Use of an anti-IgE therapeutic agent in the treatment of chronic spontaneous urticaria

(CSU) in a subject in need thereof, wherein the subject is determined to be in need thereof by (i) receiving the results of an assay performed on a sample of plasma exosomes from the subject to detect the level of one or more miRNAs relative to a reference, (ii) receiving the results of an assay performed on a sample of basophils from the subject to detect the level of one or more miRNAs relative to a reference, and/or (iii) receiving the results of a blood count indicating the number of basophils in a blood sample from the subject, and when the level of one or more of the miRNAs miR-6499-5p, miR-7848-3p, miR-4494, miR- 450a-2-3p, miR-6877-3p, and miR-3976 in plasma exosomes is increased, or the level of miR-141-3p in plasma exosomes is decreased relative to the respective reference, and/or when the level of one or more of the miRNAs miR-1200, miR-1236-3p and miR-4664-3p in basophils is increased relative to the respective reference, and/or wherein the number of basophils is greater than or equal to 21 basophils per microliter of blood.

[00253] 107. Use of a non-steroidal immunosuppressant in the treatment of chronic spontaneous urticaria (CSU) in a subject in need thereof, wherein the subject is determined to be in need thereof by (i) receiving the results of an assay performed on a sample of plasma exosomes from the subject to detect the level of one or more miRNAs relative to a reference, (ii) receiving the results of an assay performed on a sample of basophils from the subject to detect the level of one or more miRNAs relative to a reference, and/or (iii) receiving the results of a blood count indicating the number of basophils in a blood sample from the subject, and when the level of one or more of the miRNAs miR-6499-5p, miR-7848-3p, miR-4494, miR- 450a-2-3p, miR-6877-3p, and miR-3976 in plasma exosomes is not increased, or the level of miR- 141-3p in plasma exosomes is not decreased relative to the respective reference, and/or when the level of one or more of the miRNAs miR-1200, miR-1236-3p and miR-4664-3p in basophils is not increased relative to the respective reference, and/or wherein the number of basophils is not greater than or equal to 21 basophils per microliter of blood.

EXAMPLES

[00254] The methods and examples described herein are published in Al-Shaikhly, T et al. “Extracellular vesicle microRNAs as predictors of response to omalizumab in chronic spontaneous urticaria” Allergy 76(4): 1274-1277 (2021), the contents of which are incorporated herein by reference in its entirety.

EXAMPLE 1: MicroRNAs as Predictors of Response to Omalizumab in Chronic Spontaneous Urticaria

[00255] The underlying mechanisms of chronic spontaneous urticaria (CSU) remain poorly understood. ^1,2 Lesional skin biopsies in CSU patients have evidence for mast cell degranulation, tissue edema, and a mixed interstitial and perivascular infdtrate composed of lymphocytes, neutrophils, eosinophils, and basophils. ^3,4 ’ ⁷ Basophil involvement in CSU immunopathogenesis is suggested by the observed basopenia that may result from the movement of circulating basophils to the urticarial sites ¹ - Dysregulated signaling of the FceRI (i.e., high-affinity IgE receptor) pathway is seen in basophils from CSU patients compared to healthy controls. ² Targeting IgE/FcsRI-mediated mast cell and basophil activation by anti-IgE antibody has emerged as a novel treatment strategy for patients with EE-antihistamine-resistant (refractory) CSU.--

[00256] Omalizumab, a humanized IgGiK monoclonal antibody, binds to the Cs3 domain of Fc on the heavy chain of IgE leading to rapid reduction in free IgE levels and subsequent decrease in FceRI expression on basophils and mast cells.— In a proof of concept study, omalizumab resulted in clinical improvement in 11 out of 12 patients with refractory chronic autoimmune urticaria. Subsequent clinical trials ²¹¹ ’— extended this observation to patients with CSU without an autoimmune designation. However, approximately one-third of patients fail to respond to anti-IgE treatment— spurring a search for biomarkers predictive of response.— Serum autoreactivity, determined by autologous serum skin testing or serum-induced basophil histamine release assay, has been associated with a slower response to omalizumab than CSU patients without autoantibodies.—— Though, in the ASTERIA I- study of omalizumab therapy in CSU, the baseline level of thyroperoxidase antibody or the presence of a positive chronic urticaria index test that reflects the presence of autoantibodies against Fee receptors or IgE failed to predict response to omalizumab — Eosinopenia and basopenia are associated with a poor response to omalizumab in retrospective analyses— but not yet studied prospectively. Although higher IgE levels are associated with a quicker relapse after omalizumab discontinuation— and lower levels with non-response to omalizumab— in CSU, sub-analysis of median IgE levels from ASTERIA I— and ASTERIA II studies of omalizumab treatment in CSU patients found that IgE levels do not impact efficacy of omalizumab — Although current biomarkers for drug responsiveness are unsuccessful in identifying treatment-responsive phenotypes,— the utility of microRNAs as biomarkers of therapeutic response to omalizumab has not been explored.

[00257] MicroRNAs, small non-coding RNAs, are released by cells into the blood and other biofluids within small (<150 nm in diameter), stable membrane-bound microvesicles (termed exosomes),—’— making them accessible and attractive biomarkers of disease pathogenesis and response to therapeutic intervention. ^{25 27} ’ ³⁰ In our prospective cohort study of patients with refractory CSU, two groups were identified that were most discordant in their clinical response to omalizumab to explore the utility of exosomal and basophil microRNA expression profiles as predictors of clinical response.

Methods

[00258] The clinical study plan (FIG. 1A) was approved by the Western Institutional Review Board (ClinicalTrials.gov ID: NCT02814630) and conducted at Seattle Allergy and Asthma Research Institute, Seattle, WA. Inclusion and exclusion criteria are described in the Supplementary Appendix. Study participants (>18 years) provided written informed consent. After initial screening within two weeks of study start, patients received one subcutaneous injection of omalizumab at a dose of 300 mg on Days 0, 30, and 60. Blood samples were collected at baseline (Day 0, prior to omalizumab injection) for isolation of basophils and exosomes. Patients completed a twice-daily diary that was used to determine their urticaria activity score (UAS) during a 7-day period (UAS7).— To quantitate the response to omalizumab of each subject, the area-under-the-curve (AUC) for the weekly UAS7 values over the 12-week study period from a baseline value of zero (UAS7 AUC) was computed using the trapezoidal method and expressed as UAS7 x week unit (Table 1 in EXAMPLE 2). All peaks above the set baseline of zero were counted. For missing UAS7 diary scores, the last observation was carried forward. Responders were defined as those subjects with an AUC for the weekly UAS7 over the 12-week treatment period that was equal to or less than the 75 ^th percentile and achieved 90% improvement of baseline UAS7. Non-responders were defined as subjects who a) failed to achieve a 90% improvement of their baseline weekly UAS7, or b) had an AUC for the weekly UAS7 over the 12-week study period that was above the 75 ^th percentile (i.e., top quartile), or c) had an exacerbation of their symptoms necessitating the addition of an anti-inflammatory or an immunosuppressive agent.

[00259] To study the microRNA expression profile as a predictor for response to omalizumab, we examined the most discordant groups of response to the anti-IgE intervention, by determining the AUC for the weekly UAS7 over the last half of the study to identify a durable therapeutic response (FIG. IB). Responders with complete response (CR) had the lowest AUC over the last half of the study period compared to those with partial response (PR). Subjects with non-response (NR) had the highest AUC over the entire study period as well as over the last half of the study (FIG. 1, and Table 1 in EXAMPUE 2).

[00260] Exosomes and basophils were isolated from blood for microRNA isolation and microRNA-Seq profiling. After identification of a set of differentially expressed microRNAs, Ingenuity Pathway Analysis (IP A) was employed to predict mRNA targets enabling identification of canonical pathways. Additional methods (including statistical analysis) are detailed in the EXAMPLE 2 Results

Response to omalizumab in study participants

[00261] Twenty-seven subjects were screened (Day -14 visit) (FIG. 1A) and 6 either failed screening or elected not to enter the treatment phase. Twenty-one subjects received omalizumab on Day 0 and were seen for first follow-up on Day 14; of these subjects, twenty subjects received omalizumab on Days 30 and Day 60 and completed the study on the Day 90. One subject withdrew from the study on Day 30 (i.e., did not receive omalizumab at this visit) because of worsening urticaria symptoms. Based on our response definition, there were 15 responders (6 complete responders (FIG. 1C), 9 partial responders (FIG. ID)), and five non-responders (FIG. IE).

Higher basophil levels associated with clinical response to omalizumab

[00262] At study entry, the age, gender, weight, baseline UAS7, eosinophil levels in the peripheral blood, and the presence of angioedema during the week before omalizumab treatment were not statistically different between the responder and non-responder groups (FIGs. 2A, 2B). However, responders had a higher baseline basophil level in the peripheral blood compared to non-responders (mean, 28.7; 95% Cl, 20.5 to 36.83 versus mean, 15.8; 95% Cl, 12.47 to 19.13; P = 0.005). Complete responders had significantly higher baseline basophil levels compared to the non-responders (mean, 33.17; 95% confidence interval [Cl], 24.84 to 41.49 versus mean, 15.8; 95% Cl 12.47 to 19.13; P = 0.006) (FIG. 2C). Basophil levels in the partial responders were not significantly different than those of the non-responders. Baseline basophil levels correlated significantly with response to omalizumab as measured by the AUC UAS7 over the 12-week study period (R ² = 0.33, P = 0.007) (FIG. 2D). To determine a baseline basophil level that predicts response to omalizumab, a receiver operating characteristic (ROC) curve (area under the ROC curve, 0.81; 95% Cl, 0.61 to 1; P = 0.044) was generated (FIG. 2E). A baseline level >21 basophils/pL was predictive of response with 80% sensitivity (95% Cl, 54.81 to 92.95) and 100% specificity (95% Cl, 56.55 to 100). When subjects were classified based on their baseline basophil level, basophil -high subjects (defined as those with a baseline basophil level >21 basophils/pL) had a significantly better response to omalizumab with lower mean UAS7 over time compared to basophil -low (<21 basophils/pL at baseline) subjects (predicted mean difference, 11.16; ± standard error of the mean, 2.37; P <0.001) (FIG. 2F).

Exosomal microRNA profile and response to omalizumab

[00263] Seven differentially expressed exosomal microRNAs were found at baseline that were associated with a complete response to omalizumab (FDR <0.05) (FIG. 3, and Table 2 in the Supplementary Appendix). Complete responders had higher expression of six of these microRNAs: miR-6499-5p (Log fold change [logFC], 5.05; 95% Cl, 1.83 to 8.27; FDR = 0.002) (FIG. 3B), miR- 7848-3p (logFC, 4.73; 95% Cl, 1.50 to 7.97; FDR = 0.003) (FIG. 3C), miR-4494 (logFC, 4.79; 95% Cl, 1.03 to 8.57; FDR = 0.014) (FIG. 3D), miR-450a-2-3p (logFC, 4.68; 95% Cl, 0.95 to 8.84; FDR = 0.014) (FIG. 3E), miR-6877-3p (logFC, 3.56; 95% Cl, 0.59, to 6.53; FDR = 0.021) (FIG. 3F), and miR-3976 (logFC, 4.36; 95% Cl, 0.52 to 8.20; FDR = 0.037) (FIG. 3G). Complete responders had lower expression of miR-141-3p (logFC, -4.49; 95% Cl, -8.53 to -0.46; FDR = 0.04) (FIG. 3H). To further explore the utility of these microRNAs in projecting response to omalizumab, each participant was characterized as having either high or low expression (defined as higher or lower than the geometric mean expression level for each microRNA). A differential clinical response was discovered as quantified by the UAS7 score over the 12-week study period. Higher expression of miR-6499-5p (LRT = 75.2, P <0.001) (FIG. 3B), miR-7848-3p-3p (LRT = 75.2, P <0.001) (FIG. 3C), miR-4494 (LRT = 75.2, P <0.001) (FIG. 3D), miR-450a-2-3p (LRT = 75.2, P <0.001) (FIG. 3E), miR-6877-3p (LRT = 42.7, P <0.001) (FIG. 3F), and miR-3976 (LRT = 43.2, P <0.001) (FIG. 3G) was associated with favorable response to omalizumab. In contrast, there was a trend (LRT = 21.4, P = 0.07) for higher expression of miR-141-3p to be associated with poor response to omalizumab (FIG. 3H). These data indicate that an exosomal microRNA expression profile may predict response to omalizumab therapy in patients with CSU.

Basophil microRNA profile and response to omalizumab

[00264] Three differentially expressed basophil microRNAs were discovered at baseline to be associated with complete response to omalizumab (FDR <0.05) (FIG. 4A, and Table 2 in EXAMPLE 2). Complete responders had higher expression of miR-1200 (logFC, 4.68; 95% Cl, 0.90 to 8.37; FDR = 0.024) (FIG. 4B), miR-1236-3p (logFC, 4.63; 95% Cl, 0.89 to 8.37; FDR = 0.024) (FIG. 4B), and miR-4664-3p (logFC, 4.34; 95% Cl, 0.65 to 8.03; FDR = 0.033) (FIG. 4B). A positive clinical response was observed, as quantified by the mean UAS7 over the 12-week study period, among participants with high expression (above the geometric mean) of these microRNAs compared to subjects with low expression: miR-1200 (LRT = 75.2, P <0.001) (FIG. 4B), miR-1236- 3p (LTR = 34.7, P = 0.001) (FIG. 4B), and miR-4664-3p (LRT = 34.7, P = 0.001) (FIG. 4B).

Tec kinase signaling pathway modulation associated with response to omalizumab [00265] Using the QIAGEN ^® IPA microRNA Target Filter feature, 1019 mRNA targets were identified for the seven differentially expressed exosomal microRNAs between the complete responders and non-responders on Day 0 (Table 3 in the EXAMPLE 2). The differentially expressed basophil microRNAs at baseline were predicted to target the expression of 451 mRNAs. Next, canonical pathways that were significantly enriched were examined with these mRNA targets and analyzed their predicted activity using the IPA Molecule Activity Predictor feature. The seven differentially expressed exosomal microRNAs significantly modulated the activity of the Tec Kinase Signaling pathway (FDR = 0.003) (FIG. 5, and Table 4 in EXAMPLE 2). This pathway is involved in transducing signals from FceRI and other key immune receptors. This set of seven exosomal microRNAs modulated the expression of signaling molecules both upstream and downstream of the Tec kinases including serine/threonine p21 -activating kinase (PAK), protein kinase C (PKC), Wiskott-Aldrich Syndrome (WAS) protein, Rho GTPases, phosphatidylinositol-3 -kinase (PI3K) complex, Src-family tyrosine kinases, and Janus kinase (JAK)l and JAK2). IPA analysis predicted downregulation of varied cellular functions including cell adhesion, cell migration, actin reorganization, and calcium mobilization. In contrast, the effect on gene expression and cell transformation downstream of the mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription protein (STAT) transcription factors were predicted to be upregulated. [00266] Using the same differentially expressed exosomal microRNAs, no other predicted biological pathways met our pre-specified significance cutoff of FDR <0.05. However, their respective mRNA targets were less enriched (FDR 0.05-0.1) in other canonical pathways of potential relevance to CSU pathogenesis and response to omalizumab including IL-1 Signaling, IL-17a Signaling, Thl 7 Activation, Oncostatin M Signaling, CXCR4 Signaling, Role of NFAT in Regulation of the Immune Response, Glucocorticoid Receptor Signaling, and FcgRIIB Signaling in B Lymphocytes pathways (Table 4 in EXAMPLE 2). In contrast, mRNA targets identified by differentially expressed basophil microRNAs between complete responders and non-responders were not significantly enriched in any canonical pathways.

Adverse events

[00267] One participant (Subject 4) had an anaphylactic reaction two hours after omalizumab injection on Day 60 visit. Symptoms completely resolved with epinephrine and methylprednisolone treatment.

Summary of Results [00268] This study establishes a clinical and molecular phenotype in CSU characterized by high basophil levels (>21 cells/pL). unique expression profdes of exosomal and basophil microRNAs, and altered Tec kinase-dependent canonical pathways in complete responders to omalizumab.

[00269] Because a significant proportion of patients with CSU fails to respond to omalizumab—, several candidate biomarkers were examined to identify patients who would be helped from the use of this biologic therapy. A recent retrospective study found that both eosinopenia and basopenia, defined as <50 eosinophils/pL and <10 basophils/pL at baseline, predicted poor response to omalizumab with high specificity but low/moderate sensitivity.— In the prospective study, we did not find a difference in baseline eosinophil levels between the CR and NR groups; whereas, basophil levels were significantly different. A basophil level of less than 21 cell s/pL was predictive of non-response to omalizumab with 80% sensitivity and 100% specificity.

[00270] Commercial microRNA panels have been developed to identify the origin of cancers or classify malignant thyroid cancers when cytology is uncertain — The exosomal and basophil microRNA expression profiles identified in our study could possibly serve as a diagnostic panel to personalize treatment for CSU patients by selecting patients who would profit from omalizumab intervention and to potentially reduce treatment cost. In addition to serving as biomarkers, these differentially expressed microRNAs enabled the identification of a molecular phenotype associated with response to omalizumab and potential new therapeutic targets within the Tec Kinase Signaling pathway.

[00271] The Tec kinases, non-receptor protein tyrosine kinases, consist of five members: IU-2 inducible T-cell kinase (Itk), Bruton tyrosine kinase (Btk), resting lymphocyte kinase (Rlk), tyrosine kinase expressed in hepatocellular carcinoma (tec), and bone marrow kinase expressed on the X chromosome (Bmx). ³⁰ The Tec kinases are involved in many critical molecular pathways in both myeloid and lymphoid cells. They are downstream of FceRI, T-cell receptor complex, receptor tyrosine kinase, cytokine receptors, Fas ligand (FasU) receptor, and bl integrin promoting diverse biological functions. Mast cells express Tec, Btx, and Itk. ^31,32 Stem cell factor (SCF) promotes development and activation of mast cells by signaling through c-kit, a receptor tyrosine kinase. Upon stimulation with SCF, c-kit activates Tec kinase. ³³ Cross-linking of FceRI in mast cells activates Btk promoting mast cell degranulation and mediator release. ³⁰ Also activated by FceRI stimulation are Itk and Tec that regulate mast cell production of cytokines. ^31,32 The Tec kinases are important in basophils. IU-3, a cytokine promoting basophil development and activation, signals through JAK2 to activate Tec kinase. ^33,34 While FasU promotes apoptosis of basophils, IU-3 protects basophils against FasU-mediated apoptosis. ³⁵ Both mast cells and basophils express bl integrins ³⁶ that mediate adhesion of skin mast cells to extracellular matrix proteins ³⁷ and basophils to the endothelium. ³⁸ Because interruption of FceRI underlies the mechanism for the therapeutic effect of omalizumab in CSU, our findings that the differentially expressed exosomal microRNAs target the Tec kinase-dependent FceRI signaling pathway support this mechanism and suggest that this predicted altered molecular profde may facilitate response to omalizumab.

[00272] This study was limited by the small number of patients, especially non-responders. Larger studies are needed to validate our identified basophil level (>21 cells/pL) as a response biomarker and to bring to clinicians a microRNA diagnostic panel as a predictive tool for omalizumab response. Potential mRNA targets for the differentially expressed microRNAs were based on current state of knowledge and not all impacted canonical pathways may have been identified at this point in time. As studies of microRNAs are rapidly evolving, and as more experimental data become available, pathways altered by the three basophil microRNAs that were differentially expressed between the responders and non-responders may be uncovered in the future and provide further insight into the role of basophils in the immunopathogenesis of CSU.

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[00280] 8. Maurer M, Rosen K, Hsieh H-J, et al. Omalizumab for the treatment of chronic idiopathic or spontaneous urticaria. N Engl J Med 2013;368:924-35.

[00281] 9. Kaplan AP, Joseph K, Maykut RJ, Geba GP, Zeldin RK. Treatment of chronic autoimmune urticaria with omalizumab. J Allergy Clin Immunol 2008;122:569-73.

[00282] 10. Presta LG, Lahr SJ, Shields RL, et al. Humanization of an antibody directed against

IgE. J Immunol 1993;151:2623-32. [00283] 11. Kaplan A, Ledford D, Ashby M, et al. Omalizumab in patients with symptomatic chronic idiopathic/spontaneous urticaria despite standard combination therapy. J Allergy Clin Immunol 2013;132:101-9.

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[00285] 13. Kaplan A, Ferrer M, Bernstein JA, et al. Timing and duration of omalizumab response in patients with chronic idiopathic/spontaneous urticaria. J Allergy Clin Immunol 2016;137:474-81.

[00286] 14. Deza G, Ricketti PA, Gimenez-Amau AM, Casale TB. Emerging biomarkers and therapeutic pipelines for chronic spontaneous urticaria. J Allergy Clin Immunol Pract 2018;6: 1108-17. [00287] 15. Gericke J, Metz M, Ohanyan T, et al. Serum autoreactivity predicts time to response to omalizumab therapy in chronic spontaneous urticaria. J Allergy Clin Immunol 2017; 139: 1059-61. [00288] 16. Nettis E, Cegolon L, Di Leo E, Lodi Rizzini F, Detoraki A, Canonica GW.

Omalizumab in chronic spontaneous urticaria: Efficacy, safety, predictors of treatment outcome, and time to response. Ann Allergy Asthma Immunol 2018;121:474-8.

[00289] 17. Saini SS, Bindslev-Jensen C, Maurer M, et al. Efficacy and safety of omalizumab in patients with chronic idiopathic/spontaneous urticaria who remain symptomatic on HI antihistamines: a randomized, placebo-controlled study. J Invest Dermatol 2015;135:67-75.

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1 integrins that mediate adhesion to extracellular matrix proteins. J Immunol 1995;154:6058-64. [00310] 38. Bochner BS, Sterbinsky SA, Briskin M, Saini SS, MacGlashan DW, Jr. Counter receptors on human basophils for endothelial cell adhesion molecules. J Immunol 1996;157:844-50.

EXAMPLE 2: Additional methods and results

INCLUSION CRITERIA [00311] adult patients (>18 years), 2) at least 6 weeks of CSU with itching despite current use of up to x4 Hi-antihistamines ¹ and 3) an urticaria activity score (UAS) during a 7-day period (UAS7) of >16 (on a scale ranging from 0 to 42, with higher scores indicating greater activity) ² during the 7 days before the first treatment with omalizumab.

EXCLUSION CRITERIA

[00312] 1) A clearly defined underlying cause for chronic urticaria, 2) routine administration (i.e., daily or every other day for >5 consecutive days) of systemic glucocorticoids, anti-inflammatory, immunomodulatory, or immunosuppressive agents within the previous 30 days, 3) the use of any Ha- antihistamine or leukotriene-receptor antagonist within 7 days preceding the screening visit, 4) treatment with omalizumab within the previous year, or a known hypersensitivity to omalizumab, 5) a history of cancer, or 6) women who were pregnant or nursing or unable to use an effective method of contraception during dosing with omalizumab.

OMALIZUMAB TREATMENT

[00313] Twenty-seven subjects were screened (Day -14 visit, FIG. 1A) and 6 either failed screening or elected not to enter the treatment phase (i.e., not on antihistamines [N = 1], work schedule conflict [N = 1], UAS7 score less than the eligibility cutoff of 16 [N = 1], or received omalizumab [N = 1], omalizumab plus prednisone [N = 1] or prednisone [N = 1] treatment for intense urticaria symptoms prior to scheduling Day 0 visit) (i.e., not on antihistamines [N = 1], work schedule conflict [N = 1], UAS7 score less than the eligibility cutoff of 16 [N = 1] or received omalizumab [N = 1] After initial screening within two weeks of study start, patients received one subcutaneous injection of omalizumab (Xolair ^® ; Genentech, South San Francisco, CA) at a dose of 300 mg on Days 0, 30, and 60 (FIG. 1A). This dose was based on results of two international, multicenter, randomized, double-blind, placebo-controlled Phase 3 studies, ASTERIA I ³ and ASTERIA II ⁴ that demonstrated that omalizumab significantly decreased clinical symptoms and signs of chronic urticaria in patients who had remained symptomatic despite the use of Hi-antihistamines. ³ Patients were closely monitored for two hours after each dose of omalizumab with epinephrine administered if evidence of anaphylaxis per the joint task force guidelines of the American Academy of Allergy, Asthma & Immunology and the American College of Allergy, Asthma & Immunology. ⁵ Patients were provided with an epinephrine injector for self-administration of epinephrine if development of anaphylaxis after discharge from the Seattle Allergy and Asthma Research Institute (previously known as ASTHMA Inc. Clinical Research Center). Blood samples were collected at baseline (Day 0, prior to omalizumab injection) for isolation of basophils and exosomes (FIG. 1A). Patients completed a twice-daily diary that was used to determine UAS7. ⁶ Patients continued stable doses of their pretreatment period Hi -antihistamine (i.e., cetirizine, levocetirizine dihydrochloride, fexofenadine, loratadine, or desloratadine) throughout the treatment period. For the duration of the study, all patients were provided with diphenhydramine (25 mg) as rescue medication for itch relief (up to a maximum of three doses in 24 hours).

EXOSOMAL MICRORNA ISOLATION

[00314] Exosomes were isolated from the plasma of the study participants using Invitrogen’s Total Exosome Precipitation Reagent (Thermo Fisher Scientific, Inc., Waltham, MA) according to the manufacturer’s protocol. MicroRNAs were then isolated from the exosomes using the QIAGEN miRNeasy™ Serum/Plasma Kit (QIAGEN Sciences, Germantown, MD).

BASOPHIL ISOLATION

[00315] Peripheral blood samples were collected in EDTA tubes. Human nucleated cells were separated from red blood cells using HetaSep™ reagent STEMCELL Technologies, Vancouver, Canada). Basophils were isolated from nucleated cells using the STEMCELL Technologies EasySep™ Human Basophil Enrichment Kit (isolation purity, mean ± SD, 94.0 ± 2.5%). Purified basophils were centrifuged at 1,500 rpm for 10 minutes. The pellets were resuspended in 200 pL of PBS, 1 mL of Trizol was added, and the purified basophils were stored at -80°C until RNA was isolated.

BASOPHIL MICRORNA EXTRACTION

[00316] Total RNA from basophils was isolated using the QIAGEN miRNeasy™ Micro extraction kit. This kit isolates total RNA that includes microRNAs. The integrity of the total RNA samples was evaluated using a 2100 Bioanalyzer instrument (Agilent Technologies, Inc., Palo Alto, CA). A Thermo Fisher Scientific NanoDrop™ 1000 Spectrophotometer was used to determine purity of RNA samples by measuring OD260/280 and OD260/230 ratios. Only samples with an RNA Integrity Number of >7 and OD260/280 and OD260/230 ratios >1.8 were used for further analysis. Total RNA concentrations were determined using Thermo Fisher Scientific Qubit™ RNA HS Assay Kit. MICRORNA-SEQ PROFILING

[00317] MicroRNA profiling was performed at the QIAGEN Service lab in Germantown, MD. MicroRNA samples were evaluated for quality by using the QIAGEN QIAseq microRNA. Total RNA samples that included microRNAs were assessed for quality and quantity by using an Agilent TapeStation 4200 and a Thermo Fisher Scientific NanoDrop 2000c spectrophotometer respectively. Five pL exosomal microRNA and 5 pL of total basophil RNA (100 ng) were reverse transcribed into cDNA in 20 pL reactions using the QIAseq microRNA Library Kit and specially designed 3’ and 5’ adapters were ligated to mature microRNAs. The ligated microRNAs were then reverse transcribed to cDNAs using a reverse transcription primer that contains an integrated Unique Molecular Index (UMI). Following cDNA cleanup, PCR library amplification (19-22 cycles) was performed with a universal forward primer and indexing reverse primers. Following a final library cleanup, the quality of the microRNA libraries was assessed using the Agilent TapeStation 4200. MicroRNA sequencing libraries prepared with the QIAseq microRNA Library Kit were sequenced using a NextSeq 500 instrument (Illumina, Inc., San Diego, CA). Libraries were quantified, adjusted to 4 nM each and pooled. The pooled libraries were further diluted down to 1.8 pM and loaded into a flow cell on the Illumina NextSeq 500. The sequencing specifications were 1x76 bases and 12 million targeted reads per sample.

STATISTICAL ANALYSES

[00318] Descriptive statistics were performed to determine the mean and standard deviation (SD) of continuous variables and the number (N) and relative frequency (%) of categorical variables. For the UAS7 AUCs, the medians, 25 ^th and the 75 ^th percentiles were also determined. The Kolmogorov- Smimov test and histogram analyses were used to determine whether continuous variables were normally distributed. A two-tailed t-test and Analysis of Variance (ANOVA) were used for the comparisons of continuous variables between the different groups as appropriate. ^7,8 Welch’s correction that does not assume equal variance was applied when appropriate to account for unequal sample sizes. Fisher’s exact test was used for group comparison of categorical variables. For baseline clinical variables that were significantly different between responders and non-responders, a receiver operating characteristic (ROC) curve analysis was generated to determine a cutoff value predictive of response to omalizumab therapy. A mixed effects model was used to assess for differences in the mean UAS7 over the 12-week study period based on different selected baseline attributes. The mixed effects model was opted over repeated measures ANOVA to overcome missing data points. ⁹ [00319] For analysis of the miR-Seq data, raw reads were de-multiplexed and FASTQ files for each sample were generated using Illumina bcl2fastq software. FASTQ data were checked using the FastQC tool. Reads and UMI counts were generated using QIAGEN GeneGlobe pipeline. During the QIAseq microRNA Library Kit construction process, each individual microRNA molecule was tagged with an UMI. Following sequencing and trimming, reads were analyzed for the presence of UMIs. All reads containing identical insert sequence and UMI sequence (insert-UMI pair) were collapsed into a single read. These reads were passed into the analysis pipeline. Additionally, reads containing partial UMI were also passed into the analysis pipeline to allow for true quantification of the microRNAs by eliminating library amplification bias. Cutadapt™ (version 1.11) was used to remove the adapter sequences and output adapter-free sequences. The UMI counts for each microRNA were read into R (r-proj ect.org) using the Bioconductor edgeR package ¹⁰ and filtered to remove microRNAs that were either unexpressed or expressed at a consistent, unreliably low level. Samples were normalized using a trimmed mean of M-values and then counts were converted to log counts/million (logCPM) using the voomWithQualityWeights function ¹¹ from the Bioconductor limma package. ¹² This function converts the counts to logCPM and then estimates observation and sample-level weights that can be used in a conventional linear model to control for heteroscedasticity. For each subject, there were repeated measurements that were estimated using the limma duplicate Correlation function ¹³ and controlled for by using a linear mixed model when making comparisons. Comparisons between groups were made using empirical Bayes adjusted contrasts. ¹⁴ Multiplicity was controlled at a false discovery rate (FDR; Benjamini-Hochberg correction) ¹⁵ of 0.05, meaning that the expected maximum proportion of false positives is estimated to be 5%. Heatmaps for the differentially expressed microRNAs were generated after conversion to z-scores. To test for a different temporal outcome of UAS7 score over time, we used a linear mixed model that included dichotomized microRNA expression (high or low at baseline), a time coefficient (treatment week), and an interaction between the two and we tested for the significance of interaction (e.g., by dropping the interaction term from the model and computing a likelihood ratio test [LRT], which tested for a difference at one or more time points). A P value <0.05 was used to indicate statistical significance. All statistical analyses were performed using R (R Foundation for Statistical Computing, Vienna, Austria) and GraphPad Prism 8.3 (GraphPad Software, San Diego, CA).

INGENUITY PATHWAY ANALYSIS

[00320] After identification of a set of differentially expressed microRNAs, QIAGEN Ingenuity Pathway Analysis (IPA) microRNA Target Filter was employed to predict mRNA targets. The differentially expressed microRNAs were linked to their respective mRNA targets based on information in the QIAGEN Knowledge Base. Filtering was performed to prioritize only those mRNAs that were experimentally validated or had highly confident predictions of targeting based on information from TarBase, miRecords, Target Scan (human), and literature findings curated within the QIAGEN Knowledge Base.

[00321] QIAGEN IPA Expression Analyses were then performed on each mRNA dataset generated by the microRNA Target Filter Analysis. The IPA Core Analyses identified canonical pathways that were significantly enriched from the dataset. The significance of the association between the dataset and a canonical pathway was determined from a P value of overlap calculated using the right-tailed Fisher’s exact test. The Benjamini-Hochberg procedure was applied to control FDR. Significant microRNAs were added to the identified Canonical Pathways using the QIAGEN IPA Grow tool that uses information from databases and curated findings in the QIAGEN Knowledge Base to find interactions between molecules and diseases/biological functions within pathways. The QIAGEN IPA Molecule Activity Predictor (MAP) tool was used to predict the upstream and/or downstream effects of activation or inhibition of molecules in a particular pathway given one or more neighboring molecules with known activity. These predictions were based on an algorithm that incorporates the calculation of a maximum absolute z-score to assess predictions of activity of molecules, diseases, and biological functions.

Tables

Table 1. Clinical Response Classification by Area Under the Curve (AUC) of UAS7 Scores. ^*

*AUC UAS7 response is expressed as UAS7 x week unit. AUC 0-12 wk, area under the curve for the UAS7 over the 12-week of the study; AUC 6-12 wk, area under the curve for the UAS7 over weeks 6- 12 of the study period; CR, complete responders; PR, partial responders; NR, non-responders.

^† Subject 26 was excluded from the microRNA analysis because of urticaria exacerbation treated with cyclosporine A.

Table 2. Differentially Expressed Exosomal and Basophil MicroRNAs. ^*

Differential expression is between complete responder and non-responder groups on Day 0. FC, fold change.

Table 3. Number of mRNA Targets for Differentially Expressed Exosomal and Basophil MicroRNAs. ^*

FcgRIIB 0.062 CACNA2D4, DOK1, LYN, MAP2K4, miR-141-3p, miR- Pathway Target genes MicroRNAs targeting this pathway

Signaling in B MAPK10, PIK3CA, PLCG2, RAP IB, RASD2, 450a-2-3p, miR-

Lymphocytes SHC1 4494, miR-7848-3p- 3p

Reelin Signaling j 0.062 APBB1, ARPC5, DCX, FYN, ITGB2, LYN, miR-141-3p, miR- in Neurons j MAP2K4, MAP2K5, MAPK10, PIK3CA, 450a-2-3p, miR- RAC1, RAP1B, WASF1, YES1 3976, miR-4494, miR-6877-3p

Molecular j 0.062 CASP7, CASP9, CDK10, CDK8, CTNNB1, miR-141-3p, miR-

Antiproliferative I 0.062 ELK1, GNG12, GNG2, GNG3, GNG7, NOS1, miR-141-3p, miR- Role of I PIK3CA, RAP IB, RASD2, SLC39A7 450a-2-3p, miR-

Somatostatin ΐ 3976, miR-4494,

Receptor 2 ΐ miR-6877-3p, miR- 7848-3p-3p

Myc Mediated i 0.062 CASP9, FASLG, MAP2K4, MAPK10, miR-141-3p, miR-

Apoptosis i PIK3CA, RAP1B, RASD2, SHC1, YWHAG 450a-2-3p, miR-

Signaling ΐ 4494, miR-6877-3p, miR-7848-3p-3p

G Beta Gamma ΐ 0.062 CACNA2D4, GNA11, GNA12, GNAT1, miR-141-3p, miR- Signaling ΐ GNG12, GNG2, GNG3, GNG7, PLCG2, 450a-2-3p, miR- PRKACB, PRKCG, RAP1B, RASD2, SHC1 3976, miR-4494, miR-6499-5p, miR- 6877-3p, miR-7848- 3p-3p

Role of NFAT in 0.062 CACNA2D4, CSNK1A1, EP300, GNG12, miR-141-3p, miR-

Cardiac GNG2, GNG3, GNG7, IL6ST, MAP2K4, 450a-2-3p, miR-

Hypertrophy MAPK10, PIK3CA, PLCG2, PRKACB, 3976, miR-4494, PRKCG, RAP1B, RASD2, RCAN3, SHC1, miR-6499-5p, miR- Pathway FDR Target genes MicroRNAs Pathway Target genes MicroRNAs targeting this pathway

TGFB2 6877-3p, miR-7848- 3p-3p

CREB Signaling j 0.078 CACNA2D4, ELK1, EP300, GNA11, GNA12, miR-141-3p, miR- in Neurons ΐ GNAT1, GNG12, GNG2, GNG3, GNG7, 450a-2-3p, miR- PIK3CA, PLCG2, POLR2K, PRKACB, 3976, miR-4494, PRKCG, RAP1B, RASD2, SHC1 miR-6499-5p, miR- 6877-3p, miR-7848- 3p-3p

Role of NFAT in \ 0.085 CD80, CSNK1A1, FYN, GNA11, GNA12, miR-141-3p, miR- Regulation of the j GNAT1, GNG12, GNG2, GNG3, GNG7, LYN, 450a-2-3p, miR- Immune I PIK3CA, PLCG2, RAP IB, RASD2, RCAN3 3976, miR-4494,

Response I miR-6499-5p, miR- 6877-3p, miR-7848- 3p-3p fMLP Signaling i 0.085 ARPC5, ELK1, FPR3, GNG12, GNG2, miR-141-3p, miR- in Neutrophils ΐ GNG3, GNG7, PIK3CA, PRKCG, RAC1, 450a-2-3p, miR- RAP IB, RASD2 3976, miR-4494, miR-6877-3p, miR- 7848-3p-3p

HMGB1 0.085 CLCF1, ELK1, FASLG, IL17A, MAP2K4, miR-141-3p, miR-

Signaling MAP2K5, MAPK10, PIK3CA, RAC1, RAP IB, 450a-2-3p, miR- RASD2, RND3, SELE, TGFB2, TNFSF13 3976, miR-4494, miR-6499-5p, miR- 6877-3p, miR-7848- 3p-3p

Androgen Ϊ0Ό85 CACNA2D4, EP300, GNA11, GNA12, miR-141-3p, miR-

Signaling GNAT1, GNG12, GNG2, GNG3, GNG7, 450a-2-3p, miR- POLR2K, PRKACB, PRKCG, SHC1 3976, miR-4494, miR-6499-5p, miR- 7848-3p-3p

Colorectal Cancer 0.085 CASP9, CTNNB1, GNG12, GNG2, GNG3, miR-141-3p, miR-

Metastasis GNG7, IL6ST, JAK3, MAP2K4, MAPK10, 450a-2-3p, miR-

Pathway FDR Target genes MicroRNAs

[00322] ^" Ingenuity Pathway Analysis (IP A) was used to identify mRNA targets for the seven differentially expressed microRNAs (i.e., miR-450a-2-3p, miR-6877-3p, miR-4494, miR-6499-5p, miR-7848-3p-3p, miR-3976, and miR-141-3p) between the complete responder and non-responder groups on Day 0. Core pathway analysis by IPA identified pathways that were significantly enriched with these mRNA targets with an FDR <0.1.

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[00334] 12. Ritchie ME, Phipson B, Wu D, et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res 2015;43:e47.

[00335] 13. Smyth GK, Michaud J, Scott HS. Use of within-array replicate spots for assessing differential expression in microarray experiments. Bioinformatics 2005;21:2067-75.

[00336] 14. Phipson B, Lee S, Majewski IJ, Alexander WS, Smyth GK. Robust hyperparameter estimation protects against hypervariable genes and improves power to detect differential expression. Ann Appl Stat 2016;10:946-63.

[00337] 15. Benjamini Y, Hochberg Y. Controlling the false discovery rate: A practical and powerful approach to multiple testing. J R Stat Soc Series B Stat Methodol 1995;57:289-300.

EXAMPLE 3: “A three-month, single-center study to identify biomarkers/mechanism of response to Xolair® (omalizumab; 300 mg subcutaneously monthly for 3 doses) therapy in patients with chronic idiopathic urticaria: Plasma exosomal miRNAs as predictors of response/disease severity and basophil transcriptome analysis to determine mechanism of response [00338] Approximately one-third of patients with Hi-antihistamine-resistant chronic spontaneous urticaria (CSU) fails to respond to omalizumab. However, there is yet no available biomarker that accurately predicts clinical response to this biologic. The utility of blood basophil and eosinophil levels and exosomal and basophil microRNAs as biomarkers of response to omalizumab in CSU was explored. Canonical pathways, altered by differentially expressed microRNAs, were determined by Ingenuity Pathway Analysis.

[00339] Methodology: After initial screening within two weeks of study start, patients received one subcutaneous injection of omalizumab at a dose of 300 mg on Days 0, 30, and 60. Blood samples were collected at baseline (Day 0, prior to omalizumab injection) for isolation of basophils and exosomes. Patients completed a twice-daily diary that was used to determine their urticaria activity score (UAS) during a 7-day period (UAS7). Exosomes and basophils were isolated from blood for microRNA isolation and microRNA-Seq profiling. After identification of a set of differentially expressed microRNAs, Ingenuity Pathway Analysis (IP A) was employed to predict mRNA targets enabling identification of canonical pathways. The detailed study procedures are as follows:

[00340] Exosomal miRNA isolation. Exosomes were isolated from the plasma of the study participants using Invitrogen’s Total Exosome Precipitation Reagent. miRNAs were then isolated from the exosomes using the QIAGEN miRNeasy™ Serum/Plasma Kit.

[00341] Basophil isolation. Peripheral blood samples were collected in EDTA tubes. Human nucleated cells were separated from red blood cells using HetaSep reagent. Basophils were isolated from nucleated cells using the STEMCELL Technologies EasySep Human Basophil Enrichment Kit (isolation purity, mean ± SD, 94.0 ± 2.5%).

[00342] Extraction of miRNA from basophils. Total RNA from basophils was isolated using the QIAGEN miRNeasy Micro extraction kit. The integrity of the total RNA samples was evaluated using a 2100 Bioanalyzer instrument. A Thermo Fisher Scientific NanoDrop™ 1000 Spectrophotometer was used to determine purity of RNA samples by measuring OD260/280 and OD260/230 ratios. Only samples with RNA Integrity Number (RIN)s of >7 and OD260/280 and OD260/230 ratios >1.8 were used for further analysis. Total RNA concentrations were determined using Thermo Fisher Scientific Qubit RNA HS Assay Kit.

[00343] miRNA-Seq profiling. miRNA profiling was performed at the QIAGEN Service lab in Germantown, MD. miRNA samples were evaluated for quality by using the QIAGEN QIAseq miRNA. Total RNA samples were assessed for quality and quantity by using an Agilent TapeStation 4200 and a Thermo Fisher Scientific NanoDrop 2000c spectrophotometer respectively. Following cDNA cleanup, PCR library amplification was performed with a universal forward primer and indexing reverse primers. Following a final library cleanup, the quality of the miRNA libraries was assessed using an Agilent TapeStation 4200. miRNA sequencing libraries prepared with the QIAseq miRNA Library Kit were sequenced using a NextSeq 500 instrument. Libraries were quantified, adjusted to 4 nM each and pooled. The pooled libraries were further diluted down to 1.8 pM and loaded into a flow cell on an Illumina NextSeq 500. The sequencing specifications were 1x76 bases and 12 million targeted reads per sample.

[00344] Number of Patients (planned and analyzed): Twenty-seven subjects were screened to achieve the target number of twenty patients who completed the 12-week study period. Data were analyzed from these twenty study participants.

[00345] Diagnosis and Main Criteria for Inclusion and Exclusion: Study subjects were adult patients (>18 years) with chronic spontaneous urticaria (CSU) who had remained symptomatic despite the use of high dose Hi-antihistamines. Inclusion criteria were: 1) at least 6 weeks of CSU with itching despite current use of up to x4 Hi-antihistamines and 2) an urticaria activity score (UAS) during a 7-day period (UAS7) of >16 (on a scale ranging from 0 to 42, with higher scores indicating greater activity) during the 7 days before the first treatment with omalizumab. Exclusion criteria were: 1) a clearly defined underlying cause for chronic urticaria, 2) routine administration (i.e., daily or every other day for >5 consecutive days) of systemic glucocorticoids, anti-inflammatory, immunomodulatory, or immunosuppressive agents within the previous 30 days, 3) the use of any Ha- antihistamine or leukotriene-receptor antagonist within 7 days preceding the screening visit, 4) treatment with omalizumab within the previous year, or a known hypersensitivity to omalizumab, 5) a history of cancer, or 6) women who were pregnant or nursing or unable to use an effective method of contraception during dosing with omalizumab.

[00346] Test Product/Reference Therapy, Dose, and Mode of Administration: After initial screening within two weeks of study start, patients received one subcutaneous injection of omalizumab (Xolair ^® ; Genentech, South San Francisco, CA) at a dose of 300 mg on Days 0, 30, and 60.

[00347] Duration of Treatment: Twelve weeks.

[00348] Criteria for Evaluation (Endpoints)

[00349] Efficacy: To quantitate the response to omalizumab of each subject, the area-under-the-curve (AUC) for the weekly UAS7 values over the 12-week study period from a baseline value of zero (UAS7 AUC) was computed using the trapezoidal method and expressed as UAS7 x week unit. All peaks above the set baseline of zero were counted. For missing UAS7 diary scores, the last observation was carried forward. Responders were defined as those subjects with an AUC for the weekly UAS7 over the 12-week treatment period that was equal or less than the 75 ^th percentile and achieved 90% improvement of baseline UAS7. Non-Responders were defined as subjects who a) failed to achieve a 90% improvement of their baseline weekly UAS7, or b) had an AUC for the weekly UAS7 over the 12-week study period that was above the 75 ^th percentile (i.e., top quartile), or c) had an exacerbation of their symptoms necessitating the addition of an anti-inflammatory or an immunosuppressive agent. To study the miRNA expression profile as a predictor for response to omalizumab, we examined the most discordant groups of response to the anti-IgE intervention, by determining the AUC for the weekly UAS7 over the last half of the study to identify a durable therapeutic response. Responders with complete response (CR) had the lowest AUC over the last half of the study period compared to those with partial response (PR). Subjects with non-response (NR) had the highest AUC over the entire study period as well as over the last half of the study.

[00350] Safety: Patients were closely monitored for 2 hours after each dose of omalizumab with epinephrine administered if evidence of anaphylaxis per the joint task force guidelines of the American Academy of Allergy, Asthma & Immunology and the American College of Allergy, Asthma & Immunology. Patients were provided with an epinephrine injector for self-administration of epinephrine if development of anaphylaxis after discharge from the CRC. Patients continued stable doses of their pretreatment period HI -antihistamine (i.e., cetirizine, levocetirizine dihydrochloride, fexofenadine, loratadine, or desloratadine) throughout the treatment period. For the duration of the study, all patients were provided with diphenhydramine (25 mg) as rescue medication for itch relief (up to a maximum of three doses in 24 hours).

[00351] Statistical Methods: Descriptive statistics were performed to determine the mean and standard deviation (SD) of continuous variables and the number (N) and relative frequency (%) of categorical variables. For the UAS7 AUCs, the medians, 25th and the 75th percentiles were also determined. The Kolmogorov-Smimov test and histogram analyses were used to determine whether continuous variables were normally distributed. A two-tailed t-test and Analysis of Variance (ANOVA) were used for the comparisons of continuous variables between the different groups as appropriate. Welch’s correction that does not assume equal variance was applied when appropriate to account for unequal sample sizes. Fisher’s exact test was used for group comparison of categorical variables. For baseline clinical variables that were significantly different between responders and non responders, a receiver operating characteristic (ROC) curve analysis was generated to determine a cutoff value predictive of response to omalizumab therapy. A mixed effects model was used to assess for differences in the mean UAS7 over the 12-week study period based on different selected baseline attributes. The mixed effects model was opted over repeated measures ANOVA to overcome missing data points.

[00352] For analysis of the miR-Seq data, raw reads were de-multiplexed and FASTQ™ files for each sample were generated using Illumina bcl2fastq software. FASTQ™ data were checked using the FastQC™ tool. Reads and UMI counts were generated using QIAGEN GeneGlobe™ pipeline. During the QIAseq™ microRNA Fibrary Kit construction process, each individual microRNA molecule was tagged with an UMF Following sequencing and trimming, reads were analyzed for the presence of UMIs. All reads containing identical insert sequence and UMI sequence (insert-UMI pair) were collapsed into a single read. These reads were passed into the analysis pipeline. Additionally, reads containing partial UMI were also passed into the analysis pipeline to allow for true quantification of the microRNAs by eliminating library amplification bias. Cutadapt™ (version 1.11) was used to remove the adapter sequences and output adapter-free sequences. The UMI counts for each microRNA were read into R (r-proj ect.org) using the Bioconductor edgeR package and filtered to remove microRNAs that were either unexpressed or expressed at a consistent, unreliably low level. Samples were normalized using a trimmed mean of M-values and then counts were converted to log counts/million (logCPM) using the voomWithQualityWeights function from the Bioconductor limma package. This function converts the counts to logCPM and then estimates observation and sample- level weights that can be used in a conventional linear model to control for heteroscedasticity. For each subject, there were repeated measurements that were estimated using the limma duplicateCorrelation function and controlled for by using a linear mixed model when making comparisons. Comparisons between groups were made using empirical Bayes adjusted contrasts. Multiplicity was controlled at a false discovery rate (FDR; Benjamini-Hochberg correction) of 0.05, meaning that the expected maximum proportion of false positives is estimated to be 5%. Heatmaps for the differentially expressed microRNAs were generated after conversion to z-scores. To test for a different temporal outcome of UAS7 score over time, we used a linear mixed model that included dichotomized microRNA expression (high or low at baseline), a time coefficient (treatment week), and an interaction between the two and we tested for the significance of interaction (e.g., by dropping the interaction term from the model and computing a likelihood ratio test (LRT), which tested for a difference at one or more time points). A P value <0.05 was used to indicate statistical significance. All statistical analyses were performed using R (R Foundation for Statistical Computing, Vienna, Austria) and GraphPad Prism™ 8.3 (GraphPad Software, San Diego, CA).

[00353] After identification of a set of differentially expressed microRNAs, QIAGEN Ingenuity Pathway Analysis (IPA) microRNA Target Filter was employed to predict mRNA targets. The differentially expressed microRNAs were linked to their respective mRNA targets based on information in the QIAGEN Knowledge Base. Filtering was performed to prioritize only those mRNAs that were experimentally validated or had highly confident predictions of targeting based on information from TarBase, miRecords, Target Scan (human), and literature findings curated within the QIAGEN Knowledge Base.

[00354] QIAGEN IPA Expression Analyses were then performed on each mRNA dataset generated by the microRNA Target Filter Analysis. The IPA Core Analyses identified canonical pathways that were significantly enriched from the dataset. The significance of the association between the dataset and a canonical pathway was determined from a P value of overlap calculated using the right-tailed Fisher’s exact test. The Benjamini-Hochberg procedure was applied to control FDR. Significant microRNAs were added to the identified Canonical Pathways using the QIAGEN IPA Grow™ tool that uses information from databases and curated findings in the QIAGEN Knowledge Base™ to find interactions between molecules and diseases/biological functions within pathways. The QIAGEN IPA Molecule Activity Predictor (MAP) tool was used to predict the upstream and/or downstream effects of activation or inhibition of molecules in a particular pathway given one or more neighboring molecules with known activity. These predictions were based on an algorithm that incorporates the calculation of a maximum absolute z-score to assess predictions of activity of molecules, diseases, and biological functions.

Summary/Con elusions

RESPONSE TO OMALIZUMAB IN STUDY PARTICIPANTS

[00355] Twenty-seven subjects were screened (Day -14 visit) and 6 either failed screening or elected not to enter the treatment phase. Twenty-one subjects received omalizumab on Day 0 and were seen for first follow-up on Day 14; of these subjects, twenty subjects received omalizumab on Days 30 and Day 60 and completed the study on the Day 90. One subject withdrew from the study on Day 30 (i.e., did not receive omalizumab at this visit) because of worsening urticaria symptoms. Based on the response definition, there were 15 responders (6 complete responders, 9 partial responders, and five non-responders.

HIGHER BASOPHIL LEVELS ASSOCIATED WITH CLINICAL RESPONSE TO OMALIZUMAB

[00356] At study entry, the age, gender, weight, baseline UAS7, eosinophil levels in the peripheral blood, and the presence of angioedema during the week before omalizumab treatment were not statistically different between the responder and non-responder groups. However, responders had a higher baseline basophil level in the peripheral blood compared to non-responders (mean, 28.7; 95% Cl, 20.5 to 36.83 versus mean, 15.8; 95% Cl, 12.47 to 19.13; P = 0.005). Complete responders had significantly higher baseline basophil levels compared to the non-responders (mean, 33.17; 95% confidence interval [Cl], 24.84 to 41.49 versus mean, 15.8; 95% Cl 12.47 to 19.13; P = 0.006). Basophil levels in the partial responders were not significantly different than those of the non responders. Baseline basophil levels correlated significantly with response to omalizumab as measured by the AUC UAS7 over the 12-week study period (R ² = 0.33, P = 0.007). To determine a baseline basophil level that predicts response to omalizumab, we generated a receiver operating characteristic (ROC) curve (area under the ROC curve, 0.81; 95% Cl, 0.61 to 1; P = 0.044). A baseline level >21 basophils/mL was predictive of response with 80% sensitivity (95% Cl, 54.81 to 92.95) and 100% specificity (95% Cl, 56.55 to 100). When subjects were classified based on their baseline basophil level, basophil-high subjects (defined as those with a baseline basophil level >21 basophils/mL) had a significantly better response to omalizumab with lower mean UAS7 over time compared to basophil-low (<21 basophils/mL at baseline) subjects (predicted mean difference, 11.16; ± standard error of the mean, 2.37; P <0.001).

EXOSOMAL MICRORNA PROFILE AND RESPONSE TO OMALIZUMAB [00357] Seven differentially expressed exosomal microRNAs were discovered at baseline that were associated with a complete response to omalizumab (FDR <0.05). Complete responders had higher expression of six of these microRNAs: miR-6499-5p (Log fold change [logFC], 5.05; 95% Cl, 1.83 to 8.27; FDR = 0.002), miR-7848-3p (logFC, 4.73; 95% Cl, 1.50 to 7.97; FDR = 0.003), miR- 4494 (logFC, 4.79; 95% Cl, 1.03 to 8.57; FDR = 0.014), miR-450a-2-3p (logFC, 4.68; 95% Cl, 0.95 to 8.84; FDR = 0.014), miR-6877-3p (logFC, 3.56; 95% Cl, 0.59, to 6.53; FDR = 0.021), and miR- 3976 (logFC, 4.36; 95% Cl, 0.52 to 8.20; FDR = 0.037). Complete responders had lower expression of miR-141-3p (logFC, -4.49; 95% Cl, -8.53 to -0.46; FDR = 0.04). To further explore the utility of these microRNAs in projecting response to omalizumab, each participant was characterized as having either high or low expression (defined as higher or lower than the geometric mean expression level for each microRNA). The inventors found a differential clinical response as quantified by the UAS7 score over the 12-week study period. Higher expression of miR-6499-5p (LRT = 75.2, P <0.001), miR- 7848-3p-3p (LRT = 75.2, P <0.001), miR-4494 (LRT = 75.2, P <0.001), miR-450a-2-3p (LRT = 75.2, P <0.001), miR-6877-3p (LRT = 42.7, P <0.001), and miR-3976 (LRT = 43.2, P <0.001) was associated with favorable response to omalizumab. In contrast, there was a trend (LRT = 21.4, P = 0.07) for higher expression of miR-141-3p to be associated with poor response to omalizumab. These data indicate that an exosomal microRNA expression profile can predict response to omalizumab therapy in patients with CSU.

BASOPHIL MICRORNA PROFILE AND RESPONSE TO OMALIZUMAB [00358] Three differentially expressed basophil microRNAs at baseline were found to be associated with a complete response to omalizumab (FDR <0.05). Complete responders had higher expression of miR-1200 (logFC, 4.68; 95% Cl, 0.90 to 8.37; FDR = 0.024), miR-1236-3p (logFC, 4.63; 95% Cl, 0.89 to 8.37; FDR = 0.024), and miR-4664-3p (logFC, 4.34; 95% Cl, 0.65 to 8.03; FDR = 0.033). The inventors observed a positive clinical response, as quantified by the mean UAS7 over the 12-week study period, among participants with high expression (above the geometric mean) of these microRNAs compared to subjects with low expression: miR-1200 (LRT = 75.2, P <0.001), miR-1236-3p (LTR = 34.7, P = 0.001), and miR-4664-3p (LRT = 34.7, P = 0.001).

TEC KINASE SIGNALING PATHWAY MODULATION ASSOCIATED WITH RESPONSE TO OMALIZUMAB

[00359] Using the QIAGEN® IPA microRNA Target Filter feature, the inventors identified 1019 mRNA targets for the seven differentially expressed exosomal microRNAs between the complete responders and non-responders on Day 0. The differentially expressed basophil microRNAs at baseline were predicted to target the expression of 451 mRNAs. Next, canonical pathways were examined that were significantly enriched with these mRNA targets and their predicted activity was analyzed using the IPA Molecule Activity Predictor feature. The seven differentially expressed exosomal microRNAs significantly modulated the activity of the Tec Kinase Signaling pathway (FDR = 0.003). This pathway is involved in transducing signals from FcgRI and other key immune receptors. This set of seven exosomal microRNAs modulated the expression of signaling molecules both upstream and downstream of the Tec kinases including serine/threonine p21 -activating kinase (PAK), protein kinase C (PKC), Wiskott-Aldrich Syndrome (WAS) protein, Rho GTPases, phosphatidylinositol-3 -kinase (PI3K) complex, Src-family tyrosine kinases, and Janus kinase (JAK)l and JAK2). IPA analysis predicted downregulation of varied cellular functions including cell adhesion, cell migration, actin reorganization, and calcium mobilization. In contrast, the effect on gene expression and cell transformation downstream of the mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription protein (STAT) transcription factors were predicted to be upregulated.

[00360] Using the same differentially expressed exosomal microRNAs, no other predicted biological pathways met our pre-specified significance cutoff of FDR <0.05. However, their respective mRNA targets were less enriched (FDR 0.05-0.1) in other canonical pathways of potential relevance to CSU pathogenesis and response to omalizumab including IL-1 Signaling, IL-17 Signaling, Thl 7 Activation, Oncostatin M Signaling, CXCR4 Signaling, Role of NFAT in Regulation of the Immune Response, Glucocorticoid Receptor Signaling, and FcgRIIB Signaling in B Lymphocytes pathways. In contrast, mRNA targets identified by differentially expressed basophil microRNAs between complete responders and non-responders were not significantly enriched in any canonical pathways.

[00361] Safety Results: One participant had an anaphylactic reaction two hours after omalizumab injection on Day 60 visit. Symptoms completely resolved with epinephrine and methylprednisolone treatment.

[00362] Overall Conclusions: This study establishes a clinical and molecular phenotype in CSU characterized by high basophil levels (>21 cells/pL). unique expression profiles of exosomal and basophil microRNAs, and altered Tec kinase-dependent canonical pathways in complete responders to omalizumab.

[00363] Because a significant proportion of patients with CSU fails to respond to omalizumab, several candidate biomarkers were examined to identify patients who would be helped from the use of this biologic therapy. In the prospective study, the inventors did not find a difference in baseline eosinophil levels between the complete responder and non-responder groups; whereas, basophil levels were significantly different. A basophil level of less than 21 cell s/pL was predictive of non-response to omalizumab with 80% sensitivity and 100% specificity.

[00364] Commercial microRNA panels have been developed to identify the origin of cancers or classify malignant thyroid cancers when cytology is uncertain. The exosomal and basophil microRNA expression profiles identified in our study could possibly serve as a diagnostic panel to personalize treatment for CSU patients by selecting patients who would profit from omalizumab intervention and to potentially reduce treatment cost. In addition to serving as biomarkers, these differentially expressed microRNAs enabled the identification of a molecular phenotype associated with response to omalizumab and potential new therapeutic targets within the Tec Kinase Signaling pathway.

[00365] The Tec kinases, non-receptor protein tyrosine kinases, consist of five members: IL-2 inducible T-cell kinase (Itk), Bruton tyrosine kinase (Btk), resting lymphocyte kinase (Rlk), tyrosine kinase expressed in hepatocellular carcinoma (tec), and bone marrow kinase expressed on the X chromosome (Bmx). The Tec kinases are involved in many critical molecular pathways in both myeloid and lymphoid cells. They are downstream of FceRI, T-cell receptor complex, receptor tyrosine kinase, cytokine receptors, Fas ligand (FasL) receptor, and bl integrin promoting diverse biological functions. Mast cells express Tec, Btx, and Itk. Stem cell factor (SCF) promotes development and activation of mast cells by signaling through c-kit, a receptor tyrosine kinase. Upon stimulation with SCF, c-kit activates Tec kinase. Cross-linking of FceRI in mast cells activates Btk promoting mast cell degranulation and mediator release. Also activated by FceRI stimulation are Itk and Tec that regulate mast cell production of cytokines. The Tec kinases are important in basophils. IL-3, a cytokine promoting basophil development and activation, signals through JAK2 to activate Tec kinase. While FasL promotes apoptosis of basophils, IL-3 protects basophils against FasL- mediated apoptosis. Both mast cells and basophils express bl integrins that mediate adhesion of skin mast cells to extracellular matrix proteins and basophils to the endothelium. Because interruption of FceRI underlies the mechanism for the therapeutic effect of omalizumab in CSU, the findings that the differentially expressed exosomal microRNAs target the Tec kinase-dependent FceRI signaling pathway support this mechanism and indicate that this predicted altered molecular profile can facilitate response to omalizumab.

[00366] This study was limited by the small number of patients, especially non-responders. Larger studies can validate our identified basophil level (>21 cells/pL) as a response biomarker and to bring to clinicians a microR A diagnostic panel as a predictive tool for omalizumab response. The mRNA targets for the differentially expressed microRNAs were based on current state of knowledge and not all impacted canonical pathways may have been identified at this point in time. As studies of microRNAs are rapidly evolving, and as more experimental data become available, pathways altered by the three basophil microRNAs that were differentially expressed between the responders and non responders may be uncovered in the future and provide further insight into the role of basophils in the immunopathogenesis of CSU.

EXAMPLE 4: Extracellular vesicle microRNAs as predictors of response to omalizumab in chronic spontaneous urticaria

[00367] Despite the marked efficacy for omalizumab in patients with Hi-antihistamine-resistant chronic spontaneous urticaria (CSU), some patients fail to respond, and there is no available biomarker to predict response outcome. ¹ Extracellular vesicle (EV)-associated microRNAs are novel response biomarkers. ^2,3 In this IRB-approved observational study (FIG. 10), CSU patients received 300-mg omalizumab on Days 0 (baseline), 30, and 60 and their clinical response was assessed using weekly urticaria activity score (UAS7) for a 12-week period. ⁴ Blood samples for eosinophil and basophil count and EV microRNA extraction were collected at baseline (before omalizumab administration). Blood was also collected at Day 90 for EV microRNA analysis. Participants were classified as complete responders (CR), partial responders (PR), or non-responders (NR) by their Area-Under-the Curve (AUC) for UAS7 scores (Table 5, FIG. 10). Detailed methods including statistics are in online supporting information. Study outcomes were identification of 1) EV microRNA(s) that can serve as biomarkers predicting omalizumab response in CSU patients, and 2) canonical pathway(s) altered by such microRNA signature.

[00368] Nineteen subjects completed UAS7 scores over the 12-week study period for inclusion in analyses. CR (n=6) had the lowest AUC over weeks 7-12 of the study compared to PR (n=9), whereas NR (n=4) had the highest AUC (FIG. 10). Baseline clinical characteristics were not different between responders and NR (Table 6). Responders had significantly higher blood basophil levels versus NR. CR, but not PR, had significantly higher basophil levels versus NR (FIG. 11). Basophil levels correlated significantly with omalizumab response measured by AUC UAS7 while eosinophils levels did not (P = 0.58) (FIG. 11).

[00369] To identify an EV microRNA signature associated with clinical response to omalizumab, the CR and NR groups were compared with the most discordant response to omalizumab. The inventors found significant differences in the levels of seven microRNAs at baseline (FDR <0.05). CR had higher levels of miR-6499-5p, miR-7848-3p-3p, miR-4494, miR-450a-2-3p, miR-6877-3p, and miR-3976 but lower levels of miR-141-3p (FIG. 12). When participants were characterized as having either high or low expression (higher or lower than the geometric mean expression level for each microRNA), the inventors found a differential clinical response by UAS7 score over the study period (FIG. 12). The EV microRNA signature changed with omalizumab therapy (Table 7). Fevels of miR- 3976 decreased significantly by Day 90 among CR. In the NR, miR-6499-5p, miR-7848-3p-3p, miR- 6877-3p and miR-450a-2-3p levels increased by Day 90.

[00370] Using the baseline EV microRNA signature, QIAGEN ^® Ingenuity Pathway Analysis (IP A) identified 1019 mRNA targets that were enriched within the Tec Kinase Signaling pathway (FDR = 0.003). This pathway transduces signals from FcsRI and other key immune receptors (FIG. 13). The IPA Molecular Activity Predictor feature uncovered that this set of microRNAs modulated expression of signaling molecules upstream and downstream of the Tec kinases. IPA predicted downregulation of cell adhesion, cell migration, actin reorganization, and calcium mobilization. The effect on gene expression and cell transformation downstream of the mitogen-activated protein kinase and signal transducer and activator of transcription protein transcription factors was predicted to be upregulated.

[00371] In omalizumab-treated CSU patients, CR compared to NR demonstrated 1) higher basophil levels in peripheral blood as recently reported ⁵ , 2) unique EV microRNA signature that can predict response to omalizumab and personalize care for these patients, and 3) altered activity of the Tec Kinase Signaling pathway, known to regulate mast cell activation, and is downstream of FceRI indicating that this predicted altered molecular profde may facilitate response to omalizumab. ⁶ References

1. Center DM. A better IgE trap to control urticaria. N Engl J Med. 2019;381(14): 1376-1377.

2. Shah R, Patel T, Freedman JE. Circulating extracellular vesicles in human disease. N Engl J Med. 2018;379(22):2180-2181.

3. Bonneau E, Neveu B, Kostantin E, Tsongalis GJ, De Guire V. How close are miRNAs from clinical practice? A perspective on the diagnostic and therapeutic market. EJIFCC. 2019;30(2):114- 127.

4. Mathias SD, Crosby RD, Zazzali JL, Maurer M, Saini SS. Evaluating the minimally important difference of the urticaria activity score and other measures of disease activity in patients with chronic idiopathic urticaria. Ann Allergy Asthma Immunol. 2012;108(l):20-24.

5. Rijavec M, Kosnik M, Koren A, et al. A very low number of circulating basophils is predictive of a poor response to omalizumab in chronic spontaneous urticaria. Allergy. 2020.

6. Gilfillan AM, Rivera J. The tyrosine kinase network regulating mast cell activation. Immunol Rev. 2009;228(1): 149-169.

Supporting information

[00372] Study participants and plan. Prior to inclusion in the study, all participants provided written informed consent. Inclusion criteria were: 1) adult patient (>18 years) with at least 6 weeks of CSU who remained symptomatic despite the use of up to x4 Hi-antihistamines and 2) an urticaria activity score (UAS) during a 7-day period (UAS7) of >16 (on a scale ranging from 0 to 42, with higher scores indicating greater activity, based on patient diary entries completed twice daily) during the 7 days before the first treatment with omalizumab. Exclusion criteria were: 1) a clearly defined underlying cause for chronic urticaria, 2) routine administration (i.e., daily or every other day for >5 consecutive days) of systemic glucocorticoids, anti-inflammatory, immunomodulatory, or immunosuppressive agents within the previous 30 days, 3) the use of any H2-antihistamine or leukotriene-receptor antagonist within 7 days preceding the screening visit, 4) treatment with omalizumab within the previous year, or a known hypersensitivity to omalizumab, 5) a history of cancer, or 6) women who were pregnant or nursing or unable to use an effective method of contraception during dosing with omalizumab. [00373] The study plan is shown in FIG. 10. Because the study aim was to identify two groups with the most discordant response to omalizumab, response was assessed only in subjects who completed UAS7 diary scores over the entire 12-week study period. To quantitate the response to omalizumab of each subject, the area under the curve (AUC) for the weekly UAS7 values over the 12- week study period from a baseline value of zero (UAS7 AUC) was computed using the trapezoidal method and expressed as UAS7 x week unit (Table 5). All peaks above a set score of zero were counted. For the UAS7 AUCs, the medians, 25 ^th and the 75 ^th percentiles were also determined. Because the study aim was to identify two groups with the most discordant response to omalizumab, response was assessed only in subjects who completed UAS7 diary scores over the entire 12-week study period. Responders were defined as those subjects with an AUC for the weekly UAS7 over the 12-week treatment period that was equal to or less than the 75 ^th percentile and had at least a 90% improvement of baseline UAS7 at one of their weekly UAS7 diary scores. Non-response (NR) was defined as subjects who a) failed to achieve a 90% improvement of their baseline weekly UAS7, or b) had an AUC for the weekly UAS7 over the 12-week study period that was above the 75 ^th percentile (i.e., top quartile). Responders were further characterized into two groups: those with complete response (CR) and those with partial response (PR) by determining the AUC for the weekly UAS7 over the last half of the study to identify individuals with a durable therapeutic response (Table 5, FIG. 10). Baseline characteristics including blood eosinophil and basophil levels of the study participants are shown in Table 6 and FIG. 11.

TABLE 5. Clinical response classification by area under the curve (AUC) of UAS7 scores ^† Participant j 90% Improvement AUC 0-12 wk AUC 7-12 wk Response Subject 1 I Yes 21 0 CR

Subject 8 I Yes 23.5 0 CR

Subject 17 I Yes 57.5 0 CR

Subject 25 | Yes 102 0 CR

Subject 10 Yes 60 3 CR

Subject 12 I Yes 43.5 3.5 CR

Subject 22 j Yes 115 11 PR

Subject 7 I Yes 82.5 15.5 PR

Subject 4 j Yes 81.5 17.5 PR

Subject 15 i Yes 88.5 18 PR

Subject 21 I Yes 162 28 PR

Subject 5 I Yes 127.5 30.5 PR

Subject 27 | Yes 171 31.5 PR Subject 19 Yes 148 38.5 PR

Subject 16 I Yes 137.5 43 PR , area under the curve for the UAS7 over the 12-week of the study; AUC 7-12 wk, area under the curve for the UAS7 over weeks 7-12 of the study period; CR, complete responder; PR, partial responder; NR, non responder.

[00375] TABLE 6. Baseline characteristics of study participants ^†

Variable I All Subjects Responders Non-Responders P value

Subject (n) j 19 15 4

Age (yrs) ! 48 ± 13.8 49.4 ± 10.3 42.7 ± 24.6 0.63

Gender - Fe j 12 (63%) 8 (53.3%) 4 (100%) 0.25

Weight (kg) ! 90.8 ± 26.7 94.5 ± 27.7 77 ± 19.1 0.18

Baseline UAS7 § 34 ± 5.3 33.3 ± 5.1 36.7 ± 5.6 0.32

Angioedema (+) j 9 (47%) 6 (40%) 3 (75%) 0.30

Eosinophils/pL j 136.3 ± 81.4 140.6 ± 87.3 120 ± 60.7 0.60

Basophils/ pL j 26.1 ± 14 28.7 ± 14.8 16.5 ± 2.5 0.008

^† Values are presented as n (%) for categorical variables and mean ± SD for continuous variables.

Statistical differences between responders and non-responders were analyzed using Fisher exact test for categorical variables or unpaired two-tailed Welch’s t-test for continuous variables.

TABLE 7. Change in the levels of EV microRNAs by Day 90 ^†

MicroRNA I Complete Responders Non-Responders miR-6499-5p j -2.369 0.31 4.496 0.008 miR-7848-3p- i -0.721 0.88 4.606 0.001

3p ! miR-4494 | -1.272 0.84 1.407 0.76 miR-450a-2-3p j -2.476 0.32 4.506 0.016 miR-6877-3p j 0.411 0.89 3.561 0.005 miR-141-3p j -1.249 0.84 -1.728 0.63

[00376] ^† FC, fold change; FDR, false discovery rate. [00377] MicroRNA isolation and microRNA seq profiling. Extracellular vesicles (EV) were isolated from the plasma of the study participants using Invitrogen’s Total Exosome Precipitation Reagent (Thermo Fisher Scientific, Inc., Waltham, MA). MicroRNAs were isolated from the EV using the QIAGEN® miRNeasy Serum/Plasma Kit (QIAGEN Sciences, Germantown, MD) and quality assessed by the QIAGEN® QIAseq microRNA. MicroRNA seq profiling was performed at QIAGEN Sciences (Germantown, MD). Raw reads were de-multiplexed and FASTQ files for each sample were generated using Illumina bcl2fastq software. Reads and Unique Molecular Index (UMI) counts were generated using QIAGEN GeneGlobe pipeline. The UMI counts for each microRNA were read into R (r-proj ect.org) using Bioconductor edgeR package and filtered to remove microRNAs that were either unexpressed or expressed at a consistent, unreliably low level. Samples were normalized using a trimmed mean of M-values and counts converted to log counts/million (logCPM) using the voomWithQualityWeights function from the Bioconductor limma package to estimate observation and sample-level weights for use in a conventional linear model and control for heteroscedasticity. For each subject, there were repeated measurements estimated using the limma duplicateCorrelation function and controlled for by a linear mixed model when making comparisons. [00378] Statistics. Assuming a standard deviation of 0.65, which was derived from previously published experimental data ^1,2 and assuming biologically meaningful changes in microRNA expression at levels of absolute fold change greater than 1.5, a sample size of 10 was chosen for □ < 0.05 and b < 0.2 for absolute fold change > 1.5. Descriptive statistics were performed to determine the mean and standard deviation (SD) of continuous variables and the number (n) and relative frequency (%) of categorical variables. The Kolmogorov- Smirnov test and histogram analyses were used to determine whether continuous variables were normally distributed. A two-tailed t-test and Analysis of Variance (ANOVA) with Dunnett T3 correction for multiple comparisons were used for the comparisons of continuous variables between the different groups as appropriate. Welch’s correction was applied when appropriate to account for unequal sample sizes. Fisher’s exact test was used for group comparison of categorical variables. For miR-Seq data statistical analyses, comparisons between groups were made using empirical Bayes adjusted contrasts. Multiplicity was controlled at a false discovery rate (FDR; Benjamini-Hochberg correction) of 0.05 (i.e., the expected maximum proportion of false positives is estimated to be 5%). Heatmaps for differentially expressed microRNAs were generated after conversion to z-scores. To test for a different temporal outcome of UAS7 score over time, we used a linear mixed model that included dichotomized microRNA expression (high or low at baseline), a time coefficient (treatment week), and an interaction between the two; we tested for the significance of interaction by dropping the interaction term from the model and computing a likelihood ratio test (LRT), which tested for a difference at one or more time points. Analyses were performed using R (R Foundation for Statistical Computing, Vienna, Austria) and GraphPad Prism 8.3 (GraphPad Software, San Diego, CA). [00379] Ingenuity pathway analysis (IPA). QIAGEN ^® IPA microRNA Target Filter was employed using high stringency filters to predict the corresponding mRNA targets of differentially expressed exosomal microRNAs. Only mRNA targets that were experimentally validated or had high confidence predictions were selected using the TarBase, miRecords, and Target Scan (human) databases and literature findings curated within the QIAGEN ^® Knowledge Base. Predicted mRNA targets were analyzed with the IPA core analysis feature to identify significantly enriched canonical pathways. Significance of canonical pathways was determined using a right-tailed Fisher’s exact test. Benjamini-Hochberg procedure was applied to control False Discovery Rate (FDR). IPA Grow tool visualized the relationship between individual microRNAs and their predicted canonical pathway targets. QIAGEN ^® IPA Molecule Activity Predictor tool predicted the upstream and/or downstream effects of activation or inhibition of molecules in a pathway given one or more neighboring molecules with known activity. Predictions were based on an algorithm that incorporates calculation of a maximum absolute z-score to predict activity of molecules, diseases, and biological functions. References

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[00381] 2. Ach RA, Wang H, Curry B. Measuring microRNAs: comparisons of microarray and quantitative PCR measurements, and of different total RNA prep methods. BMC Biotechnol. 2008;8:69.