Biomarkers for Chronic Fatigue Syndrome and Long COVID and Their Use

Abstract

The present disclosure provides methods and kits using certain biomarkers in the prediction and monitoring of post-viral syndromes (e.g., chronic fatigue syndrome (CFS) and / or long COVID), the selective treatment of such syndromes, and the evaluation of clinical sensitivity and treatment responsiveness to treatment. Determining the expression level of a biomarker in a sample from a subject is disclosed.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 346,698, filed May 27, 2022, the content of which is hereby incorporated by reference in its entirety.

[0002] 1. Field The present disclosure provides methods and kits using certain biomarkers in the prediction and monitoring of post - viral syndromes (e.g., chronic fatigue syndrome (CFS) and / or long COVID), the selective treatment of such syndromes, and the evaluation of clinical sensitivity and treatment responsiveness to treatment.

Background Art

[0003] 2. Background Post - viral syndromes, which may include chronic fatigue syndrome (CFS) and long COVID, are complex medical conditions that can be associated with physical, cognitive, emotional, and neurological difficulties. These conditions can lead to symptoms including fatigue, post - exertional malaise, cognitive impairment, sensorimotor symptoms, headache, memory impairment, insomnia, myalgia, palpitations, shortness of breath, dizziness and balance disorders, speech disorders, joint pain, and chest pressure. These symptoms can persist for weeks, months, or longer after the clearance of the viral infection. In some cases, affected patients may become withdrawn and / or bedridden.

[0004] CFS, also known as myalgic encephalomyelitis (ME), is a debilitating medical condition that can cause severe impairment of physical, psychological, cognitive, social, and occupational functioning. The cause of CFS remains controversial and complex, but CFS can develop after a viral infection. Currently, there is no clinical diagnostic test for CFS. Although several treatments are used off - label to manage the symptoms of CFS, existing treatments specific to CFS are limited, if any.

[0005] Some of those infected with the virus that causes COVID-19 may experience the effects of their infection over a long period of time, which is known as long COVID. Long COVID is also known as post-COVID conditions, long-haul COVID, post-acute COVID-19, long-term effects of COVID, and chronic COVID. Those suffering from long COVID may have a wide range of symptoms that can persist for weeks, months, or even years after infection. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0006] In the art, there is still a need for methods for identifying, characterizing, and treating chronic fatigue syndrome (CFS), long COVID, and post-viral fatigue syndrome. MEANS FOR SOLVING THE PROBLEMS

[0007] 3. SUMMARY OF THE INVENTION In one aspect, provided herein is a method of identifying a subject having chronic fatigue syndrome (CFS) or confirming CFS in a subject, the method comprising: (a) determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a cereblon (CRBN)-associated protein (CAP) selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2, and CD3D; and (b) identifying or confirming that the subject has CFS if the expression level of the biomarker is higher than a reference expression level of the biomarker.

[0008] In some embodiments, the subject complains of chronic debilitating fatigue, non-restorative sleep, mental and / or physical pain, neurological and cognitive impairments, and / or autoimmunity or immunodeficiency.

[0009] In another aspect, provided herein is a method for determining the severity of CFS in a subject, comprising: (a) determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a cereblon (CRBN)-associated protein (CAP) selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2, and CD3D; (b) comparing the expression level of the biomarker to a reference expression level of the biomarker; and (c) determining the severity of the subject's CFS based on the comparison of step (b).

[0010] In some embodiments, the severity of CFS is determined to be severe if the expression level of the biomarker is higher than the reference expression level of the biomarker.

[0011] In some embodiments, the reference expression level of the biomarker is the expression level of the biomarker in a subject having mild CFS or a cohort of subjects having mild CFS.

[0012] In another aspect, provided herein is a method for identifying a subject who is likely or unlikely to respond to treatment of CFS, or for predicting whether a subject will respond to treatment of CFS, comprising: (a) determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a cereblon (CRBN)-associated protein (CAP) selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2, and CD3D; and (b) identifying or predicting that the subject is likely to respond to treatment if the expression level of the biomarker is higher than the reference expression level of the biomarker.

[0013] In some embodiments, the method further comprises administering treatment to a subject identified or predicted to be likely to respond to treatment.

[0014] In another aspect, provided herein is a method for selectively treating a subject having or suspected of having CFS, the method comprising: (a) determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a cereblon (CRBN)-associated protein (CAP) selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2, and CD3D; (b) identifying or predicting that the subject is likely to respond to treatment for CFS if the expression level of the biomarker is higher than a reference expression level of the biomarker; and (c) administering treatment to a subject identified or predicted to be likely to respond to treatment.

[0015] In some embodiments, the reference expression level of the biomarker is a predetermined expression level of the biomarker. In some embodiments, the reference expression level of the biomarker is the expression level of the biomarker in a subject without CFS or a cohort of subjects without CFS. In some embodiments, the reference expression level of the biomarker is the expression level of the biomarker in a healthy subject or a cohort of healthy subjects. In some embodiments, the reference expression level of the biomarker is the expression level of the biomarker in a subject with mild CFS or a cohort of subjects with mild CFS.

[0016] In some embodiments, the biomarker is HSPA8 or ABCE1, and optionally, the reference expression level of the biomarker is the expression level of the biomarker in a healthy subject or a subject without CFS, or a cohort of healthy subjects or subjects without CFS.

[0017] In some embodiments, the method comprises determining the expression levels of 2, 3, 4, 5, or all of the biomarkers selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2, and CD3D. In some embodiments, the method comprises determining the expression levels of: (i) IKZF2 and at least 1, 2, 3, or 4 of IKZF3, ABCE1, BACH2, CD3D, and HSPA8; (ii) IKZF3 and at least 1, 2, 3, or 4 of IKZF2, ABCE1, BACH2, CD3D, and HSPA8; (iii) ABCE1 and at least 1, 2, 3, or 4 of IKZF2, IKZF3, BACH2, CD3D, and HSPA8; (iv) BACH2 and at least 1, 2, 3, or 4 of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8; (v) CD3D and at least 1, 2, 3, or 4 of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8; or (vi) HSPA8 and at least 1, 2, 3, or 4 of IKZF2, IKZF3, ABCE1, BACH2, and CD3D. In some embodiments, the method comprises determining the expression levels of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8.

[0018] In some embodiments, the method comprises comparing the expression level of each biomarker to its respective reference expression level. In some embodiments, the method comprises obtaining an overall score based on the expression levels of the biomarkers and comparing the overall score to a reference score determined from the reference expression levels of the biomarkers.

[0019] In another aspect, provided herein is a method for determining or monitoring the effectiveness of treatment in a subject having CFS, the method comprising: (a) determining a first expression level of a biomarker in a first sample obtained from the subject before administering treatment to the subject, wherein the biomarker is a cereblon (CRBN)-associated protein (CAP) selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2, and CD3D; (b) administering treatment to the subject; (c) determining a second expression level of the biomarker in a second sample obtained from the subject after administering treatment to the subject; and (d) determining the effectiveness of the treatment based on a comparison of the first expression level and the second expression level.

[0020] In some embodiments, the method comprises determining that the treatment is effective if the second expression level is lower than the first expression level. In some embodiments, the method further comprises determining or adjusting the dosage of treatment for the subject.

[0021] In another aspect, provided herein is a method for screening the effectiveness of a treatment in treating CFS, the method comprising: (a) determining a first expression level of a biomarker in a sample before administering treatment to the sample, wherein the biomarker is a cereblon (CRBN)-associated protein (CAP) selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2, and CD3D; (b) administering treatment to the sample; (c) determining a second expression level of the biomarker in the sample after administering treatment to the sample; (d) comparing the first expression level with the second expression level; and (e) selecting the treatment if the second expression level is lower than the first expression level.

[0022] In some embodiments, the biomarker is HSPA8 or ABCE1.

[0023] In some embodiments, the method comprises determining first and second expression levels of two, three, four, five, or all of the biomarkers selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2, and CD3D. In some embodiments, the method comprises determining first and second expression levels for: (i) IKZF2 and at least one, two, three, or four of IKZF3, ABCE1, BACH2, CD3D, and HSPA8; (ii) IKZF3 and at least one, two, three, or four of IKZF2, ABCE1, BACH2, CD3D, and HSPA8; (iii) ABCE1 and at least one, two, three, or four of IKZF2, IKZF3, BACH2, CD3D, and HSPA8; (iv) BACH2 and at least one, two, three, or four of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8; (v) CD3D and at least one, two, three, or four of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8; or (vi) HSPA8 and at least one, two, three, or four of IKZF2, IKZF3, ABCE1, BACH2, and CD3D. In some embodiments, the method comprises determining first and second expression levels of all of the biomarkers in the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2, and CD3D.

[0024] In some embodiments, the method comprises comparing the first expression level of each biomarker with its respective second expression level. In some embodiments, the method comprises obtaining a first composite score based on the first expression level of the biomarker and a second composite score based on the second expression level of the biomarker, and comparing the first composite score with the second composite score.

[0025] In some embodiments, the treatment includes an immunomodulatory drug (IMiD). In some embodiments, the treatment includes a cereblon (CRBN) modulator or a compound having the ability to bind to CRBN and / or induce a conformational change thereof. In some embodiments, the treatment includes an agent that depletes B cells (e.g., an anti-CD20 antibody, e.g., rituximab).

[0026] In some embodiments, CFS is associated with an autoimmune disease or a viral infection.

[0027] In another aspect, provided herein is a method of identifying a subject having long COVID or confirming long COVID in a subject, the method comprising: (a) determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a cereblon (CRBN)-associated protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; and (b) identifying or confirming that the subject has long COVID if the expression level of the biomarker is higher than the reference expression level of the biomarker.

[0028] In another aspect, provided herein is a method of identifying a subject who is likely or unlikely to respond to treatment of long COVID or predicting whether a subject will respond to treatment of long COVID, the method comprising: (a) determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a cereblon (CRBN)-associated protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; and (b) identifying or predicting that the subject is likely to respond to treatment if the expression level of the biomarker is higher than the reference expression level of the biomarker.

[0029] In some embodiments, the method further comprises administering treatment to a subject identified or predicted to be likely to respond to treatment.

[0030] In another aspect, provided herein is a method for selectively treating a subject having or suspected of having long COVID, the method comprising: (a) determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a cereblon (CRBN)-associated protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; (b) identifying or predicting that the subject is likely to respond to treatment for long COVID if the expression level of the biomarker is higher than a reference expression level of the biomarker; and (c) administering treatment to the subject identified or predicted to be likely to respond to treatment.

[0031] In some embodiments, the reference expression level of the biomarker is a predetermined expression level of the biomarker. In some embodiments, the reference expression level of the biomarker is the expression level of the biomarker in a cohort of subjects without long COVID or subjects suspected of not having long COVID. In some embodiments, the reference expression level of the biomarker is the expression level of the biomarker in a cohort of healthy subjects or subjects suspected of being healthy. In some embodiments, the reference expression level of the biomarker is the expression level of the biomarker in a cohort of subjects with acute COVID or subjects suspected of having acute COVID.

[0032] In some embodiments, the biomarker is HSPA8 or IKZF3, and optionally the reference expression level of the biomarker is the expression level of the biomarker in a cohort of healthy subjects or subjects without long COVID, or healthy subjects or subjects suspected of not having long COVID.

[0033] In some embodiments, the method comprises determining the expression levels of 2, 3, 4, 5, or all of the biomarkers selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D. In some embodiments, the method comprises determining the expression levels of: (i) IKZF2 and at least 1, 2, 3, or 4 of IKZF3, ABCE1, BACH2, CD3D, and HSPA8; (ii) IKZF3 and at least 1, 2, 3, or 4 of IKZF2, ABCE1, BACH2, CD3D, and HSPA8; (iii) ABCE1 and at least 1, 2, 3, or 4 of IKZF2, IKZF3, BACH2, CD3D, and HSPA8; (iv) BACH2 and at least 1, 2, 3, or 4 of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8; (v) CD3D and at least 1, 2, 3, or 4 of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8; or (vi) HSPA8 and at least 1, 2, 3, or 4 of IKZF2, IKZF3, ABCE1, BACH2, and CD3D. In some embodiments, the method comprises determining the expression levels of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D.

[0034] In some embodiments, the method comprises comparing the expression level of each biomarker to its respective reference expression level. In some embodiments, the method comprises obtaining an overall score based on the expression levels of the biomarkers and comparing the overall score to a reference score derived from the reference expression levels of the biomarkers.

[0035] In another aspect, provided herein is a method for determining or monitoring the effectiveness of a treatment in a subject having long COVID, the method comprising: (a) determining a first expression level of a biomarker in a first sample obtained from the subject before administering the treatment to the subject, wherein the biomarker is a cereblon (CRBN)-associated protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; (b) administering the treatment to the subject; (c) determining a second expression level of the biomarker in a second sample obtained from the subject after administering the treatment to the subject; and (d) determining the effectiveness of the treatment based on a comparison of the first expression level and the second expression level.

[0036] In some embodiments, the method comprises determining that the treatment is effective if the second expression level is lower than the first expression level. In some embodiments, the method further comprises determining or adjusting a dosage of the treatment for the subject.

[0037] In another aspect, provided herein is a method for screening the effectiveness of a treatment for treating long COVID, the method comprising: (a) determining a first expression level of a biomarker in a sample before administering the treatment to the sample, wherein the biomarker is a cereblon (CRBN)-associated protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; (b) administering the treatment to the sample; (c) determining a second expression level of the biomarker in the sample after administering the treatment to the sample; (d) comparing the first expression level to the second expression level; and (e) selecting the treatment if the second expression level is lower than the first expression level.

[0038] In some embodiments, the biomarker is HSPA8 or IKZF3.

[0039] In some embodiments, the method includes determining first and second expression levels of two, three, four, five, or all of the biomarkers selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D. In some embodiments, the method includes (i) for IKZF2 and at least one, two, three, or four of IKZF3, ABCE1, BACH2, CD3D, and HSPA8; (ii) for IKZF3 and at least one, two, three, or four of IKZF2, ABCE1, BACH2, CD3D, and HSPA8; (iii) for ABCE1 and at least one, two, three, or four of IKZF2, IKZF3, BACH2, CD3D, and HSPA8; (iv) for BACH2 and at least one, two, three, or four of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8; (v) for CD3D and at least one, two, three, or four of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8; or (vi) for HSPA8 and at least one, two, three, or four of IKZF2, IKZF3, ABCE1, BACH2, and CD3D, determining first and second expression levels. In some embodiments, the method includes determining first and second expression levels of all of the biomarkers in the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D.

[0040] In some embodiments, the method includes comparing the first expression level of each biomarker with its respective second expression level. In some embodiments, the method includes obtaining a first composite score based on the first expression level of the biomarker and a second composite score based on the second expression level of the biomarker, and comparing the first composite score with the second composite score.

[0041] In some embodiments, the treatment includes an immunomodulatory drug (IMiD). In some embodiments, the treatment includes a CRBN modulator or a compound having the ability to bind to CRBN and / or induce a conformational change thereof. In some embodiments, the treatment includes an agent that depletes B cells (e.g., an anti-CD20 antibody, e.g., rituximab).

[0042] In some embodiments, the subject has been infected with coronavirus disease 2019 (COVID-19).

[0043] In some embodiments, the expression level of a biomarker is determined by measuring the mRNA level of the biomarker. In some embodiments, the mRNA level is determined by using quantitative reverse transcriptase PCR (RT-qPCR), microarray, Northern blot, or RNA sequencing. In some embodiments, the expression level of a biomarker is determined by measuring the protein level of the biomarker. In some embodiments, the protein level of the biomarker is determined by using mass spectrometry (MS), liquid chromatography-tandem mass spectrometry (LC MS / MS), immunoassay, flow cytometry, immunohistochemistry, Western blot, or enzyme-linked immunosorbent assay (ELISA).

[0044] In one aspect, provided herein is a kit for performing any of the above-described methods that includes an agent for determining the expression level of at least one biomarker selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D.

[0045] In some embodiments, the kit further includes an instrument for obtaining a sample.

[0046] In some embodiments, the kit further includes instructions regarding the interpretation of the determined expression level.

[0047] In some embodiments, the kit further includes a reference expression level of a biomarker.

Brief Description of the Drawings

[0048] 4. Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0049] 5. DETAILED DESCRIPTION OF THE INVENTION This disclosure is based, in part, on the finding that there are significant symptom similarities between Long COVID and CFS, and that the levels of certain cereblon (CRBN)-associated proteins (CAPs) biomarkers (e.g., the mRNA, cDNA, or protein of these biomarkers in Figure 7) correlate with CFS and Long COVID and their severity.

[0050] 5.1 Definitions Unless otherwise defined herein, scientific and technical terms used in this specification have the meanings commonly understood by one of ordinary skill in the art. Where appropriate, terms used in the singular shall also include the plural, and vice versa. In the event of any conflict between the description of any term shown and any document incorporated herein by reference, the description of the term shown below shall prevail.

[0051] As used herein, the term "biomarker" is a substance whose detection, quantity, change, or any other characterization indicates a particular biological state, such as a disease state or its progression. In some embodiments, biomarkers can be determined individually. In other embodiments, several biomarkers can be measured simultaneously.

[0052] As used herein, the term "expression" refers to transcription from a gene that produces an RNA nucleic acid molecule that is at least partially complementary to a region of one of the two nucleic acid strands of the gene. The term "expression" also refers to translation from an RNA molecule that produces a protein, polypeptide, or a portion thereof, as used herein.

[0053] As used herein in the context of a biomarker, the term "level" refers to the amount, accumulation, or rate of a biomarker molecule (e.g., the mRNA or protein expression level of a gene or organic acid). The level may be represented, for example, by the amount or rate of synthesis of messenger RNA (mRNA) encoded by a gene, the amount or rate of synthesis of a polypeptide or protein encoded by a gene, or the amount or rate of synthesis of a biomolecule that accumulates in a cell or biological fluid. As used herein, a level can be the absolute amount or relative amount of a molecule in a sample, determined under steady-state or non-steady-state conditions. In some embodiments, the expression level of a biomarker is the expression level after normalization. In some embodiments, the expression level after normalization can be obtained by obtaining the expression level of a housekeeping gene (e.g., ACTB, GAPDH, or TFRC) and normalizing the expression level of the biomarker by the expression level of the housekeeping gene (e.g., ACTB, GAPDH, or TFRC) to determine the expression level of the biomarker after normalization.

[0054] In this specification, any normalization method known in the art can be used. Generally speaking, the normalization methods for gene expression data can be divided into three categories, namely, data-driven reference, external reference, and whole gene set reference. For data-driven procedures, first, a subset of genes with no or minimal variation between samples is identified as the data-driven housekeeping genes for normalizing the data set. For external controls, several experiments are designed with external controls such as spike-in controls. These natural controls can be used as external references for gene expression data normalization. For the whole gene set, values or some values for data normalization are determined using all the genes in the experiment. In some algorithms, the entire genome is regarded as a reference for data normalization. Another exemplary normalization method is "global" normalization, which means that the entire gene panel (e.g., the whole transcriptome) is used, and the RFU value of the gene of interest is divided by the median or average RFU of the whole panel. In some embodiments, the normalization method for gene expression profiling data assumes that the majority of genes in the genome are equally expressed in each experimental unit and that the genes are symmetrically distributed between overexpression and underexpression. Non-normalized data provides another method when the assumptions for the above normalization do not apply, for example, when a large partition of genes with expression differences is included in the data.

[0055] The normalized values can be scaled. An exemplary scaling procedure for gene expression profiling data is provided below: The relationship between gene expression data observed on different technology platforms cannot be assumed to be the same in the state of its raw data. In order to compare data between platforms, it is assumed that the ranges of the observed data are similar. For example, in scaling, it can be assumed that the observed average value data is the same, and the simplest function for scaling the data range from one platform to another can be performed based on a logarithmic scale. Scaling enables the centering of the data point average value and the alignment of the measurement ranges between platforms, thus enabling the comparison of gene expression profiling data from different platforms. In a specific exemplary method, the raw RFU is used and divided by the geometric mean of a set of housekeeping genes. In one embodiment, this set of genes includes, or consists of, the three housekeeping genes ACTB, GAPDH, and TFRC. The Log2 of the normalized value can be calculated, whereby the value is scaled to the range of -1 to 1. This enables all genes to be plotted together in one graph for comparison.

[0056] The term "reference level" refers to the level of a biomarker that is useful for the purpose of comparison (e.g., the expression level of a gene (e.g., mRNA or protein expression level) or the level of an organic acid). The reference level of a biomarker (e.g., the reference expression level of a gene (e.g., mRNA or protein expression level) or the level of an organic acid) can be determined in a subject by any one of the methods provided herein. In some embodiments, the reference level of a biomarker is a predetermined level of said biomarker. In some embodiments, the predetermined expression level can be found in a public database. In some embodiments, the reference level is the expression level of a gene in the blood. In some embodiments, the reference level is the level of an organic acid in urine. In some embodiments, the reference level of a biomarker is the level (e.g., the expression level of a gene (e.g., mRNA or protein expression level) or the level of an organic acid) of said biomarker measured at different time points or from different samples in the same subject. In some embodiments, the reference level of a biomarker is the level (e.g., the expression level of a gene (e.g., mRNA or protein expression level) or the level of an organic acid) in the corresponding tissue of a control subject (e.g., a healthy subject, a subject without CFS, a subject with mild CFS, a subject without long COVID, a subject with acute COVID) in which said biomarker was measured. In some embodiments, the reference level of a biomarker is the level (e.g., the median or average of the expression level of a gene (e.g., mRNA or protein expression level) or the median or average of the level of an organic acid) in the corresponding tissue of a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without CFS, a cohort of subjects with mild CFS (used synonymously with moderate CFS herein), a cohort of subjects without long COVID, a cohort of subjects with acute COVID) in which said biomarker was measured. In some embodiments, the reference level of a biomarker (e.g., the reference expression level of a gene (e.g., mRNA or protein expression level) or the level of an organic acid) is the level of said biomarker in a sample obtained from a subject prior to treatment of the subject and / or administration of a compound.

[0057] As used herein, the terms "treat", "treatment", "treating", "attempt to treat", "alleviate", "alleviation", "alleviating", or "attempt to alleviate" refer to therapeutic measures aimed at curing, mitigating, alleviating symptoms of, and / or arresting the progression of a pathological condition or disorder.

[0058] As used herein, and unless otherwise specified, the term "therapeutically effective amount" of a compound is an amount sufficient to provide a therapeutic benefit in the treatment or management of a disease or disorder (e.g., CFS or Long COVID), or to delay or minimize one or more symptoms associated with the presence of the disease or disorder (e.g., CFS or Long COVID). A therapeutically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease or disorder (e.g., CFS or Long COVID). The term "therapeutically effective amount" can encompass an amount that improves the overall therapy, an amount that reduces or avoids the symptoms or causes of a disease or disorder (e.g., CFS or Long COVID), or an amount that enhances the therapeutic effectiveness of another therapeutic agent. This term also refers to an amount of a compound sufficient to elicit a biological or medical response that is sought by a researcher, veterinarian, physician, or clinician in a biomolecule (e.g., protein, enzyme, RNA, DNA, or organic acid), cell, tissue, system, animal, or human.

[0059] As used herein, the terms "prevent", "prevention", or "preventing" refer to the partial or total inhibition of the onset, recurrence, development, or spread of a disease, disorder, or medical condition, or its symptoms, in a subject.

[0060] As used in connection with any of the methods provided herein, the terms "responsive" or "responsiveness" refer to the degree of effectiveness of a treatment in alleviating or reducing the symptoms of a disease, such as CFS or Long COVID.

[0061] The term "predict" or "predicting" generally means to determine or convey in advance. When the term "predicting" is used in relation to any one of the methods provided herein, it means that the likelihood of the outcome of a particular action, e.g., treatment, is determined initially before the treatment is carried out.

[0062] The term "obtaining", when used herein in connection with the level of a biomarker, refers to an act or step regarding obtaining information about the level of the biomarker. In some embodiments, the term "obtaining" includes steps such as "determining", "measuring", "evaluating", "assessing", and / or "assaying". In some embodiments, "obtaining" involves ordering a determination, evaluation, assessment, and / or assay performed by a third party, or using the results of such determination, evaluation, assessment, and / or assay. In some embodiments, the term "obtaining" also includes the step of obtaining a sample from a subject. In other embodiments, the term may also include steps necessary to prepare the sample in a state suitable for an assay for measuring the nucleic acid or protein of the biomarker within the sample.

[0063] The terms "determining", "measuring", "evaluating", "assessing", and "assaying" are used interchangeably herein to refer to a form of measurement value, including determining whether an element is present or not. The measurement value can be a quantitative determination and / or a qualitative determination. "Determining the expression level of ~" can include measuring the amount of something present, as well as determining whether it is present or not.

[0064] As used herein, the term "monitoring" generally refers to the supervision, management, regulation, observation, tracking, or surveillance of activities. For example, the term "monitoring the effectiveness of a compound" refers to tracking the effectiveness of the treatment of a disease or disorder (e.g., CFS or Long COVID) in a patient. The term "monitoring the effectiveness of a compound" also refers to tracking the effectiveness (i.e., reduction of fatigue) of treating CFS or Long COVID in a patient. Similarly, the term "monitoring" refers to tracking or verifying that a patient is actually taking the drug under study as prescribed when used in relation to patient compliance, either as seen personally or in a clinical trial. Monitoring can be performed, for example, by tracking the expression levels of mRNA or protein biomarkers or the levels of urinary organic acids.

[0065] The term "likely" or "likelihood" generally refers to an increase in the probability of an event. The term "likelihood", when used in relation to the effectiveness of a treatment in a subject, generally contemplates an increase in the probability that the progression or extent of the disease will decrease. The term "likelihood", when used in relation to the effectiveness of the treatment of CFS or Long COVID, may generally mean a reduction in the symptoms of CFS or Long COVID (e.g., severe fatigue).

[0066] As used herein, the term "sample" refers to a material or mixture of materials containing one or more components of interest, for example, in fluid or solid form. The sample may be a biological sample obtained from a biological subject, including samples of biological tissue or body fluid obtained, accessed, or collected in vivo or in situ. Such samples can be, but are not limited to, organs, tissues, and cells isolated from mammals. Exemplary biological samples include, but are not limited to, cell lysates, cell cultures, cell lines, tissues, oral tissues, gastrointestinal tissues, organs, organelles, biological fluids, blood samples, urine samples, skin samples, etc.

[0067] The terms "polypeptide" and "protein" are used synonymously herein and refer to a polymer in which three or more amino acids are linked by peptide bonds and are arranged in sequence. The term "polypeptide" includes proteins, protein fragments, protein analogs, oligopeptides, and the like. The term "polypeptide" may also refer to a peptide when used herein. The amino acids that make up a polypeptide may be of natural origin or synthetic. A polypeptide can be purified from a biological sample. Polypeptides, proteins, or peptides also include modified polypeptides, proteins, and peptides, such as glycopolypeptides, glycoproteins, or glycopeptides; or lipopolypeptides, lipoproteins, or lipopeptides.

[0068] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to describe a polymer of nucleotides of any length, for example, deoxyribonucleotides or ribonucleotides, or a synthetically made compound that can hybridize to a naturally occurring nucleic acid in a sequence-specific manner similar to two naturally occurring nucleic acids, e.g., capable of participating in Watson-Crick base pairing interactions. When used in connection with a polynucleotide sequence, the term "bases" (or "base") is synonymous with "nucleotides" (or "nucleotide"), i.e., the monomeric subunits of a polynucleotide. The terms "nucleoside" and "nucleotide" are intended to include moieties having not only the known purine and pyrimidine bases but also other heterocyclic bases that are modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles. In addition, the terms "nucleoside" and "nucleotide" are intended to include moieties having not only the conventional ribose and deoxyribose sugars but also other sugars. Modified nucleosides or nucleotides also include modifications to the sugar moiety, e.g., where one or more of the hydroxyl groups are replaced by a halogen atom or an aliphatic group, or are functionalized as ethers, amines, etc. "Analog" refers to a molecule recognized in the literature by terms such as mimetic, derivative, having a similar structure, or other similar terms, e.g., polynucleotides incorporating non-natural nucleotides, nucleotide mimetics such as 2'-modified nucleosides, peptide nucleic acids, oligomeric nucleoside phosphonic acids, and any polynucleotide to which substituents such as protecting groups or linking moieties have been added.

[0069] The terms "about" or "approximately" mean an acceptable error as determined by one of ordinary skill in the art for a particular value, which in part depends on how that value is measured or determined. In certain embodiments, the terms "about" or "approximately" mean within 1, 2, 3, or 4 standard deviations. In certain embodiments, the terms "about" or "approximately" mean within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0070] As used herein in phrases such as "A and / or B," the term "and / or" is intended to include both A and B; A or B; A alone; and B alone. Similarly, when used in phrases such as "A, B, and / or C," the term "and / or" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A alone; B alone; and C alone.

[0071] As used in this disclosure and the claims, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise.

[0072] Whenever embodiments are described herein with the term "comprising," it is to be understood that other similar embodiments are also provided which are described with the terms "consisting of" and / or "consisting essentially of." Also, whenever embodiments are described herein with the phrase "consisting essentially of," it is to be understood that other similar embodiments are also provided which are described with the term "consisting of."

[0073] 5.2 Biomarkers and Methods of Using the Same In one aspect, provided herein is a method of using the level of at least one cereblon-associated protein (CAP) biomarker (e.g., gene expression level (e.g., mRNA or protein expression level)) identified herein as being associated with post-viral syndrome characterized by fatigue. In some embodiments, the fatigue is fatigue that persists for at least 3 months after infection with a virus. In some embodiments, the fatigue is fatigue that persists for at least 6 months after infection with a virus. In some embodiments, the fatigue is debilitating. In some embodiments, the fatigue interferes with activities of daily living. In some embodiments, a patient suffering from post-viral syndrome is withdrawn, bedridden, or both. In some embodiments, post-viral syndrome includes one or more of the following: post-exertional malaise, cognitive dysfunction, sensorimotor symptoms, headache, memory impairment, insomnia, myalgia, palpitations, shortness of breath, dizziness and balance disorders, speech disorders, arthralgia, chest pressure. In certain embodiments, post-viral syndrome is CFS. In certain embodiments, post-viral syndrome is long COVID.

[0074] "Cereblon-associated protein" or "CAP" refers to a protein that interacts, either directly or indirectly, with or binds to CRBN. In certain embodiments, CAP is any protein that binds directly to cereblon, as well as any protein that is an indirect downstream effector of the cereblon pathway. In certain embodiments, "cereblon-associated protein" or "CAP" is a substrate of CRBN, e.g., a protein substrate of the E3 ubiquitin ligase complex involving CRBN, or a downstream substrate thereof. In certain embodiments, CAP is IKAROS family zinc finger 2 (IKZF2), IKAROS family zinc finger 3 (IKZF3), ATP-binding cassette subfamily E member 1 (ABCE1), BTB domain and CNC homology 2 (BACH2), CD3 delta subunit of the T cell receptor (CD3D), or heat shock protein family A member 8 (HSPA8).

[0075] In some embodiments, provided herein is a method of identifying a subject having post-viral infection syndrome, comprising obtaining an expression level of a biomarker in a sample from the subject, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8, and identifying that the subject has post-viral infection syndrome if the expression level of the biomarker is higher than a reference expression level of the biomarker.

[0076] In some embodiments, provided herein is a method of confirming post-viral infection syndrome in a subject, comprising obtaining an expression level of a biomarker in a sample from the subject, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8; and confirming that the subject has post-viral infection syndrome if the expression level of the biomarker is higher than a reference expression level of the biomarker.

[0077] In some embodiments, provided herein is a method of determining the severity of post-viral infection syndrome in a subject, comprising obtaining an expression level of a biomarker in a sample from the subject, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8, and determining the severity of post-viral infection syndrome in the subject based on the expression level. In some embodiments, the method further comprises comparing the expression level of the biomarker to a reference expression level of the biomarker. In some embodiments, a higher expression level of the biomarker indicates a higher severity of post-viral infection syndrome.

[0078] In some embodiments, provided herein is a method of monitoring the progression of a post-viral infection syndrome in a subject, the method comprising obtaining an expression level of a biomarker in a sample from the subject, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8, and determining the progression of the post-viral infection syndrome based on the expression level of the biomarker. In certain embodiments, an elevated expression level of the biomarker when compared to a reference expression level of the biomarker (e.g., the expression level of the biomarker at an earlier time point) indicates that the post-viral infection syndrome is progressing. In certain embodiments, an elevated expression level of the biomarker when compared to a reference expression level of the biomarker (e.g., the expression level of the biomarker at an earlier time point) indicates that the post-viral infection syndrome is regressing.

[0079] In some embodiments, provided herein is a method of identifying a subject who is likely or unlikely to respond to treatment of a post-viral infection syndrome, or predicting whether a subject will respond to treatment of a post-viral infection syndrome, the method comprising obtaining an expression level of a biomarker in a sample from the subject, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8; and identifying or predicting that the subject is likely to respond to treatment of the post-viral infection syndrome if the expression level of the biomarker is higher than a reference expression level of the biomarker.

[0080] In some embodiments, provided herein is a method for selectively treating a subject having or suspected of having post-viral syndrome, the method comprising obtaining an expression level of a biomarker in a sample from the subject, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8; identifying or predicting that the subject is likely to respond to treatment for post-viral syndrome if the expression level of the biomarker is higher than a reference expression level of the biomarker; and administering treatment to a subject identified or predicted to be likely to respond to treatment.

[0081] In some embodiments, the reference expression level of the biomarker is a predetermined expression level of the biomarker from a public database. In some embodiments, the reference expression level of the biomarker is the expression level of the biomarker in a subject without post-viral syndrome or a healthy subject. In some embodiments, the reference expression level of the biomarker is the expression level of the biomarker in a subject having mild post-viral syndrome or having acute viral infection. In some embodiments, the reference expression level of the biomarker is the expression level of the biomarker determined based on a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without post-viral syndrome, a cohort of subjects having mild post-viral syndrome, or a cohort of subjects having acute viral infection). In some embodiments, the reference expression level of the biomarker is the median or mean of the expression levels of the biomarker in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without post-viral syndrome, a cohort of subjects having mild post-viral syndrome, or a cohort of subjects having acute viral infection).

[0082] In certain embodiments, the method comprises using two or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, when multiple biomarkers are used, the expression level of each biomarker is compared to a reference expression level of that biomarker. In other embodiments, when multiple biomarkers are used, an overall score is calculated based on the multiple biomarkers and compared to a reference overall score. In some embodiments, the overall score is calculated using the median Z-score method. In other embodiments, the overall score is calculated using the single sample gene set enrichment (ssGSEA) method.

[0083] In some embodiments, provided herein is a method for determining or monitoring the effectiveness of treatment in a subject having a post-viral syndrome, the method comprising obtaining a first expression level of a biomarker in a first sample from the subject, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8; administering treatment to the subject; obtaining a second expression level of the biomarker in a second sample obtained from the subject after administering the treatment to the subject; and determining the effectiveness of the treatment based on a comparison of the first expression level and the second expression level. In some embodiments, the method comprises determining that the treatment is effective if the second expression level is lower than the first expression level. In some embodiments, the method comprises determining that the treatment is not effective if the second expression level is not lower than the first expression level. In some embodiments, the method comprises determining or adjusting (e.g., increasing) the dosage of the treatment or administering a different treatment to the subject if the second expression level is not lower than the first expression level.

[0084] In some embodiments, provided herein is a method of screening for the effectiveness of a treatment in treating post-viral syndrome for a certain treatment, comprising: (a) determining a first expression level of a biomarker in a sample before administering a compound to the sample, wherein the biomarker is a cereblon (CRBN)-associated protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; (b) administering the treatment to the sample; (c) determining a second expression level of the biomarker in the sample after administering the treatment to the sample; (d) comparing the first expression level with the second expression level; and (e) selecting the treatment if the second expression level is lower than the first expression level.

[0085] In certain embodiments, the method comprises using two or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, when multiple biomarkers are used, the second expression level of each biomarker is compared with the first expression level of that biomarker, and the method comprises determining that the treatment is effective if the second expression level of each biomarker is lower than the first expression level of that biomarker. In other embodiments, when multiple biomarkers are used, an overall score is calculated based on the multiple biomarkers and compared with a reference overall score. In some embodiments, the overall score is calculated using the median Z-score method. In other embodiments, the overall score is calculated using the single-sample gene set enrichment (ssGSEA) method.

[0086] In some embodiments, the treatment of post-viral syndrome disclosed herein includes immunomodulatory drugs (IMiDs) as disclosed in Section 5.2. In some embodiments, the treatment of post-viral syndrome includes a cereblon (CRBN) modulator or a compound having the ability to bind to CRBN and / or induce a conformational change thereof as disclosed in Section 5.2. In some embodiments, the treatment of post-viral syndrome includes an agent that depletes B cells (e.g., an anti-CD20 antibody such as rituximab) as disclosed in Section 5.2.

[0087] 5.2.1 Biomarkers of CFS "Chronic fatigue syndrome" or "CFS" as used herein refers to a disorder with fatigue symptoms that persist for a period of time, such as at least 4 weeks. In some embodiments, the period is at least 6 weeks. In some embodiments, the period is at least 2 months, 3 months, 4 months, 5 months, or 6 months. In some embodiments, the period is at least 6 months. The fatigue symptoms can be persistent or intermittent during this period. In some embodiments, the fatigue symptoms persist during the said period. In some embodiments, due to the fatigue symptoms, the patient becomes bedridden. In some embodiments, the disorder includes a combination of symptoms, which can include one or more of the following: debilitating fatigue, post-exertional malaise, non-restorative sleep or sleep disorder (or both), and / or cognitive difficulties. In some embodiments, the combination of symptoms includes debilitating fatigue, post-exertional malaise, non-restorative sleep or sleep disorder (or both), and cognitive difficulties. In some embodiments, such a combination of symptoms is seen over a period of time as disclosed herein. In some embodiments, the period is at least 6 weeks in adults and at least 4 weeks in children. Most often, the fatigue symptoms persist for more than 6 months. In certain cases, the disorder can be accompanied by various other symptoms such as non-restorative sleep, mental and physical pain, neurological and cognitive impairments, and autoimmunity or immunodeficiency.

[0088] In some embodiments, CFS is a post-viral syndrome. In some embodiments, CFS occurs in the absence of viral infection and is thus not a post-viral syndrome.

[0089] In some embodiments, provided herein is a method of identifying a subject having CFS, the method comprising obtaining an expression level of a biomarker in a sample from the subject, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8, and identifying that the subject has CFS if the expression level of the biomarker is higher than a reference expression level of the biomarker. In some embodiments, the biomarker is IKZF2. In some embodiments, the biomarker is IKZF3. In some embodiments, the biomarker is ABCE1. In some embodiments, the biomarker is BACH2. In some embodiments, the biomarker is CD3D. In some embodiments, the biomarker is HSPA8.

[0090] In some embodiments, the reference expression level of a biomarker is a predetermined expression level of the biomarker. In some embodiments, the reference expression level of a biomarker is a predetermined expression level of the biomarker obtained from a public database. In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker in a healthy subject or a subject without CFS. In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker in a subject with moderate CFS. In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker determined based on a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without CFS, or a cohort of subjects with moderate CFS). In some embodiments, the reference expression level of a biomarker is the median or average value of the expression levels of the biomarker in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without CFS, a cohort of subjects with moderate CFS). In some embodiments, the biomarker is HSPA8 or ABCE1, and the reference expression level of the biomarker is the expression level of the biomarker in a healthy subject or a subject without CFS, or in a cohort of healthy subjects or subjects without CFS.

[0091] In some embodiments, the method includes identifying that a subject has CFS if the expression level of a biomarker is at least 5% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying that a subject has CFS if the expression level of a biomarker is at least 10% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying that a subject has CFS if the expression level of a biomarker is at least 20% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying that a subject has CFS if the expression level of a biomarker is at least 30% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying that a subject has CFS if the expression level of a biomarker is at least 40% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying that a subject has CFS if the expression level of a biomarker is at least 50% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying that a subject has CFS if the expression level of a biomarker is at least 60% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying that a subject has CFS if the expression level of a biomarker is at least 70% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying that a subject has CFS if the expression level of a biomarker is at least 80% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying that a subject has CFS if the expression level of a biomarker is at least 90% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying that a subject has CFS if the expression level of a biomarker is at least twice the reference expression level of the biomarker. In some embodiments, the method includes identifying that a subject has CFS if the expression level of a biomarker is at least three times the reference expression level of the biomarker.In some embodiments, the method includes identifying that a subject has CFS if the expression level of the biomarker is at least 4 times the reference expression level of the biomarker. In some embodiments, the method includes identifying that a subject has CFS if the expression level of the biomarker is at least 5 times the reference expression level of the biomarker. In some embodiments, the method includes identifying that a subject has CFS if the expression level of the biomarker is at least 6 times the reference expression level of the biomarker. In some embodiments, the method includes identifying that a subject has CFS if the expression level of the biomarker is at least 7 times the reference expression level of the biomarker. In some embodiments, the method includes identifying that a subject has CFS if the expression level of the biomarker is at least 8 times the reference expression level of the biomarker. In some embodiments, the method includes identifying that a subject has CFS if the expression level of the biomarker is at least 9 times the reference expression level of the biomarker. In some embodiments, the method includes identifying that a subject has CFS if the expression level of the biomarker is at least 10 times the reference expression level of the biomarker. The expression level of the biomarker can be determined using any known method in the art, and exemplary methods are described in further detail in Section 5.3 below. In some embodiments, the expression level of the biomarker is determined to be higher than the reference level if the level is higher (e.g., statistically significantly higher) than the reference level when observed by a measurement assay.

[0092] In certain embodiments, the method includes identifying a subject having CFS using two or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method includes identifying a subject having CFS using three or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method includes identifying a subject having CFS using four or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method includes identifying a subject having CFS using five or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method includes identifying a subject having CFS using all of the biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the expression levels of IKZF2 and IKZF3 are determined. In some embodiments, the expression levels of IKZF2 and ABCE1 are determined. In some embodiments, the expression levels of IKZF2 and BACH2 are determined. In some embodiments, the expression levels of IKZF2 and CD3D are determined. In some embodiments, the expression levels of IKZF2 and HSPA8 are determined. In some embodiments, the expression levels of IKZF3 and ABCE1 are determined. In some embodiments, the expression levels of IKZF3 and BACH2 are determined. In some embodiments, the expression levels of IKZF3 and CD3D are determined. In some embodiments, the expression levels of IKZF3 and HSPA8 are determined. In some embodiments, the expression levels of ABCE1 and BACH2 are determined. In some embodiments, the expression levels of ABCE1 and CD3D are determined. In some embodiments, the expression levels of ABCE1 and HSPA8 are determined. In some embodiments, the expression levels of BACH2 and CD3D are determined.In some embodiments, the expression levels of BACH2 and HSPA8 are determined. In some embodiments, the expression levels of CD3D and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3 and ABCE1 are determined. In some embodiments, the expression levels of IKZF2, IKZF3 and BACH2 are determined. In some embodiments, the expression levels of IKZF2, IKZF3 and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3 and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1 and BACH2 are determined. In some embodiments, the expression levels of IKZF2, ABCE1 and CD3D are determined. In some embodiments, the expression levels of IKZF2, ABCE1 and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, BACH2 and CD3D are determined. In some embodiments, the expression levels of IKZF2, BACH2 and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, CD3D and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1 and BACH2 are determined. In some embodiments, the expression levels of IKZF3, ABCE1 and CD3D are determined. In some embodiments, the expression levels of IKZF3, ABCE1 and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, BACH2 and CD3D are determined. In some embodiments, the expression levels of IKZF3, BACH2 and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, CD3D and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, BACH2 and CD3D are determined. In some embodiments, the expression levels of ABCE1, BACH2 and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, CD3D and HSPA8 are determined. In some embodiments, the expression levels of BACH2, CD3D and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, BACH2, CD3D and HSPA8 are determined.In some embodiments, the expression levels of IKZF3, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, CD3D, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, and CD3D are determined.In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, when multiple biomarkers are used, the expression level of each biomarker is compared to the reference expression level of that biomarker, and the method includes identifying that the subject has CFS if the expression level of each biomarker is higher than the reference expression level of that biomarker. In other embodiments, when multiple biomarkers are used, an overall score is calculated based on the multiple biomarkers and compared to a reference overall score. In some embodiments, the method includes identifying that the subject has CFS if the overall score is higher than the reference overall score. In some embodiments, the overall score is calculated using the median Z-score method. Briefly, the median Z-score is obtained by first calculating the mean value of each gene from all samples within the gene expression matrix. Next, this mean value is subtracted from each corresponding gene for all samples, and then scaling is performed by dividing the value by its standard deviation. The median of the scaled values from the target multiple genes constitutes the overall score. Another exemplary method for calculating the overall score is the single-sample gene set enrichment (ssGSEA) method. The single-sample gene score represents the degree to which genes within a particular gene set are coordinately upregulated or downregulated within a sample. This score is calculated by adjusting the cumulative sum statistic based on a decreasing walk through the ranked expression list. The enrichment score is the maximum deviation from zero encountered during the walk; this corresponds to a weighted Kolmogorov-Smirnov-like statistic (see, for example, Subramanian et al., PNAS, 102(43):15545-15550 (2005); and Barbie et al., Nature, 462(7269):108-112).

[0093] In other embodiments, provided herein is a method of ascertaining the CFS of a subject, comprising obtaining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8; and ascertaining that the subject has CFS if the expression level of the biomarker is higher than the reference expression level of the biomarker. In some embodiments, the subject complains of symptoms associated with CFS. Thus, in some embodiments, provided herein is a method of ascertaining the CFS of a subject, comprising selecting a subject who complains of symptoms associated with CFS; obtaining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8; and ascertaining that the subject has CFS if the expression level of the biomarker is higher than the reference expression level of the biomarker. Exemplary symptoms associated with CFS include, but are not limited to, chronic debilitating fatigue, non-restorative sleep, mental and / or physical pain, neurological and cognitive impairments, and / or autoimmunity or immunodeficiency. In some embodiments, the biomarker is IKZF2. In some embodiments, the biomarker is IKZF3. In some embodiments, the biomarker is ABCE1. In some embodiments, the biomarker is BACH2. In some embodiments, the biomarker is CD3D. In some embodiments, the biomarker is HSPA8.

[0094] In some embodiments, the reference expression level of a biomarker is a predetermined expression level of the biomarker from a public database. In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker in a subject without CFS. In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker in a subject with moderate CFS. In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker determined based on a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without CFS, or a cohort of subjects with moderate CFS). In some embodiments, the reference expression level of a biomarker is the median or average value of the expression levels of the biomarker in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without CFS, or a cohort of subjects with moderate CFS). In some embodiments, the biomarker is HSPA8 or ABCE1, and the reference expression level of the biomarker is the expression level of the biomarker in a healthy subject or a subject without CFS, or in a cohort of healthy subjects or subjects without CFS. In some embodiments, the biomarker is HSPA8 or ABCE1, and the reference expression level of the biomarker is the expression level of the biomarker in a healthy subject or a subject without CFS, or in a cohort of healthy subjects or subjects without CFS.

[0095] In some embodiments, the method includes determining that the subject has CFS if the expression level of the biomarker is at least 5% higher than the reference expression level of the biomarker. In some embodiments, the method includes determining that the subject has CFS if the expression level of the biomarker is at least 10% higher than the reference expression level of the biomarker. In some embodiments, the method includes determining that the subject has CFS if the expression level of the biomarker is at least 20% higher than the reference expression level of the biomarker. In some embodiments, the method includes determining that the subject has CFS if the expression level of the biomarker is at least 30% higher than the reference expression level of the biomarker. In some embodiments, the method includes determining that the subject has CFS if the expression level of the biomarker is at least 40% higher than the reference expression level of the biomarker. In some embodiments, the method includes determining that the subject has CFS if the expression level of the biomarker is at least 50% higher than the reference expression level of the biomarker. In some embodiments, the method includes determining that the subject has CFS if the expression level of the biomarker is at least 60% higher than the reference expression level of the biomarker. In some embodiments, the method includes determining that the subject has CFS if the expression level of the biomarker is at least 70% higher than the reference expression level of the biomarker. In some embodiments, the method includes determining that the subject has CFS if the expression level of the biomarker is at least 80% higher than the reference expression level of the biomarker. In some embodiments, the method includes determining that the subject has CFS if the expression level of the biomarker is at least 90% higher than the reference expression level of the biomarker. In some embodiments, the method includes determining that the subject has CFS if the expression level of the biomarker is at least twice the reference expression level of the biomarker. In some embodiments, the method includes determining that the subject has CFS if the expression level of the biomarker is at least three times the reference expression level of the biomarker.In some embodiments, the method includes determining that a subject has CFS if the expression level of a biomarker is at least 4 times the reference expression level of the biomarker. In some embodiments, the method includes determining that a subject has CFS if the expression level of a biomarker is at least 5 times the reference expression level of the biomarker. In some embodiments, the method includes determining that a subject has CFS if the expression level of a biomarker is at least 6 times the reference expression level of the biomarker. In some embodiments, the method includes determining that a subject has CFS if the expression level of a biomarker is at least 7 times the reference expression level of the biomarker. In some embodiments, the method includes determining that a subject has CFS if the expression level of a biomarker is at least 8 times the reference expression level of the biomarker. In some embodiments, the method includes determining that a subject has CFS if the expression level of a biomarker is at least 9 times the reference expression level of the biomarker. In some embodiments, the method includes determining that a subject has CFS if the expression level of a biomarker is at least 10 times the reference expression level of the biomarker. The expression level of the biomarker can be determined using any known method in the art, and exemplary methods are described in more detail in Section 5.3 below. In some embodiments, if the level is higher than the reference level (e.g., statistically significantly higher than it) when observed by a measurement assay, the level is determined to be higher than the reference level.

[0096] In certain embodiments, the method comprises identifying a subject having CFS using two or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises identifying a subject having CFS using three or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises identifying a subject having CFS using four or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises identifying a subject having CFS using five or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises identifying a subject having CFS using all of the biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the expression levels of IKZF2 and IKZF3 are determined. In some embodiments, the expression levels of IKZF2 and ABCE1 are determined. In some embodiments, the expression levels of IKZF2 and BACH2 are determined. In some embodiments, the expression levels of IKZF2 and CD3D are determined. In some embodiments, the expression levels of IKZF2 and HSPA8 are determined. In some embodiments, the expression levels of IKZF3 and ABCE1 are determined. In some embodiments, the expression levels of IKZF3 and BACH2 are determined. In some embodiments, the expression levels of IKZF3 and CD3D are determined. In some embodiments, the expression levels of IKZF3 and HSPA8 are determined. In some embodiments, the expression levels of ABCE1 and BACH2 are determined. In some embodiments, the expression levels of ABCE1 and CD3D are determined. In some embodiments, the expression levels of ABCE1 and HSPA8 are determined. In some embodiments, the expression levels of BACH2 and CD3D are determined.In some embodiments, the expression levels of BACH2 and HSPA8 are determined. In some embodiments, the expression levels of CD3D and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and ABCE1 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and BACH2 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF2, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF3, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, BACH2, CD3D, and HSPA8 are determined.In some embodiments, the expression levels of IKZF3, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, CD3D, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, and CD3D are determined.In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, when multiple biomarkers are used, the expression level of each biomarker is compared to the reference expression level of that biomarker, and the method includes determining that the subject has CFS if the expression level of each biomarker is higher than the reference expression level of that biomarker. In other embodiments, when multiple biomarkers are used, an aggregate score is calculated based on the multiple biomarkers and compared to a reference aggregate score. In some embodiments, the method includes determining that the subject has CFS if the aggregate score is higher than the reference aggregate score. In some embodiments, the aggregate score is calculated using the median Z-score method. In other embodiments, the aggregate score is calculated using the single-sample gene set enrichment (ssGSEA) method.

[0097] The present disclosure is also, in part, based on the finding that the expression levels of certain CAP biomarkers are related to the severity of CFS. Thus, in other embodiments, provided herein is a method of determining the severity of CFS in a subject, the method comprising obtaining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8, and determining the severity of CFS in the subject based on the expression level. In some embodiments, the method further comprises comparing the expression level of the biomarker to a reference expression level of the biomarker. In some embodiments, a high expression level of the biomarker indicates a high severity of CFS. "Severe CFS" as used herein refers to a type of CFS in which the patient is more severely affected and is housebound and / or bedridden. In certain embodiments, a patient with severe CFS spends most of their time housebound and / or bedridden. In certain embodiments, a patient with severe CFS (e.g., a very severe CFS patient) is completely bedridden and requires assistance with basic activities including nutrition and hydration. "Mild CFS" and "moderate CFS" are used interchangeably herein and refer to a type of CFS in which the patient is not housebound or bedridden.

[0098] In some embodiments, the biomarker is IKZF2. In some embodiments, the biomarker is IKZF3. In some embodiments, the biomarker is ABCE1. In some embodiments, the biomarker is BACH2. In some embodiments, the biomarker is CD3D. In some embodiments, the biomarker is HSPA8.

[0099] In some embodiments, the reference expression level of a biomarker is a predetermined expression level of the biomarker from a public database. In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker in a healthy subject or a subject without CFS. In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker determined based on a cohort of healthy subjects. In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker in a subject in which the severity of CFS has been determined and is known. In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker in a subject having moderate CFS or a cohort of subjects having moderate CFS. In some embodiments, the reference expression level of a biomarker is the median or average value of the expression levels of the biomarker in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects having moderate CFS, or a cohort of subjects in which the severity of CFS has been determined and is known).

[0100] In some embodiments, the biomarker is HSPA8 or ABCE1, and the reference expression level of the biomarker is the expression level of the biomarker in a healthy subject or a subject without CFS, or a cohort of healthy subjects or subjects without CFS. In some embodiments, the biomarker is selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8, and the reference expression level of the biomarker is the expression level of the biomarker in a subject having mild CFS or a cohort of subjects having mild CFS.

[0101] In yet other embodiments, provided herein is a method of monitoring the progression of CFS in a subject, the method comprising obtaining an expression level of a biomarker in a sample from the subject, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8, and determining the progression of CFS based on the expression level of the biomarker. In some embodiments, the expression level of the biomarker is obtained at two or more time points, for example, obtained periodically. In some embodiments, the biomarker is IKZF2. In some embodiments, the biomarker is IKZF3. In some embodiments, the biomarker is ABCE1. In some embodiments, the biomarker is BACH2. In some embodiments, the biomarker is CD3D. In some embodiments, the biomarker is HSPA8. In some embodiments, the reference expression level of the biomarker is the expression level of the biomarker at an earlier time point in the same subject. In certain embodiments, that the expression level of the biomarker is high when compared to the reference expression level of the biomarker (e.g., the expression level of the biomarker at an earlier time point) indicates that CFS is progressing. In certain embodiments, that the expression level of the biomarker is low when compared to the reference expression level of the biomarker (e.g., the expression level of the biomarker at an earlier time point) indicates that CFS is regressing.

[0102] The expression level of the biomarker can be determined using any known method in the art, and exemplary methods are described in more detail in Section 5.3 below. In some embodiments, when the level is observed by a measurement assay to be higher than the reference level (e.g., statistically significantly higher than it), the level is determined to be higher than the reference level. In some embodiments, when the level is observed by a measurement assay to be lower than the reference level (e.g., statistically significantly) the level is determined to be lower than the reference level.

[0103] In certain embodiments, the method comprises using two or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises using three or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises using four or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises using five or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises using all of the biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the expression levels of IKZF2 and IKZF3 are determined. In some embodiments, the expression levels of IKZF2 and ABCE1 are determined. In some embodiments, the expression levels of IKZF2 and BACH2 are determined. In some embodiments, the expression levels of IKZF2 and CD3D are determined. In some embodiments, the expression levels of IKZF2 and HSPA8 are determined. In some embodiments, the expression levels of IKZF3 and ABCE1 are determined. In some embodiments, the expression levels of IKZF3 and BACH2 are determined. In some embodiments, the expression levels of IKZF3 and CD3D are determined. In some embodiments, the expression levels of IKZF3 and HSPA8 are determined. In some embodiments, the expression levels of ABCE1 and BACH2 are determined. In some embodiments, the expression levels of ABCE1 and CD3D are determined. In some embodiments, the expression levels of ABCE1 and HSPA8 are determined. In some embodiments, the expression levels of BACH2 and CD3D are determined. In some embodiments, the expression levels of BACH2 and HSPA8 are determined. In some embodiments, the expression levels of CD3D and HSPA8 are determined.In some embodiments, the expression levels of IKZF2, IKZF3, and ABCE1 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and BACH2 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF2, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF3, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, CD3D, and HSPA8 are determined.In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, CD3D, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8 are determined.In some embodiments, when multiple biomarkers are used, the expression level of each biomarker is compared to the reference level of that biomarker. In other embodiments, when multiple biomarkers are used, an overall score is calculated based on the multiple biomarkers and compared to a reference overall score. In some embodiments, the overall score is calculated using the median Z-score method. In other embodiments, the overall score is calculated using the single sample gene set enrichment (ssGSEA) method.

[0104] In another aspect, the biomarkers provided herein are used to predict whether a subject will respond to treatment for CFS. In some embodiments, provided herein is a method of identifying a subject likely or unlikely to respond to treatment for CFS or predicting whether a subject will respond to treatment for CFS, the method comprising obtaining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8; and identifying or predicting that the subject is likely to respond to treatment for CFS if the expression level of the biomarker is higher than the reference expression level of the biomarker. In some embodiments, the biomarker is IKZF2. In some embodiments, the biomarker is IKZF3. In some embodiments, the biomarker is ABCE1. In some embodiments, the biomarker is BACH2. In some embodiments, the biomarker is CD3D. In some embodiments, the biomarker is HSPA8.

[0105] In some embodiments, the reference expression level of a biomarker is a predetermined expression level of the biomarker obtained from a public database. In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker in a healthy subject or a subject without CFS. In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker in a subject with moderate CFS. In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker determined based on a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without CFS, or a cohort of subjects with moderate CFS). In some embodiments, the reference expression level of a biomarker is the median or average value of the expression levels of the biomarker in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without CFS, or a cohort of subjects with moderate CFS). In some embodiments, the biomarker is HSPA8 or ABCE1, and the reference expression level of the biomarker is the expression level of the biomarker in a healthy subject or a subject without CFS, or in a cohort of healthy subjects or subjects without CFS.

[0106] In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of the biomarker is at least 5% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of the biomarker is at least 10% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of the biomarker is at least 20% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of the biomarker is at least 30% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of the biomarker is at least 40% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of the biomarker is at least 50% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of the biomarker is at least 60% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of the biomarker is at least 70% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of the biomarker is at least 80% higher than the reference expression level of the biomarker. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of the biomarker is at least 90% higher than the reference expression level of the biomarker.In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of a biomarker is at least 2 times the reference expression level of the biomarker. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of a biomarker is at least 3 times the reference expression level of the biomarker. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of a biomarker is at least 4 times the reference expression level of the biomarker. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of a biomarker is at least 5 times the reference expression level of the biomarker. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of a biomarker is at least 6 times the reference expression level of the biomarker. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of a biomarker is at least 7 times the reference expression level of the biomarker. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of a biomarker is at least 8 times the reference expression level of the biomarker. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of a biomarker is at least 9 times the reference expression level of the biomarker. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the expression level of a biomarker is at least 10 times the reference expression level of the biomarker. The expression level of the biomarker can be determined using any known method in the art, and exemplary methods are described in more detail in Section 5.3 below.In some embodiments, if the level is higher than a reference level (e.g., statistically significantly higher than it) when observed by a measurement assay, the level is determined to be higher than the reference level.

[0107] In certain embodiments, the method comprises using two or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises using three or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises using four or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises using five or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises using all of the biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the expression levels of IKZF2 and IKZF3 are determined. In some embodiments, the expression levels of IKZF2 and ABCE1 are determined. In some embodiments, the expression levels of IKZF2 and BACH2 are determined. In some embodiments, the expression levels of IKZF2 and CD3D are determined. In some embodiments, the expression levels of IKZF2 and HSPA8 are determined. In some embodiments, the expression levels of IKZF3 and ABCE1 are determined. In some embodiments, the expression levels of IKZF3 and BACH2 are determined. In some embodiments, the expression levels of IKZF3 and CD3D are determined. In some embodiments, the expression levels of IKZF3 and HSPA8 are determined. In some embodiments, the expression levels of ABCE1 and BACH2 are determined. In some embodiments, the expression levels of ABCE1 and CD3D are determined. In some embodiments, the expression levels of ABCE1 and HSPA8 are determined. In some embodiments, the expression levels of BACH2 and CD3D are determined. In some embodiments, the expression levels of BACH2 and HSPA8 are determined. In some embodiments, the expression levels of CD3D and HSPA8 are determined.In some embodiments, the expression levels of IKZF2, IKZF3, and ABCE1 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and BACH2 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF2, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF3, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, CD3D, and HSPA8 are determined.In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, CD3D, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8 are determined.In some embodiments, when multiple biomarkers are used, the level of each biomarker is compared to a reference level of that biomarker, and the method includes identifying or predicting that the subject is likely to respond to treatment if the level of each biomarker is higher than the reference level of that biomarker. In other embodiments, when multiple biomarkers are used, an overall score is calculated based on the multiple biomarkers, compared to a reference overall score, and the method includes identifying or predicting that the subject is likely to respond to treatment for CFS if the overall score is higher than the reference overall score. In some embodiments, the overall score is calculated using the median Z-score method. In other embodiments, the overall score is calculated using the single-sample gene set enrichment (ssGSEA) method.

[0108] In another aspect, provided herein is a selective treatment method including performing treatment for CFS on a subject identified or predicted to be likely to respond to treatment according to the methods provided herein. Specifically, in some embodiments, provided herein is a method for selectively treating a subject having or suspected of having CFS, the method including obtaining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8; identifying or predicting that the subject is likely to respond to treatment for CFS if the expression level of the biomarker is higher than the reference expression level of the biomarker; and performing treatment on the subject identified or predicted to be likely to respond to treatment.

[0109] Treatment for CFS in the methods described above includes any treatment that reduces any symptom associated with CFS. In some embodiments, the treatment includes an immunomodulatory compound.

[0110] In some specific embodiments, the treatment of CFS includes immunomodulatory drugs (IMiDs). IMiDs include a group of compounds that can be useful for the treatment of several types of human diseases, including certain cancers. As used herein, unless otherwise indicated, the term "immunomodulatory compound" can include certain organic small molecules that inhibit LPS-induced monocyte TNF-α, IL-1β, IL-12, IL-6, MIP-1α, MCP-1, GM-CSF, G-CSF, and COX-2 production. These compounds can be prepared synthetically or obtained commercially. The inflammatory cytokine TNF-α produced by macrophages and monocytes during acute inflammation causes a variety of intracellular signaling events. Without being limited by a particular theory, one of the biological effects exerted by the immunomodulatory compounds disclosed herein is the reduction of TNF-α production by myeloid cells. The immunomodulatory compounds disclosed herein can enhance the degradation of TNF-α mRNA. Furthermore, without being limited by theory, the immunomodulatory compounds disclosed herein are also potent co-stimulatory factors for T cells and can dramatically increase cell proliferation in a dose-dependent manner. The immunomodulatory compounds disclosed herein can also have a higher co-stimulatory effect on the CD8+ T cell subset than on the CD4+ T cell subset. In addition, such compounds can have anti-inflammatory properties against myeloid cell responses, but can also efficiently co-stimulate T cells to produce larger amounts of IL-2 and IFN-γ, enhancing T cell proliferation and the cytotoxic activity of CD8+ T cells. Furthermore, without being limited by a particular theory, the immunomodulatory compounds disclosed herein have both the ability to act indirectly through cytokine activation and the ability to act directly on natural killer ("NK") cells and natural killer T ("NKT") cells, increasing the ability of NK cells to produce beneficial cytokines such as, but not limited to, IFN-γ, and the ability to enhance the cytotoxic activity of NK and NKT cells. The various immunomodulatory compounds disclosed herein have one or more chiral centers and can exist as a racemic mixture of enantiomers or a mixture of diastereomers.Accordingly, the present specification also provides for the use of such compounds in stereoisomerically pure form, as well as the use of mixtures of those forms. For example, mixtures containing enantiomers of a particular immunomodulatory compound in equal or unequal amounts may be used. Such isomers may be synthesized asymmetrically or resolved using standard techniques such as chiral columns or chiral resolving agents. See, for example, Jacques, J., et al., Enantiomers, Racemates and Resolutions (Wiley-Interscience, New York, 1981); Wilen, S.H., et al., Tetrahedron 33:2725 (1977); Eliel, E.L., Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S.H., Tables of Resolving Agents and Optical Resolutions p.268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0111] In other embodiments, the treatment of CFS provided herein includes a CRBN modulator. In some embodiments, the CRBN modulator is an agent that can directly or indirectly modulate at least one of the biological activities of CRBN. In some embodiments, the CRBN modulator is an agent that can physically bind to CRBN. In other embodiments, the CRBN modulator does not directly bind to CRBN, but can act in other ways through the CRBN-mediated pathway. Cereblon (CRBN), a component of the DDB1-CUL4a-Roc1 ubiquitin ligase complex, has been identified as a target for certain immunomodulatory compounds, such as thalidomide, lenalidomide, pomalidomide, and iberdomide (Lopez-Girona et al., Leukemia volume 26, pages 2326-2335 (2012); Bjorklund et al., Leukemia. 2020; 34(4):1197-1201). It is thought that the interaction of CRBN with certain immunomodulatory compounds mediates its anti-proliferative effect in multiple myeloma (MM) cells (Lopez-Girona et al., Leukemia 2012; Zhu et al., Blood 2011, 118, Abstract 127). CRBN is encoded by a 25 kb gene on chromosome 6 consisting of 11 exons and 10 introns. Thus, through the process of alternative splicing, multiple functional proteins and variants of a protein from a single gene with different structural compositions and functional activities can potentially arise. Truncated proteins that have lost interaction domains or critically important functional amino acid residues can create non-functional or abnormal CRBN proteins, thus interfering with the function of the full-length CRBN protein and potentially reducing or altering the therapeutic activity of therapeutic compounds that act through their interaction with the full-length CRBN protein. There are at least two isoforms of the protein cereblon (CRBN), 442 and 441 amino acids in length, respectively, and CRBN is conserved from plants to humans. In humans, the CRBN gene has been identified as a candidate gene for autosomal recessive nonsyndromic mental retardation (ARNSMR).See Higgins, J. J. et al., Neurology, 2004, 63: 1927-1931. CRBN was initially characterized as a novel RGS-containing protein that interacts with the calcium-activated potassium channel protein (SLO1) in the rat brain, but was later shown to interact with the voltage-gated chloride channel (CIC-2) in the retina that has AMPK1 and DDB1. See Jo, S. et al., J. Neurochem, 2005, 94: 1212-1224; Hohberger B. et al., FEBS Lett, 2009, 583: 633-637; Angers S. et al., Nature, 2006, 443: 590-593. DDB1 was initially identified as a nucleotide excision repair protein associated with damaged DNA binding protein 2 (DDB2). Deficiency of its activity causes repair deficiency in patients with xeroderma pigmentosum complementation group E (XPE). DDB1 also appears to function as a component of numerous different DCX (DDB1-CUL4-X box) E3 ubiquitin-protein ligase complexes that mediate ubiquitination of target proteins and subsequent proteasomal degradation. CRBN has also been identified as a target for the development of therapeutic agents for diseases of the cerebral cortex. See WO 2010 / 137547 A1 pamphlet. In some embodiments, the beneficial effects of certain therapeutic compounds provided herein require binding to CRBN or one or more CRBN substrates. In some embodiments, the compounds provided herein for the treatment of CFS can induce CRBN to undergo a conformational change. In some embodiments, using the therapeutic compounds provided herein leads to a characteristic conformational change on the surface of CRBN or other modification of its properties, resulting in a characteristic phenotypic response. In certain embodiments, the CRBN modulator is an immunomodulatory compound. In other embodiments, CRBN is not an immunomodulatory compound.

[0112] In some embodiments, the treatment of CFS disclosed herein comprises an agent that depletes B cells. In some embodiments, the agent that depletes B cells is an antibody that specifically binds to an antigen of B cells. In some embodiments, the antigen of B cells is CD20, CD19, CD22, CD38, or B cell activating factor (BAFF). In some embodiments, the agent that depletes B cells is an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is rituximab, ocrelizumab, or ofatumumab. The agent that depletes B cells is an anti-CD19 antibody. In some embodiments, the anti-CD19 antibody is inebilizumab. In some embodiments, the agent that depletes B cells is an anti-BAFF antibody. In some embodiments, the anti-BAFF antibody is belimumab.

[0113] In some embodiments, the therapeutic compound in the selective treatment methods herein is formulated into a pharmaceutical composition comprising the therapeutic compound provided herein and a pharmaceutically acceptable excipient. The pharmaceutical composition comprising the therapeutic compound provided herein comprises the compound provided herein and an optional physiologically acceptable excipient (e.g., Remington, Remington’s Pharmaceutical Sciences (18 th(see also Remington, ed. 1980) and are prepared for storage in the form of an aqueous solution or in a lyophilized or other dried form by mixing. The therapeutic compounds of the present disclosure can be formulated in any form suitable for delivery to target cells / tissues, for example, as microcapsules or macroemulsions (Remington, supra; Park et al., 2005, Molecules 10:146 - 61; Malik et al., 2007, Curr. Drug. Deliv. 4:141 - 51), as sustained - release formulations (Putney and Burke, 1998, Nature Biotechnol. 16:153 - 57), or in liposomes (Maclean et al., 1997, Int. J. Oncol. 11:325 - 32; Kontermann, 2006, Curr. Opin. Mol. Ther. 8:39 - 45). The therapeutic compounds provided herein can also be encapsulated, for example, in microcapsules prepared by coacervation techniques or by interfacial polymerization, such as hydroxy - methylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed, for example, in Remington, supra. A variety of compositions and delivery systems are known and can be used with the compounds as described herein. In some embodiments, the composition can be provided as a controlled - release system or a sustained - release system. In one embodiment, a pump can be used to achieve controlled or sustained release (see, for example, Langer, supra; Sefton, 1987, Crit. Ref. Biomed. Eng. 14:201 - 40; Buchwald et al., 1980, Surgery 88:507 - 16; and Saudek et al., 1989, N. Engl. J. Med. 321:569 - 74).In another embodiment, a polymeric material can be used to achieve controlled or sustained release of the prophylactic or therapeutic agents or compositions provided herein (see, e.g., 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, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126; Levy et al., 1985, Science 228:190-92; During et al., 1989, Ann. Neurol. 25:351-56; Howard et al., 1989, J. Neurosurg. 71:105-12; U.S. Patent Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; and 5,128,326; International Publication Nos. 99 / 15154 and 99 / 20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained release formulation is inert, contains no leachable impurities, is stable upon storage, is sterile, and is biodegradable.In yet another embodiment, the controlled or sustained release system can be placed in proximity to a particular target tissue, e.g., the nasal passages or the lungs, and thus only a fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984)). Controlled release systems are discussed, for example, by Langer, 1990, Science 249:1527-33. Sustained release formulations containing the therapeutic compounds described herein can be made using any technique known to those of skill in the art (see, e.g., U.S. Patent No. 4,526,938, International Publication Nos. WO 91 / 05548 and WO 96 / 20698, Ning et al., 1996, Radiotherapy & Oncology 39:179-89; Song et al., 1995, PDA J. of Pharma. Sci. & Tech. 50:372-97; Cleek et al., 1997, Pro. Int’l. Symp. Control. Rel. Bioact. Mater. 24:853-54; and Lam et al., 1997, Proc. Int’l. Symp. Control Rel. Bioact. Mater. 24:759-60). A variety of delivery systems are known and can be used for administration of the therapeutic compounds provided herein.

[0114] In yet another aspect of the present disclosure, whether a subject responds to a therapeutic compound can be determined by changes in the expression levels of the biomarkers provided herein. For example, a decrease in the expression level of a biomarker provided herein with treatment indicates that the subject is responsive to the treatment; while no change in the expression level of the biomarker indicates that the subject is non-responsive. The degree of change in the biomarker can also be used to indicate the degree of response. Next, treatment and dosage adjustments can be planned accordingly. Thus, in some embodiments, provided herein is a method of determining or monitoring the effectiveness of treatment in a subject having CFS, the method comprising obtaining a first expression level of a biomarker in a first sample from the subject, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8; administering treatment to the subject; obtaining a second expression level of the biomarker in a second sample obtained from the subject after administering treatment to the subject; and determining the effectiveness of the treatment based on a comparison of the first expression level and the second expression level. In some embodiments, the method comprises determining that the treatment is effective if the second expression level is lower than the first expression level. In some embodiments, the method comprises determining that the treatment is not effective if the second expression level is not lower than the first expression level. In some embodiments, the method comprises determining or adjusting (e.g., increasing) the dosage of the treatment, or administering a different treatment to the subject, if the second expression level is not lower than the first expression level

[0115] In some embodiments, the method includes determining that the treatment is effective if the second expression level is at least 10% lower than the first expression level. In some embodiments, the method includes determining that the treatment is effective if the second expression level is at least 20% lower than the first expression level. In some embodiments, the method includes determining that the treatment is effective if the second expression level is at least 30% lower than the first expression level. In some embodiments, the method includes determining that the treatment is effective if the second expression level is at least 40% lower than the first expression level. In some embodiments, the method includes determining that the treatment is effective if the second expression level is at least 50% lower than the first expression level. In some embodiments, the method includes determining that the treatment is effective if the second expression level is at least 60% lower than the first expression level. In some embodiments, the method includes determining that the treatment is effective if the second expression level is at least 70% lower than the first expression level. In some embodiments, the method includes determining that the treatment is effective if the second expression level is at least 80% lower than the first expression level. In some embodiments, the method includes determining that the treatment is effective if the second expression level is at least 90% lower than the first expression level. In some embodiments, the treatment includes an immunomodulatory drug (IMiD). In other embodiments, the treatment includes a CRBN modulator. In some embodiments, the treatment includes an agent that depletes B cells (e.g., an anti-CD20 antibody, e.g., rituximab).

[0116] Based on the comparison between the first expression level and the second expression level, in one or more subsequent treatment cycles, different treatments or different dosage regimens may be implemented for the subject. In some embodiments, the biomarker is IKZF2. In some embodiments, the biomarker is IKZF3. In some embodiments, the biomarker is ABCE1. In some embodiments, the biomarker is BACH2. In some embodiments, the biomarker is CD3D. In some embodiments, the biomarker is HSPA8. The expression level of the biomarker can be determined using any known method in the art, and exemplary methods are described in more detail in Section 5.3 below. In some embodiments, if the level is higher than the second level (e.g., statistically significantly higher than it) when observed by a measurement assay, the level is determined to be higher than the second level. In some embodiments, if the level is lower than the second level (e.g., statistically significantly) when observed by a measurement assay, the level is determined to be lower than the second level.

[0117] In certain embodiments, the method comprises using two or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises using three or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises using four or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises using five or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises using all of the biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the expression levels of IKZF2 and IKZF3 are determined. In some embodiments, the expression levels of IKZF2 and ABCE1 are determined. In some embodiments, the expression levels of IKZF2 and BACH2 are determined. In some embodiments, the expression levels of IKZF2 and CD3D are determined. In some embodiments, the expression levels of IKZF2 and HSPA8 are determined. In some embodiments, the expression levels of IKZF3 and ABCE1 are determined. In some embodiments, the expression levels of IKZF3 and BACH2 are determined. In some embodiments, the expression levels of IKZF3 and CD3D are determined. In some embodiments, the expression levels of IKZF3 and HSPA8 are determined. In some embodiments, the expression levels of ABCE1 and BACH2 are determined. In some embodiments, the expression levels of ABCE1 and CD3D are determined. In some embodiments, the expression levels of ABCE1 and HSPA8 are determined. In some embodiments, the expression levels of BACH2 and CD3D are determined. In some embodiments, the expression levels of BACH2 and HSPA8 are determined. In some embodiments, the expression levels of CD3D and HSPA8 are determined.In some embodiments, the expression levels of IKZF2, IKZF3, and ABCE1 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and BACH2 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF2, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF3, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, CD3D, and HSPA8 are determined.In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, CD3D, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8 are determined.In some embodiments, when multiple biomarkers are used, the second expression level of each biomarker is compared to the first expression level of that biomarker, and the method includes determining that the treatment is effective if the second expression level of each biomarker is lower than the first expression level of that biomarker. In other embodiments, when multiple biomarkers are used, an aggregate score is calculated based on the multiple biomarkers and compared to a reference aggregate score. In some embodiments, the aggregate score is calculated using the median Z-score method. In other embodiments, the aggregate score is calculated using the single sample gene set enrichment (ssGSEA) method.

[0118] The present disclosure also includes the use of the biomarkers provided herein for screening the effectiveness of a compound in treating CFS. Thus, in some embodiments, provided herein is a method for screening the effectiveness of a compound in treating CFS, the method comprising obtaining a first expression level of a biomarker in a sample, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8; administering the compound to the sample; obtaining a second expression level of the biomarker in the sample after administering the compound to the sample; comparing the first expression level with the second expression level; and selecting the compound if the second expression level is lower than the first expression level. In some embodiments, the method includes selecting the compound if the second expression level is at least 10% lower than the first expression level. In some embodiments, the method includes selecting the compound if the second expression level is at least 20% lower than the first expression level. In some embodiments, the method includes selecting the compound if the second expression level is at least 30% lower than the first expression level. In some embodiments, the method includes selecting the compound if the second expression level is at least 40% lower than the first expression level. In some embodiments, the method includes selecting the compound if the second expression level is at least 50% lower than the first expression level. In some embodiments, the method includes selecting the compound if the second expression level is at least 60% lower than the first expression level. In some embodiments, the method includes selecting the compound if the second expression level is at least 70% lower than the first expression level. In some embodiments, the method includes selecting the compound if the second expression level is at least 80% lower than the first expression level. In some embodiments, the method includes selecting the compound if the second expression level is at least 10% lower than the first expression level.In some embodiments, the method comprises selecting the compound if the second expression level is at least 90% lower than the first expression level. In some embodiments, the therapeutic compound is an immunomodulatory drug (IMiD). In other embodiments, the therapeutic compound is a CRBN modulator or a compound having the ability to bind to CRBN and / or induce a conformational change thereof. In some embodiments, the therapeutic compound is an agent that depletes B cells (e.g., an anti-CD20 antibody, e.g., rituximab). In some embodiments, the biomarker is IKZF2. In some embodiments, the biomarker is IKZF3. In some embodiments, the biomarker is ABCE1. In some embodiments, the biomarker is BACH2. In some embodiments, the biomarker is CD3D. In some embodiments, the biomarker is HSPA8. In certain embodiments, the method comprises using two or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises using three or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises using four or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises using five or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the method comprises using all of the biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the expression levels of IKZF2 and IKZF3 are determined. In some embodiments, the expression levels of IKZF2 and ABCE1 are determined. In some embodiments, the expression levels of IKZF2 and BACH2 are determined. In some embodiments, the expression levels of IKZF2 and CD3D are determined.In some embodiments, the expression levels of IKZF2 and HSPA8 are determined. In some embodiments, the expression levels of IKZF3 and ABCE1 are determined. In some embodiments, the expression levels of IKZF3 and BACH2 are determined. In some embodiments, the expression levels of IKZF3 and CD3D are determined. In some embodiments, the expression levels of IKZF3 and HSPA8 are determined. In some embodiments, the expression levels of ABCE1 and BACH2 are determined. In some embodiments, the expression levels of ABCE1 and CD3D are determined. In some embodiments, the expression levels of ABCE1 and HSPA8 are determined. In some embodiments, the expression levels of BACH2 and CD3D are determined. In some embodiments, the expression levels of BACH2 and HSPA8 are determined. In some embodiments, the expression levels of CD3D and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3 and ABCE1 are determined. In some embodiments, the expression levels of IKZF2, IKZF3 and BACH2 are determined. In some embodiments, the expression levels of IKZF2, IKZF3 and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3 and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1 and BACH2 are determined. In some embodiments, the expression levels of IKZF2, ABCE1 and CD3D are determined. In some embodiments, the expression levels of IKZF2, ABCE1 and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, BACH2 and CD3D are determined. In some embodiments, the expression levels of IKZF2, BACH2 and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, CD3D and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1 and BACH2 are determined. In some embodiments, the expression levels of IKZF3, ABCE1 and CD3D are determined. In some embodiments, the expression levels of IKZF3, ABCE1 and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, BACH2 and CD3D are determined.In some embodiments, the expression levels of IKZF3, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and BACH2 are determined.In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, CD3D, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, when multiple biomarkers are used, the second expression level of each biomarker is compared to the first expression level of that biomarker, and the method includes selecting the compound if the second expression level of each biomarker is lower than the first expression level of that biomarker. In other embodiments, when multiple biomarkers are used, an overall score is calculated based on the multiple biomarkers, compared to a reference overall score, and the compound is selected if the overall score is higher than the reference overall score. In some embodiments, the method includes obtaining a first overall score based on the first expression level of the biomarker and a second overall score based on the second expression level of the biomarker, and comparing the first overall score to the second overall score. In some embodiments, the overall score is calculated using the median Z-score method. In other embodiments, the overall score is calculated using the single-sample gene set enrichment (ssGSEA) method.

[0119] In another aspect, the present disclosure also provides methods and kits based on the unexpected finding that there is a correlation between CSF and certain organic acids. As shown in the Examples section, urinary organic acid profiling reveals differences between CFS and normal cohorts. More specifically, in the profile of urinary organic acids, 23 organic acids - hippuric acid, 3-hydroxypropionic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, α-keto-β-methylvaleric acid, α-hydroxybutyric acid, glycolic acid, pyruvic acid, citramalic acid, lactic acid, α-ketoadipic acid, citric acid, malic acid, kynurenic acid, xanthurenic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanillylmandelic acid, methylmalonic acid, glyceric acid - showed statistically significant (p < 0.05) differences between CFS patients and normal subjects (see FIGS. 6A - 6F). Among these, 12 organic acids had a median fold change > 1.4 (decrease in CFS) and p < 0.05, and they were xanthurenic acid, glycolic acid, pyruvic acid, hippuric acid, isovalerylglycine, kynurenic acid, 3-hydroxyisovaleric acid, vanillylmandelic acid, pyroglutamic acid, 3-hydroxypropionic acid, glyceric acid, and α-ketoadipic acid. In addition, there was a significant difference in vanillylmandelic acid levels between bedridden CFS subjects and non-bedridden subjects. Thus, in the various methods described above, one or more of these organic acids can be used in combination with IKZF2, IKZF3, ABCE1, BACH2, CD3D, and / or HSPA8.In other embodiments, one or more of these organic acids, independently of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and / or HSPA8, can be used in various ways, as described in embodiments 56-94 below, for example, to identify a subject having CFS or to confirm CFS in a subject, to determine the severity of CFS in a subject, to identify a subject likely or unlikely to respond to treatment of CFS or to predict whether a subject will respond to treatment of CFS, to selectively treat a subject having or suspected of having CFS in treatment, and / or to determine or monitor the effectiveness of treatment in a subject having CFS and to screen for the effectiveness of a compound in treating CFS.

[0120] In some embodiments, provided herein is a method of identifying a subject having chronic fatigue syndrome (CFS) or a method of confirming CFS in a subject, the method comprising obtaining the level of urinary organic acids in a sample from the subject, wherein the urinary organic acids are selected from the group consisting of hippuric acid, 3-hydroxypropionic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, α-keto-β-methylvaleric acid, α-hydroxybutyric acid, glycolic acid, pyruvic acid, citramalic acid, lactic acid, α-ketoadipic acid, citric acid, malic acid, kynurenic acid, xanthurenic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanilmandelic acid, methylmalonic acid, and glyceric acid; and identifying or confirming that the subject has CFS if the level of urinary organic acids in the sample is different from the reference level of urinary organic acids. In some embodiments, the urinary organic acids are selected from the group consisting of xanthurenic acid, glycolic acid, pyruvic acid, hippuric acid, isovalerylglycine, kynurenic acid, 3-hydroxyisovaleric acid, vanilmandelic acid, pyroglutamic acid, 3-hydroxypropionic acid, glyceric acid, and α-ketoadipic acid. In some embodiments, the organic acid is hippuric acid. In some embodiments, the organic acid is 3-hydroxypropionic acid. In some embodiments, the organic acid is α-ketoisocaproic acid. In some embodiments, the organic acid is α-keto-β-methylvaleric acid. In some embodiments, the organic acid is α-hydroxybutyric acid. In some embodiments, the organic acid is glycolic acid. In some embodiments, the organic acid is pyruvic acid. In some embodiments, the organic acid is glycolic acid. In some embodiments, the organic acid is citramalic acid. In some embodiments, the organic acid is lactic acid. In some embodiments, the organic acid is α-ketoadipic acid. In some embodiments, the organic acid is citric acid. In some embodiments, the organic acid is malic acid. In some embodiments, the organic acid is kynurenic acid. In some embodiments, the organic acid is xanthurenic acid.In some embodiments, the organic acid is isovalerylglycine. In some embodiments, the organic acid is 3-hydroxyisovaleric acid. In some embodiments, the organic acid is isocitric acid. In some embodiments, the organic acid is cis-aconitic acid. In some embodiments, the organic acid is pyroglutamic acid. In some embodiments, the organic acid is vanilmandelic acid. In some embodiments, the organic acid is methylmalonic acid. In some embodiments, the organic acid is glyceric acid. In some embodiments, the reference level of the organic acid is a predetermined level of the organic acid obtained from a public database. In some embodiments, the reference level of the organic acid is the level of the organic acid in a healthy subject or a subject without CFS. In some embodiments, the reference level of the organic acid is the level of the organic acid in a subject with moderate CFS. In some embodiments, the reference level of the organic acid is the level of the organic acid determined based on a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without CFS, or a cohort of subjects with moderate CFS). In some embodiments, the reference level of the organic acid is the median or average value of the levels of the organic acid in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without CFS, or a cohort of subjects with moderate CFS). In some embodiments, the method includes identifying or confirming that the subject has CFS if the level of the organic acid in the urine is lower than the reference level of the organic acid in the urine. In some embodiments, the method includes identifying that the subject has CFS if the level of the organic acid is at least 5% lower than the reference level of the organic acid. In some embodiments, the method includes identifying that the subject has CFS if the level of the organic acid is at least 10% lower than the reference level of the organic acid. In some embodiments, the method includes identifying that the subject has CFS if the level of the organic acid is at least 20% lower than the reference level of the organic acid. In some embodiments, the method includes identifying that the subject has CFS if the level of the organic acid is at least 30% lower than the reference level of the organic acid.In some embodiments, the method includes identifying that the subject has CFS if the level of the organic acid is at least 40% lower than the reference level of the organic acid. In some embodiments, the method includes identifying that the subject has CFS if the level of the organic acid is at least 50% lower than the reference level of the organic acid. In some embodiments, the method includes identifying that the subject has CFS if the level of the organic acid is at least 60% lower than the reference level of the organic acid. In some embodiments, the method includes identifying that the subject has CFS if the level of the organic acid is at least 70% lower than the reference level of the organic acid. In some embodiments, the method includes identifying that the subject has CFS if the level of the organic acid is at least 80% lower than the reference level of the organic acid. In some embodiments, the method includes identifying that the subject has CFS if the level of the organic acid is at least 90% lower than the reference level of the organic acid. In some embodiments, the method includes identifying that the subject has CFS if the level of the organic acid is at least one half as compared to the reference level of the organic acid. In some embodiments, the method includes identifying that the subject has CFS if the level of the organic acid is at least one third as compared to the reference level of the organic acid. In some embodiments, the method includes identifying that the subject has CFS if the level of the organic acid is at least one fourth as compared to the reference level of the organic acid. In some embodiments, the method includes identifying that the subject has CFS if the level of the organic acid is at least one fifth as compared to the reference level of the organic acid. In some embodiments, the method includes identifying that the subject has CFS if the level of the organic acid is at least one sixth as compared to the reference level of the organic acid. In some embodiments, the method includes identifying that the subject has CFS if the level of the organic acid is at least one seventh as compared to the reference level of the organic acid. In some embodiments, the method includes identifying that the subject has CFS if the level of the organic acid is at least one eighth as compared to the reference level of the organic acid.In some embodiments, the method includes identifying that a subject has CFS if the level of organic acids is at least one-ninth of the reference level of organic acids. In some embodiments, the method includes identifying that a subject has CFS if the level of organic acids is at least one-tenth of the reference level of organic acids. The level of organic acids can be determined using any known method in the art, and exemplary methods are described in more detail in Section 5.3 below. In some embodiments, the level of organic acids is determined to be lower than the reference level if the level is lower (e.g., statistically significantly) than the reference level when observed by a measurement assay. In certain embodiments, the method includes identifying a subject having CFS using two or more organic acids selected from the group consisting of hippuric acid, 3-hydroxypropionic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, α-keto-β-methylvaleric acid, α-hydroxybutyric acid, glycolic acid, pyruvic acid, citramalic acid, lactic acid, α-ketoadipic acid, citric acid, malic acid, kynurenic acid, xanthurenic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanilmandelic acid, methylmalonic acid, and glyceric acid. In some embodiments, the method includes identifying or confirming that a subject has CFS if the level of urinary organic acids in a sample is lower than the reference level of urinary organic acids. In some embodiments, CFS is associated with an autoimmune disease or an infectious disease.

[0121] In some embodiments, provided herein is a method for determining the severity of CFS in a subject, the method comprising: obtaining the levels of urinary organic acids in a sample from the subject, wherein the urinary organic acids are selected from the group consisting of hippuric acid, 3-hydroxypropionic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, α-keto-β-methylvaleric acid, α-hydroxybutyric acid, glycolic acid, pyruvic acid, citramalic acid, lactic acid, α-ketoadipic acid, citric acid, malic acid, kynurenic acid, xanthurenic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanilmandelic acid, methylmalonic acid, and glyceric acid; comparing the levels of urinary organic acids in the sample to a reference level of urinary organic acids; and determining the severity of CFS in the subject based on the comparison of the levels of urinary organic acids in the sample to the reference level of urinary organic acids. In some embodiments, the urinary organic acids are selected from the group consisting of xanthurenic acid, glycolic acid, pyruvic acid, hippuric acid, isovalerylglycine, kynurenic acid, 3-hydroxyisovaleric acid, vanilmandelic acid, pyroglutamic acid, 3-hydroxypropionic acid, glyceric acid, and α-ketoadipic acid. In some embodiments, the method further comprises comparing the levels of the organic acids to a reference level of the organic acids. In some embodiments, a low level of the organic acids indicates a high severity of CFS.In yet other embodiments, provided herein is a method of monitoring the progression of CFS in a subject, the method comprising obtaining the level of an organic acid in a sample from the subject, wherein the organic acid is selected from the group consisting of hippuric acid, 3-hydroxypropionic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, α-keto-β-methylvaleric acid, α-hydroxybutyric acid, glycolic acid, pyruvic acid, citramalic acid, lactic acid, α-ketoadipic acid, citric acid, malic acid, kynurenic acid, xanthurenic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanilmandelic acid, methylmalonic acid, and glyceric acid, and determining the progression of CFS based on the level of the biomarker. In some embodiments, the organic acid is hippuric acid. In some embodiments, the organic acid is 3-hydroxypropionic acid. In some embodiments, the organic acid is α-ketoisocaproic acid. In some embodiments, the organic acid is α-keto-β-methylvaleric acid. In some embodiments, the organic acid is α-hydroxybutyric acid. In some embodiments, the organic acid is glycolic acid. In some embodiments, the organic acid is pyruvic acid. In some embodiments, the organic acid is glycolic acid. In some embodiments, the organic acid is citramalic acid. In some embodiments, the organic acid is lactic acid. In some embodiments, the organic acid is α-ketoadipic acid. In some embodiments, the organic acid is citric acid. In some embodiments, the organic acid is malic acid. In some embodiments, the organic acid is kynurenic acid. In some embodiments, the organic acid is xanthurenic acid. In some embodiments, the organic acid is isovalerylglycine. In some embodiments, the organic acid is 3-hydroxyisovaleric acid. In some embodiments, the organic acid is isocitric acid. In some embodiments, the organic acid is cis-aconitic acid. In some embodiments, the organic acid is pyroglutamic acid. In some embodiments, the organic acid is vanilmandelic acid.In some embodiments, the organic acid is methylmalonic acid. In some embodiments, the organic acid is glyceric acid. In certain embodiments, the method comprises identifying a subject having CFS using two or more organic acids selected from the group consisting of hippuric acid, 3-hydroxypropionic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, α-keto-β-methylvaleric acid, α-hydroxybutyric acid, glycolic acid, pyruvic acid, citramalic acid, lactic acid, α-ketoadipic acid, citric acid, malic acid, kynurenic acid, xanthurenic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanilmandelic acid, methylmalonic acid, and glyceric acid. In some embodiments, the reference level of the organic acid is a predetermined level of the organic acid obtained from a public database. In some embodiments, the reference level of the organic acid is the level of the organic acid in a healthy subject or a subject without CFS. In some embodiments, the reference level of the organic acid is the level of the organic acid in a subject having moderate CFS. In some embodiments, the reference level of the organic acid is the level of the organic acid determined based on a cohort of healthy subjects. In some embodiments, the reference level of the organic acid is the level of the organic acid in a subject in which the severity of CFS has been determined and is known. In some embodiments, the reference level of the organic acid is the median or mean value of the level of the organic acid in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects having moderate CFS, or a cohort of subjects in which the severity of CFS has been determined and is known). In other embodiments, the reference level of the organic acid is the level of the organic acid measured at different time points while being the same subject. The level of the organic acid can be determined using any known method in the art, and exemplary methods are described in more detail in Section 5.3 below. In some embodiments, when the level is higher (e.g., statistically significantly higher) than the reference level as observed by a measurement assay, the level is determined to be higher than the reference level.In some embodiments, when the level is lower (e.g., statistically significantly) than the reference level as observed by a measurement assay, the level is determined to be lower than the reference level. In certain embodiments, the method includes identifying a subject having CFS using two or more organic acids selected from the group consisting of hippuric acid, 3-hydroxypropionic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, α-keto-β-methylvaleric acid, α-hydroxybutyric acid, glycolic acid, pyruvic acid, citramalic acid, lactic acid, α-ketoadipic acid, citric acid, malic acid, kynurenic acid, xanthurenic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanilmandelic acid, methylmalonic acid, and glyceric acid. In some embodiments, the CFS is associated with an autoimmune disease or an infectious disease.

[0122] In some embodiments, provided herein is a method of identifying a subject who is likely or unlikely to respond to treatment of CFS, or predicting whether a subject will respond to treatment of CFS, the method comprising obtaining levels of urinary organic acids in a sample from the subject, wherein the urinary organic acids are selected from the group consisting of hippuric acid, 3-hydroxypropionic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, α-keto-β-methylvaleric acid, α-hydroxybutyric acid, glycolic acid, pyruvic acid, citramalic acid, lactic acid, α-ketoadipic acid, citric acid, malic acid, kynurenic acid, xanthurenic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanilmandelic acid, methylmalonic acid, and glyceric acid; and identifying or predicting that the subject is likely to respond to treatment of CFS if the levels of urinary organic acids in the sample differ from a reference level of urinary organic acids. In some embodiments, the urinary organic acids are selected from the group consisting of xanthurenic acid, glycolic acid, pyruvic acid, hippuric acid, isovalerylglycine, kynurenic acid, 3-hydroxyisovaleric acid, vanilmandelic acid, pyroglutamic acid, 3-hydroxypropionic acid, glyceric acid, and α-ketoadipic acid. In some embodiments, the organic acid is hippuric acid. In some embodiments, the organic acid is 3-hydroxypropionic acid. In some embodiments, the organic acid is α-ketoisocaproic acid. In some embodiments, the organic acid is α-keto-β-methylvaleric acid. In some embodiments, the organic acid is α-hydroxybutyric acid. In some embodiments, the organic acid is glycolic acid. In some embodiments, the organic acid is pyruvic acid. In some embodiments, the organic acid is glycolic acid. In some embodiments, the organic acid is citramalic acid. In some embodiments, the organic acid is lactic acid. In some embodiments, the organic acid is α-ketoadipic acid. In some embodiments, the organic acid is citric acid. In some embodiments, the organic acid is malic acid. In some embodiments, the organic acid is kynurenic acid.In some embodiments, the organic acid is xanthurenic acid. In some embodiments, the organic acid is isovalerylglycine. In some embodiments, the organic acid is 3-hydroxyisovaleric acid. In some embodiments, the organic acid is isocitric acid. In some embodiments, the organic acid is cis-aconitic acid. In some embodiments, the organic acid is pyroglutamic acid. In some embodiments, the organic acid is vanilmandelic acid. In some embodiments, the organic acid is methylmalonic acid. In some embodiments, the organic acid is glyceric acid. In some embodiments, the reference level of the organic acid is a predetermined level of the organic acid obtained from a public database. In some embodiments, the reference level of the organic acid is the level of the organic acid in a healthy subject or a subject without CFS. In some embodiments, the reference level of the organic acid is the level of the organic acid in a subject with moderate CFS. In some embodiments, the reference level of the organic acid is the level of the organic acid determined based on a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without CFS, or a cohort of subjects with moderate CFS). In some embodiments, the reference level of the organic acid is the median or average value of the levels of the organic acid in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without CFS, or a cohort of subjects with moderate CFS). In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is at least 5% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is at least 10% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is at least 20% lower than the reference level of the organic acid.In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS when the level of the organic acid is at least 30% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS when the level of the organic acid is at least 40% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS when the level of the organic acid is at least 50% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS when the level of the organic acid is at least 60% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS when the level of the organic acid is at least 70% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS when the level of the organic acid is at least 80% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS when the level of the organic acid is at least 90% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS when the level of the organic acid is at least one half compared to the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS when the level of the organic acid is at least one third compared to the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS when the level of the organic acid is at least one fourth compared to the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS when the level of the organic acid is at least one fifth compared to the reference level of the organic acid.In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of organic acid is at least one-sixth compared to the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of organic acid is at least one-seventh compared to the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of organic acid is at least one-eighth compared to the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of organic acid is at least one-ninth compared to the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of organic acid is at least one-tenth compared to the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of urinary organic acid is lower than the reference level of urinary organic acid. In some embodiments, the method includes administering treatment to a subject identified or predicted to be likely to respond to treatment. In certain embodiments, the method includes identifying a subject having CFS using two or more organic acids selected from the group consisting of hippuric acid, 3-hydroxypropionic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, α-keto-β-methylvaleric acid, α-hydroxybutyric acid, glycolic acid, pyruvic acid, citramalic acid, lactic acid, α-ketoadipic acid, citric acid, malic acid, kynurenic acid, xanthurenic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanilmandelic acid, methylmalonic acid, and glyceric acid. In some embodiments, the CFS is associated with an autoimmune disease or an infectious disease.

[0123] In some embodiments, provided herein is a method for selectively treating a subject having or suspected of having CFS, the method comprising obtaining levels of urinary organic acids in a sample from the subject, wherein the urinary organic acids are selected from the group consisting of hippuric acid, 3-hydroxypropionic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, α-keto-β-methylvaleric acid, α-hydroxybutyric acid, glycolic acid, pyruvic acid, citramalic acid, lactic acid, α-ketoadipic acid, citric acid, malic acid, kynurenic acid, xanthurenic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanilmandelic acid, methylmalonic acid, and glyceric acid; identifying or predicting that the subject is likely to respond to treatment for CFS if the level of urinary organic acids in the sample is different from a reference level of urinary organic acids; and administering treatment to a subject identified or predicted to be likely to respond to treatment. In some embodiments, the urinary organic acids are selected from the group consisting of xanthurenic acid, glycolic acid, pyruvic acid, hippuric acid, isovalerylglycine, kynurenic acid, 3-hydroxyisovaleric acid, vanilmandelic acid, pyroglutamic acid, 3-hydroxypropionic acid, glyceric acid, and α-ketoadipic acid. In some embodiments, the organic acid is hippuric acid. In some embodiments, the organic acid is 3-hydroxypropionic acid. In some embodiments, the organic acid is α-ketoisocaproic acid. In some embodiments, the organic acid is α-keto-β-methylvaleric acid. In some embodiments, the organic acid is α-hydroxybutyric acid. In some embodiments, the organic acid is glycolic acid. In some embodiments, the organic acid is pyruvic acid. In some embodiments, the organic acid is glycolic acid. In some embodiments, the organic acid is citramalic acid. In some embodiments, the organic acid is lactic acid. In some embodiments, the organic acid is α-ketoadipic acid. In some embodiments, the organic acid is citric acid. In some embodiments, the organic acid is malic acid. In some embodiments, the organic acid is kynurenic acid.In some embodiments, the organic acid is xanthurenic acid. In some embodiments, the organic acid is isovalerylglycine. In some embodiments, the organic acid is 3-hydroxyisovaleric acid. In some embodiments, the organic acid is isocitric acid. In some embodiments, the organic acid is cis-aconitic acid. In some embodiments, the organic acid is pyroglutamic acid. In some embodiments, the organic acid is vanilmandelic acid. In some embodiments, the organic acid is methylmalonic acid. In some embodiments, the organic acid is glyceric acid. In some embodiments, the reference level of the organic acid is a predetermined level of the organic acid obtained from a public database. In some embodiments, the reference level of the organic acid is the level of the organic acid in a healthy subject or a subject without CFS. In some embodiments, the reference level of the organic acid is the level of the organic acid in a subject with moderate CFS. In some embodiments, the reference level of the organic acid is the level of the organic acid determined based on a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without CFS, or a cohort of subjects with moderate CFS). In some embodiments, the reference level of the organic acid is the median or average value of the level of the organic acid in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without CFS, or a cohort of subjects with moderate CFS). In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is at least 5% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is at least 10% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is at least 20% lower than the reference level of the organic acid.In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is at least 30% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is at least 40% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is at least 50% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is at least 60% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is at least 70% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is at least 80% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is at least 90% lower than the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is at least one half compared to the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is at least one third compared to the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is at least one fourth compared to the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of the organic acid is at least one fifth compared to the reference level of the organic acid.In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of organic acid is at least one-sixth of the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of organic acid is at least one-seventh of the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of organic acid is at least one-eighth of the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of organic acid is at least one-ninth of the reference level of the organic acid. In some embodiments, the method includes identifying or predicting that a subject is likely to respond to treatment for CFS if the level of organic acid is at least one-tenth of the reference level of the organic acid. In certain embodiments, the method includes identifying a subject having CFS using two or more organic acids selected from the group consisting of hippuric acid, 3-hydroxypropionic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, α-keto-β-methylvaleric acid, α-hydroxybutyric acid, glycolic acid, pyruvic acid, citramalic acid, lactic acid, α-ketoadipic acid, citric acid, malic acid, kynurenic acid, xanthurenic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanilmandelic acid, methylmalonic acid, and glyceric acid. In some embodiments, CFS is associated with an autoimmune disease or an infectious disease.

[0124] In some embodiments, provided herein is a method of determining or monitoring the effectiveness of treatment in a subject having CFS, comprising obtaining a first level of urinary organic acids in a first sample from the subject prior to administering treatment to the subject, wherein the urinary organic acids are selected from the group consisting of hippuric acid, 3-hydroxypropionic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, α-keto-β-methylvaleric acid, α-hydroxybutyric acid, glycolic acid, pyruvic acid, citramalic acid, lactic acid, α-ketoadipic acid, citric acid, malic acid, kynurenic acid, xanthurenic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanilmandelic acid, methylmalonic acid, and glyceric acid; administering treatment to the subject, and obtaining a second level of urinary organic acids in a second sample obtained from the subject after administering treatment to the subject; and determining the effectiveness of treatment based on a comparison of the first level and the second level. In some embodiments, the urinary organic acids are selected from the group consisting of xanthurenic acid, glycolic acid, pyruvic acid, hippuric acid, isovalerylglycine, kynurenic acid, 3-hydroxyisovaleric acid, vanilmandelic acid, pyroglutamic acid, 3-hydroxypropionic acid, glyceric acid, and α-ketoadipic acid. In some embodiments, the urinary organic acids are selected from the group consisting of xanthurenic acid, glycolic acid, pyruvic acid, hippuric acid, isovalerylglycine, kynurenic acid, 3-hydroxyisovaleric acid, vanilmandelic acid, pyroglutamic acid, 3-hydroxypropionic acid, glyceric acid, and α-ketoadipic acid. In some embodiments, the organic acid is hippuric acid. In some embodiments, the organic acid is 3-hydroxypropionic acid. In some embodiments, the organic acid is α-ketoisocaproic acid. In some embodiments, the organic acid is α-keto-β-methylvaleric acid. In some embodiments, the organic acid is α-hydroxybutyric acid. In some embodiments, the organic acid is glycolic acid. In some embodiments, the organic acid is pyruvic acid. In some embodiments, the organic acid is glycolic acid.In some embodiments, the organic acid is citramalic acid. In some embodiments, the organic acid is lactic acid. In some embodiments, the organic acid is α-ketoadipic acid. In some embodiments, the organic acid is citric acid. In some embodiments, the organic acid is malic acid. In some embodiments, the organic acid is quinic acid. In some embodiments, the organic acid is xanthurenic acid. In some embodiments, the organic acid is isovalerylglycine. In some embodiments, the organic acid is 3-hydroxyisovaleric acid. In some embodiments, the organic acid is isocitric acid. In some embodiments, the organic acid is cis-aconitic acid. In some embodiments, the organic acid is pyroglutamic acid. In some embodiments, the organic acid is vanilmandelic acid. In some embodiments, the organic acid is methylmalonic acid. In some embodiments, the organic acid is glyceric acid. In some embodiments, the method includes determining that the treatment is effective when the second level is higher than the first level. In some embodiments, the method includes determining that the treatment is effective when the second level is at least 10% higher than the first level. In some embodiments, the method includes determining that the treatment is effective when the second level is at least 20% higher than the first level. In some embodiments, the method includes determining that the treatment is effective when the second level is at least 30% higher than the first level. In some embodiments, the method includes determining that the treatment is effective when the second level is at least 40% higher than the first level. In some embodiments, the method includes determining that the treatment is effective when the second level is at least 50% higher than the first level. In some embodiments, the method includes determining that the treatment is effective when the second level is at least 60% higher than the first level. In some embodiments, the method includes determining that the treatment is effective when the second level is at least 70% higher than the first level. In some embodiments, the method includes determining that the treatment is effective when the second level is at least 80% higher than the first level.In some embodiments, the method includes determining that the treatment is effective if the second level is at least 90% higher than the first level. In some embodiments, the method includes determining or adjusting a dosage of treatment for a subject. In some embodiments, the method includes determining the levels of two or more urinary organic acids and comparing each level of the urinary organic acids to its respective reference level. In some embodiments, the reference level of the organic acid is a predetermined level of the organic acid from a public database. In some embodiments, the reference level is the level of the organic acid in a healthy subject or a subject without CFS. In some embodiments, the reference level of the organic acid is the level of the organic acid in a subject with moderate CFS. In some embodiments, the reference level of the organic acid is the level of the organic acid determined based on a cohort of healthy subjects. In some embodiments, the reference level of the organic acid is the level of the organic acid in a first sample obtained from the subject. In some embodiments, the reference level of the organic acid is the median or average value of the levels of the organic acid in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects with moderate CFS, or a cohort of first samples obtained from the subject). In some embodiments, CFS is associated with an autoimmune disease or an infectious disease.

[0125] In some embodiments, provided herein is a method for screening the effectiveness of a compound in treating CFS, comprising obtaining a first level of urinary organic acids in a sample, wherein (i) the urinary organic acids are selected from the group consisting of hippuric acid, 3-hydroxypropionic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, α-keto-β-methylvaleric acid, α-hydroxybutyric acid, glycolic acid, pyruvic acid, citramalic acid, lactic acid, α-ketoadipic acid, citric acid, malic acid, kynurenic acid, xanthurenic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanilmandelic acid, methylmalonic acid, and glyceric acid; (ii) xanthurenic acid, glycolic acid, pyruvic acid, hippuric acid, isovalerylglycine, kynurenic acid, 3-hydroxyisovaleric acid, vanilmandelic acid, pyroglutamic acid, 3-hydroxypropionic acid, glyceric acid, and α-ketoadipic acid; and / or (iii) the urinary organic acid is vanilmandelic acid; administering the compound to the sample; obtaining a second level of urinary organic acids in the sample after administering the compound to the sample; comparing the first level with the second level; and selecting the compound if the second level is different from the first level, wherein the compound is selected if the second level is higher than the first level. In some embodiments, the organic acid is hippuric acid. In some embodiments, the organic acid is 3-hydroxypropionic acid. In some embodiments, the organic acid is α-ketoisocaproic acid. In some embodiments, the organic acid is α-keto-β-methylvaleric acid. In some embodiments, the organic acid is α-hydroxybutyric acid. In some embodiments, the organic acid is glycolic acid. In some embodiments, the organic acid is pyruvic acid. In some embodiments, the organic acid is glycolic acid. In some embodiments, the organic acid is citramalic acid. In some embodiments, the organic acid is lactic acid. In some embodiments, the organic acid is α-ketoadipic acid. In some embodiments, the organic acid is citric acid.In some embodiments, the organic acid is malic acid. In some embodiments, the organic acid is quinolinic acid. In some embodiments, the organic acid is xanthurenic acid. In some embodiments, the organic acid is isovalerylglycine. In some embodiments, the organic acid is 3-hydroxyisovaleric acid. In some embodiments, the organic acid is isocitric acid. In some embodiments, the organic acid is cis-aconitic acid. In some embodiments, the organic acid is pyroglutamic acid. In some embodiments, the organic acid is vanilmandelic acid. In some embodiments, the organic acid is methylmalonic acid. In some embodiments, the organic acid is glyceric acid. In some embodiments, the method includes selecting the compound if the second level is higher than the first level. In some embodiments, the method includes selecting the compound if the second level is 10% higher than the first level. In some embodiments, the method includes selecting the compound if the second level is 20% higher than the first level. In some embodiments, the method includes selecting the compound if the second level is 30% higher than the first level. In some embodiments, the method includes selecting the compound if the second level is 40% higher than the first level. In some embodiments, the method includes selecting the compound if the second level is 50% higher than the first level. In some embodiments, the method includes selecting the compound if the second level is 60% higher than the first level. In some embodiments, the method includes selecting the compound if the second level is 70% higher than the first level. In some embodiments, the method includes selecting the compound if the second level is 80% higher than the first level. In some embodiments, the method includes selecting the compound if the second level is 90% higher than the first level. In some embodiments, the method includes determining the levels of two or more urinary organic acids and comparing each level of the urinary organic acids to its respective reference level.In some embodiments, the reference level of the organic acid is a predetermined level of the organic acid from a public database. In some embodiments, the reference level is the level of the organic acid in a healthy subject or a subject without CFS. In some embodiments, the reference level of the organic acid is the level of the organic acid in a subject with moderate CFS. In some embodiments, the reference level of the organic acid is the level of the organic acid determined based on a cohort of healthy subjects. In some embodiments, the reference level of the organic acid is the level of the organic acid in a first sample obtained from the subject. In some embodiments, the reference level of the organic acid is the median or average value of the level of the organic acid in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects with moderate CFS, or a cohort of first samples obtained from the subject). In some embodiments, CFS is associated with an autoimmune disease or an infectious disease.

[0126] In some embodiments, the treatment of CFS includes any treatment for reducing any symptoms associated with CFS. In some embodiments, the treatment includes an immunomodulatory compound. In some embodiments, the treatment includes an immunomodulatory drug (IMiD). IMiD includes a group of compounds that may be useful for the treatment of several types of human diseases, including certain cancers. As used herein, unless otherwise indicated, the term "immunomodulatory compound" may include certain organic small molecules that inhibit LPS-induced monocyte TNF-α, IL-1β, IL-12, IL-6, MIP-1α, MCP-1, GM-CSF, G-CSF, and COX-2 production. These compounds can be prepared synthetically or obtained commercially. In other embodiments, the treatment of CFS provided herein includes a CRBN regulator. In some embodiments, the CRBN regulator is an agent that can directly or indirectly regulate at least one of the biological activities of CRBN. In some embodiments, the CRBN regulator is an agent that can physically bind to CRBN. In other embodiments, the CRBN regulator does not directly bind to CRBN but can act in other ways through CRBN-mediated pathways. In some embodiments, the treatment of CFS provided herein includes an agent that depletes B cells. In some embodiments, the agent that depletes B cells is an antibody that specifically binds to an antigen of B cells. In some embodiments, the antigen of B cells is CD20, CD19, CD22, CD38, or B cell-activating factor (BAFF). In some embodiments, the agent that depletes B cells is an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is rituximab, ocrelizumab, or ofatumumab. The agent that depletes B cells is an anti-CD19 antibody. In some embodiments, the anti-CD19 antibody is inebilizumab. In some embodiments, the agent that depletes B cells is an anti-BAFF antibody. In some embodiments, the anti-BAFF antibody is belimumab.

[0127] In some embodiments, the therapeutic compound is formulated into a pharmaceutical composition comprising the therapeutic compound provided herein and a pharmaceutically acceptable excipient. In some embodiments, CFS is associated with an autoimmune disease or an infectious disease.

[0128] In some embodiments, when multiple organic acids are used, a second level of each organic acid is compared to a first level of that organic acid, and the method includes identifying that the subject has CFS if the second level of each organic acid is lower than the first level of that organic acid. In some embodiments, when two or more organic acids are used, an overall score is calculated based on the multiple organic acids and compared to a reference overall score. In some embodiments, the method includes determining first and second levels of two or more urinary organic acids, obtaining a first overall score based on the first levels of the urinary organic acids and a second overall score based on the second levels of the urinary organic acids, and comparing the first overall score to the second overall score. In some embodiments, the overall score is calculated using the median Z-score method. In other embodiments, the overall score is calculated using the single-sample gene set enrichment (ssGSEA) method.

[0129] In some embodiments, provided herein is a kit comprising an agent for determining the level of at least one urinary organic acid selected from the group consisting of hippuric acid, 3-hydroxypropionic acid, α-ketoisocaproic acid, α-ketoisovaleric acid, α-keto-β-methylvaleric acid, α-hydroxybutyric acid, glycolic acid, pyruvic acid, citramalic acid, lactic acid, α-ketoadipic acid, citric acid, malic acid, kynurenic acid, xanthurenic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanilmandelic acid, methylmalonic acid, and glyceric acid. In some embodiments, the organic acid is hippuric acid. In some embodiments, the organic acid is 3-hydroxypropionic acid. In some embodiments, the organic acid is α-ketoisocaproic acid. In some embodiments, the organic acid is α-keto-β-methylvaleric acid. In some embodiments, the organic acid is α-hydroxybutyric acid. In some embodiments, the organic acid is glycolic acid. In some embodiments, the organic acid is pyruvic acid. In some embodiments, the organic acid is glycolic acid. In some embodiments, the organic acid is citramalic acid. In some embodiments, the organic acid is lactic acid. In some embodiments, the organic acid is α-ketoadipic acid. In some embodiments, the organic acid is citric acid. In some embodiments, the organic acid is malic acid. In some embodiments, the organic acid is kynurenic acid. In some embodiments, the organic acid is xanthurenic acid. In some embodiments, the organic acid is isovalerylglycine. In some embodiments, the organic acid is 3-hydroxyisovaleric acid. In some embodiments, the organic acid is isocitric acid. In some embodiments, the organic acid is cis-aconitic acid. In some embodiments, the organic acid is pyroglutamic acid. In some embodiments, the organic acid is vanilmandelic acid. In some embodiments, the organic acid is methylmalonic acid. In some embodiments, the organic acid is glyceric acid.In some embodiments, the kit includes an instrument for obtaining a sample. In some embodiments, the kit includes instructions regarding one or more determined levels of interpretation. In some embodiments, CFS is associated with an autoimmune disease or an infectious disease.

[0130] 5.2.2 Biomarkers of Long COVID Long COVID is also known as post-COVID conditions, long-haul COVID, post-acute COVID-19, long-term effects of COVID, and chronic COVID. Long COVID is a wide-ranging, new, recurring, or ongoing health problem seen in people after infection with the virus that causes COVID-19 (see, e.g., Thaweethai et al., JAMA (published online May 25, 2023) “Development of a Definition of Postacute Sequelae of SARS-CoV-2 Infection”). Since most people with COVID-19 recover within days to weeks after infection, long COVID can first be identified at least 3 months after infection. Symptoms of long COVID can include fatigue or tiredness that interferes with daily life, shortness of breath, cough, chest pain, heart palpitations, difficulty thinking or concentrating (sometimes called “brain fog”), headache, sleep problems, dizziness when standing up (vertigo), pins and needles sensations, changes in smell or taste, depression or anxiety, diarrhea, stomach pain, joint or muscle pain, rash, and changes in the menstrual cycle. In certain embodiments, a long COVID patient has at least one long COVID symptom at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 18 months, or at least 2 years after the initial infection with the virus that causes COVID-19. In certain embodiments, a long COVID patient has at least one long COVID symptom at least 6 months after infection with the virus that causes COVID-19. In some embodiments, the long COVID symptoms are selected from fatigue, post-exertional malaise, cognitive dysfunction, sensorimotor symptoms, headache, memory impairment, insomnia, myalgia, heart palpitations, shortness of breath, dizziness and balance disorders, speech disorders, joint pain, and chest pressure.

[0131] In some embodiments, provided herein is a method of identifying a subject having long COVID or a method of confirming long COVID in a subject, the method comprising: (a) determining an expression level of a biomarker in a sample from the subject, wherein the biomarker is a cereblon (CRBN)-associated protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; and (b) identifying or confirming that the subject has long COVID if the expression level of the biomarker is higher than a reference expression level of the biomarker.

[0132] In some embodiments, provided herein is a method of identifying a subject that is likely or unlikely to respond to treatment for long COVID or a method of predicting whether a subject will respond to treatment for long COVID, the method comprising: (a) determining an expression level of a biomarker in a sample from the subject, wherein the biomarker is a cereblon (CRBN)-associated protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; and (b) identifying or predicting that the subject is likely to respond to treatment if the expression level of the biomarker is higher than a reference expression level of the biomarker. In certain embodiments, the method further comprises administering treatment to a subject identified or predicted to be likely to respond to treatment.

[0133] In some embodiments, provided herein is a method for selectively treating a subject having or suspected of having long COVID, the method comprising: (a) determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a cereblon (CRBN)-associated protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; (b) identifying or predicting that the subject is likely to respond to treatment for long COVID if the expression level of the biomarker is higher than a reference expression level of the biomarker; and (c) administering treatment to a subject identified or predicted to be likely to respond to treatment.

[0134] In some embodiments, the biomarker is HSPA8. In some embodiments, the biomarker is IKZF3. In some embodiments, the biomarker is ABCE1. In some embodiments, the biomarker is BACH2. In some embodiments, the biomarker is CD3D. In some embodiments, the biomarker is IKZF2.

[0135] In some embodiments, the reference expression level of a biomarker is a predetermined expression level of the biomarker. In some embodiments, the reference expression level of a biomarker is a predetermined expression level of the biomarker obtained from a public database. In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker in a healthy subject or a subject without long COVID. In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker in a subject with acute COVID (e.g., severe acute COVID). In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker determined based on a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without long COVID, or a cohort of subjects with acute COVID (e.g., severe acute COVID)). In some embodiments, the reference expression level of a biomarker is the median or average value of the expression levels of the biomarker in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without long COVID, a cohort of subjects with acute COVID (e.g., severe acute COVID)).

[0136] In some embodiments, the biomarker is HSPA8 or IKZF3, and the reference expression level of the biomarker is the expression level of the biomarker in a healthy subject or a subject without long COVID, or in a cohort of healthy subjects or subjects without long COVID. In some embodiments, the biomarker is selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D, and the reference expression level of the biomarker is the expression level of the biomarker in a subject with acute COVID (e.g., severe acute COVID) or in a cohort of subjects with acute COVID (e.g., severe acute COVID).

[0137] As used herein, a subject having acute COVID refers to a subject who has been confirmed to be infected with SARS-CoV-2 and is in the acute phase of the disease that begins with the onset of initial symptoms associated with SARS-CoV-2 infection and ends within 15 days. A subject having severe acute COVID refers to a subject having acute COVID with dyspnea as defined by the need for oxygen supplementation, although mechanical ventilation is not required. In certain embodiments, a subject having severe acute COVID was hospitalized (or awaiting hospitalization in the ED).

[0138] In some embodiments, the method includes identifying that a subject has long COVID if the expression level of a biomarker is at least about 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% higher than the reference expression level of the biomarker. The expression level of the biomarker can be determined using any known method in the art, and exemplary methods are described in further detail in Section 5.3 below. In some embodiments, a level is determined to be higher than the reference level if the level is higher (e.g., statistically significantly higher) than the reference level when observed by a measurement assay.

[0139] In certain embodiments, the method comprises identifying or confirming a subject having long COVID, or identifying or predicting that a subject is likely to respond to long COVID treatment, using two, three, four, five, or all six biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the expression levels of IKZF2 and IKZF3 are determined. In some embodiments, the expression levels of IKZF2 and ABCE1 are determined. In some embodiments, the expression levels of IKZF2 and BACH2 are determined. In some embodiments, the expression levels of IKZF2 and CD3D are determined. In some embodiments, the expression levels of IKZF2 and HSPA8 are determined. In some embodiments, the expression levels of IKZF3 and ABCE1 are determined. In some embodiments, the expression levels of IKZF3 and BACH2 are determined. In some embodiments, the expression levels of IKZF3 and CD3D are determined. In some embodiments, the expression levels of IKZF3 and HSPA8 are determined. In some embodiments, the expression levels of ABCE1 and BACH2 are determined. In some embodiments, the expression levels of ABCE1 and CD3D are determined. In some embodiments, the expression levels of ABCE1 and HSPA8 are determined. In some embodiments, the expression levels of BACH2 and CD3D are determined. In some embodiments, the expression levels of BACH2 and HSPA8 are determined. In some embodiments, the expression levels of CD3D and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and ABCE1 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and BACH2 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, and CD3D are determined.In some embodiments, the expression levels of IKZF2, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF3, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, and HSPA8 are determined.In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, CD3D, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8 are determined.

[0140] In some embodiments, when multiple biomarkers are used, the expression level of each biomarker is compared to the reference expression level of that biomarker, and the method includes identifying or confirming a subject with long COVID or identifying or predicting that the subject is likely to respond to long COVID treatment if the expression level of each biomarker is higher than the reference expression level of that biomarker. In other embodiments, when multiple biomarkers are used, an overall score is calculated based on the multiple biomarkers and compared to a reference overall score. In some embodiments, the method includes identifying or confirming a subject with long COVID or identifying or predicting that the subject is likely to respond to long COVID treatment if the overall score is higher than the reference overall score. In some embodiments, the overall score is calculated using the median Z-score method. Briefly, the median Z-score is obtained by first calculating the mean value of each gene from all samples within the gene expression matrix. Next, this mean value is subtracted from each corresponding gene for all samples, and then scaling is performed by dividing the value by its standard deviation. The median of the scaled values from the multiple genes of interest forms the overall score. Another exemplary method for calculating the overall score is the single-sample gene set enrichment (ssGSEA) method. The single-sample gene score represents the degree to which genes within a particular gene set are coordinately upregulated or downregulated within a sample. This score is calculated by adjusting the cumulative sum statistic based on a decreasing walk through the ranked expression list. The enrichment score is the maximum deviation from zero encountered during the walk; this corresponds to a weighted Kolmogorov-Smirnov-like statistic (see, for example, Subramanian et al., PNAS, 102(43):15545-15550 (2005); and Barbie et al., Nature, 462(7269):108-112).

[0141] In some embodiments, provided herein is a method for determining or monitoring the effectiveness of a treatment in a subject having long COVID, the method comprising: (a) determining a first expression level of a biomarker in a first sample obtained from the subject prior to administering the treatment to the subject, wherein the biomarker is a cereblon (CRBN)-associated protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; (b) administering the treatment to the subject; (c) determining a second expression level of the biomarker in a second sample obtained from the subject after administering the treatment to the subject; and (d) determining the effectiveness of the treatment based on a comparison of the first expression level and the second expression level. In certain embodiments, the method comprises determining that the treatment is effective if the second expression level is lower than the first expression level. In certain embodiments, the method comprises determining that the treatment is ineffective if the second expression level is not lower than the first expression level. In certain embodiments, the method further comprises determining or adjusting (e.g., increasing) the dosage of the treatment or administering a different long COVID treatment to the subject if the second expression level is not lower than the first expression level.

[0142] In some embodiments, provided herein is a method for screening the effectiveness of a treatment for treating long COVID, the method comprising: (a) determining a first expression level of a biomarker in a sample prior to administering a compound to the sample, wherein the biomarker is a cereblon (CRBN)-associated protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; (b) administering the treatment to the sample; (c) determining a second expression level of the biomarker in the sample after administering the treatment to the sample; (d) comparing the first expression level to the second expression level; and (e) selecting the treatment if the second expression level is lower than the first expression level.

[0143] In some embodiments, the method includes determining that the treatment is effective if the second expression level is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower than the first expression level. Based on the comparison of the first expression level and the second expression level, in one or more subsequent treatment cycles, a different treatment or a different dosage regimen may be administered to the subject.

[0144] In some embodiments, the biomarker is IKZF2. In some embodiments, the biomarker is IKZF3. In some embodiments, the biomarker is ABCE1. In some embodiments, the biomarker is BACH2. In some embodiments, the biomarker is CD3D. In some embodiments, the biomarker is HSPA8.

[0145] The expression level of the biomarker can be determined using any known method in the art, and exemplary methods are described in more detail in Section 5.3 below. In some embodiments, if the level is higher (e.g., statistically significantly higher) than the second level when observed by a measurement assay, the level is determined to be higher than the second level. In some embodiments, if the level is lower (e.g., statistically significantly higher) than the second level when observed by a measurement assay, the level is determined to be lower than the second level.

[0146] In certain embodiments, the method comprises using two, three, four, five, or all six biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the expression levels of IKZF2 and IKZF3 are determined. In some embodiments, the expression levels of IKZF2 and ABCE1 are determined. In some embodiments, the expression levels of IKZF2 and BACH2 are determined. In some embodiments, the expression levels of IKZF2 and CD3D are determined. In some embodiments, the expression levels of IKZF2 and HSPA8 are determined. In some embodiments, the expression levels of IKZF3 and ABCE1 are determined. In some embodiments, the expression levels of IKZF3 and BACH2 are determined. In some embodiments, the expression levels of IKZF3 and CD3D are determined. In some embodiments, the expression levels of IKZF3 and HSPA8 are determined. In some embodiments, the expression levels of ABCE1 and BACH2 are determined. In some embodiments, the expression levels of ABCE1 and CD3D are determined. In some embodiments, the expression levels of ABCE1 and HSPA8 are determined. In some embodiments, the expression levels of BACH2 and CD3D are determined. In some embodiments, the expression levels of BACH2 and HSPA8 are determined. In some embodiments, the expression levels of CD3D and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and ABCE1 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and BACH2 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF2, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, BACH2, and CD3D are determined.In some embodiments, the expression levels of IKZF2, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF3, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF3, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, CD3D, and HSPA8 are determined.In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, and BACH2 are determined. In some embodiments, the expression levels of IKZF3, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, ABCE1, BACH2, CD3D, and HSPA8 are determined. In some embodiments, the expression levels of IKZF2, IKZF3, BACH2, CD3D, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8 are determined. In other embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, and CD3D are determined. In some embodiments, the expression levels of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8 are determined.

[0147] In some embodiments, when multiple biomarkers are used, the second expression level of each biomarker is compared to the first expression level of that biomarker, and the method includes determining that the treatment is effective if the second expression level of each biomarker is lower than the first expression level of that biomarker. In other embodiments, when multiple biomarkers are used, an aggregate score is calculated based on the multiple biomarkers and compared to a reference aggregate score. In some embodiments, the aggregate score is calculated using the median Z-score method. In other embodiments, the aggregate score is calculated using the single-sample gene set enrichment (ssGSEA) method.

[0148] In certain embodiments, the disclosed treatments for long COVID include immunomodulatory drugs (IMiDs). IMiDs include a group of compounds that can be useful in the treatment of several types of human diseases, including certain cancers. As used herein, unless otherwise indicated, the term “immunomodulatory compound” can include certain small organic molecules that inhibit LPS-induced monocyte TNF-α, IL-1β, IL-12, IL-6, MIP-1α, MCP-1, GM-CSF, G-CSF, and COX-2 production. These compounds can be synthetically prepared or obtained as commercial products. The inflammatory cytokine TNF-α, produced by macrophages and monocytes during acute inflammation, causes diverse intracellular signaling events. Without being limited by a particular theory, one of the biological effects exerted by the immunomodulatory compounds disclosed herein is the reduction of TNF-α production by myeloid cells. The immunomodulatory compounds disclosed herein can enhance the degradation of TNF-α mRNA. Further, without being limited by theory, the immunomodulatory compounds disclosed herein are also potent co-stimulators of T cells and can dramatically increase cell proliferation in a dose-dependent manner. The immunomodulatory compounds disclosed herein can also have a higher co-stimulatory effect on the CD8+ T cell subset than on the CD4+ T cell subset. In addition, such compounds can have anti-inflammatory properties against myeloid cell responses, but can further efficiently co-stimulate T cells to produce increased amounts of IL-2 and IFN-γ, enhancing T cell proliferation and the cytotoxic activity of CD8+ T cells. Further, without being limited by a particular theory, the immunomodulatory compounds disclosed herein have both the ability to act indirectly through cytokine activation and the ability to act directly on natural killer (“NK”) cells and natural killer T (“NKT”) cells, increasing the ability of NK cells to produce beneficial cytokines such as, but not limited to, IFN-γ, and the ability to enhance the cytotoxic activity of NK and NKT cells. The various immunomodulatory compounds disclosed herein have one or more chiral centers and can exist as racemic mixtures of enantiomers or mixtures of diastereomers.Accordingly, the present specification also provides for the use of such compounds in stereoisomerically pure form, as well as the use of mixtures of those forms. For example, mixtures containing enantiomers of certain immunomodulatory compounds in equal or unequal amounts may be used. Such isomers may be asymmetrically synthesized or resolved using standard techniques such as chiral columns or chiral resolving agents. See, for example, Jacques, J., et al., Enantiomers, Racemates and Resolutions (Wiley-Interscience, New York, 1981); Wilen, S.H., et al., Tetrahedron 33:2725 (1977); Eliel, E.L., Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, S.H., Tables of Resolving Agents and Optical Resolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).

[0149] In certain embodiments, the treatment of long COVID disclosed herein includes a cereblon (CRBN) modulator. In some embodiments, the CRBN modulator is an agent that can directly or indirectly modulate at least one of the biological activities of CRBN. In some embodiments, the CRBN modulator is an agent that can physically bind to CRBN. In other embodiments, the CRBN modulator does not directly bind to CRBN but can otherwise act through CRBN-mediated pathways. Cereblon (CRBN), a component of the DDB1-CUL4a-Roc1 ubiquitin ligase complex, has been identified as a target for certain immunomodulatory compounds, such as thalidomide, lenalidomide, pomalidomide, and iberdomide (Lopez-Girona et al., Leukemia volume 26, pages 2326-2335 (2012); Bjorklund et al., Leukemia. 2020; 34(4):1197-1201). It is thought that the anti-proliferative effects of CRBN in multiple myeloma (MM) cells are mediated by its interaction with certain immunomodulatory compounds (Lopez-Girona et al., Leukemia 2012; Zhu et al., Blood 2011, 118, Abstract 127). CRBN is encoded by a 25 kb gene on chromosome 6 consisting of 11 exons and 10 introns. Thus, through the process of alternative splicing, multiple functional proteins from a single gene with different structural configurations and functional activities, as well as variants of a protein, may potentially result. Truncated proteins that have lost interaction domains or critically important functional amino acid residues can create non-functional or abnormal CRBN proteins, thus interfering with the function of the full-length CRBN protein and potentially reducing or altering the therapeutic activity of therapeutic compounds that act through their interaction with the full-length CRBN protein. There are at least two isoforms of the protein cereblon (CRBN), 442 and 441 amino acids in length respectively, and CRBN is conserved from plants to humans.In humans, the CRBN gene has been identified as a candidate gene for autosomal recessive nonsyndromic mental retardation (ARNSMR). See Higgins, J.J. et al., Neurology, 2004, 63:1927-1931. CRBN was initially characterized as a novel RGS-containing protein that interacts with the calcium-activated potassium channel protein (SLO1) in the rat brain, but was later shown to interact with the voltage-gated chloride channel (CIC-2) in the retina, which has AMPK1 and DDB1. See Jo, S. et al., J. Neurochem, 2005, 94:1212-1224; Hohberger B. et al., FEBS Lett, 2009, 583:633-637; Angers S. et al., Nature, 2006, 443:590-593. DDB1 was initially identified as a nucleotide excision repair protein associated with the damaged DNA binding protein 2 (DDB2). Deficiency of its activity causes repair deficiency in patients with xeroderma pigmentosum complementation group E (XPE). DDB1 also appears to function as a component of numerous different DCX (DDB1-CUL4-X box) E3 ubiquitin-protein ligase complexes that mediate ubiquitination of target proteins and subsequent proteasomal degradation. CRBN has also been identified as a target for the development of therapeutic agents for the treatment of cerebral cortex diseases. See International Publication No. WO 2010 / 137547 A1 pamphlet. In some embodiments, the beneficial effects of certain therapeutic compounds provided herein require binding to CRBN or one or more CRBN substrates. In some embodiments, the therapeutic compounds provided herein can induce CRBN to undergo a conformational change. In some embodiments, the use of the therapeutic compounds provided herein leads to a characteristic conformational change on the surface of CRBN or other modification of its properties, resulting in a characteristic phenotypic response. In certain embodiments, the CRBN modulator is an immunomodulatory compound. In other embodiments, CRBN is not an immunomodulatory compound.

[0150] In some embodiments, the treatment of long COVID disclosed herein includes an agent that depletes B cells. In some embodiments, the agent that depletes B cells is an antibody that specifically binds to an antigen of B cells. In some embodiments, the antigen of B cells is CD20, CD19, CD22, CD38, or B cell activating factor (BAFF). In some embodiments, the agent that depletes B cells is an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is rituximab, ocrelizumab, or ofatumumab. The agent that depletes B cells is an anti-CD19 antibody. In some embodiments, the anti-CD19 antibody is inebilizumab. In some embodiments, the agent that depletes B cells is an anti-BAFF antibody. In some embodiments, the anti-BAFF antibody is belimumab.

[0151] In some embodiments, the therapeutic compounds provided herein are formulated into pharmaceutical compositions comprising the therapeutic compounds provided herein and a pharmaceutically acceptable excipient. Pharmaceutical compositions comprising the therapeutic compounds provided herein are prepared for storage, in aqueous solution form, or in lyophilized or other dry forms, by mixing the compounds provided herein with an optional physiologically acceptable excipient (see, e.g., Remington, Remington’s Pharmaceutical Sciences (18th ed. 1980)). The therapeutic compounds of the present disclosure can be formulated in any form suitable for delivery to target cells / tissues, such as, for example, as microcapsules or macroemulsions (Remington, supra; Park et al., 2005, Molecules 10:146 - 61; Malik et al., 2007, Curr. Drug. Deliv. 4:141 - 51), as sustained-release formulations (Putney and Burke, 1998, Nature Biotechnol. 16:153 - 57), or in liposomes (Maclean et al., 1997, Int. J. Oncol. 11:325 - 32; Kontermann, 2006, Curr. Opin. Mol. Ther. 8:39 - 45). The therapeutic compounds provided herein can also be encapsulated, for example, in microcapsules prepared by coacervation techniques or by interfacial polymerization, such as, for example, hydroxyethylmethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed, for example, in Remington, supra. A variety of compositions and delivery systems are known and can be used with the compounds as described herein. In some embodiments, the composition can be provided as a controlled-release system or a sustained-release system.In one embodiment, a pump can be used to achieve controlled or sustained release (see, e.g., Langer, supra; Sefton, 1987, Crit. Ref. Biomed. Eng. 14:201-40; Buchwald et al., 1980, Surgery 88:507-16; and Saudek et al., 1989, N. Engl. J. Med. 321:569-74). In another embodiment, a polymeric material can be used to achieve controlled or sustained release of a prophylactic or therapeutic agent or composition provided herein (see, e.g., 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, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61-126; Levy et al., 1985, Science 228:190-92; During et al., 1989, Ann. Neurol. 25:351-56; Howard et al., 1989, J. Neurosurg. 71:105-12; U.S. Patent Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; and 5,128,326; International Publication Nos. 99 / 15154 and 99 / 20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters.In one embodiment, the polymers used in the sustained release formulations are inert, contain no leachable impurities, are stable upon storage, are sterile, and are biodegradable. In yet another embodiment, the controlled release or sustained release system can be placed in proximity to a particular target tissue, such as the nasal passages or the lung, and thus only a fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release Vol. 2, 115-38 (1984)). Controlled release systems are discussed, for example, in Langer, 1990, Science 249:1527-33. Sustained release formulations containing the therapeutic compounds described herein can be made using any technique known to those of skill in the art (see, e.g., U.S. Patent No. 4,526,938, International Publication Nos. 91 / 05548 and 96 / 20698, Ning et al., 1996, Radiotherapy & Oncology 39:179-89; Song et al., 1995, PDA J. of Pharma. Sci. & Tech. 50:372-97; Cleek et al., 1997, Pro. Int’l. Symp. Control. Rel. Bioact. Mater. 24:853-54; and Lam et al., 1997, Proc. Int’l. Symp. Control Rel. Bioact. Mater. 24:759-60). A variety of delivery systems are known and can be used for administration of the therapeutic compounds provided herein.

[0152] 5.3. Methods for Detecting and Quantifying Biomarkers In this method, any method and technique known in the art for detecting or quantifying biomarkers can be used. In certain embodiments, the expression level of a biomarker is determined by measuring the nucleic acid level of the biomarker. In certain embodiments, the expression level of the biomarkers provided herein is determined by measuring the mRNA level of the biomarker. In other embodiments, the expression level of the biomarkers provided herein is determined by measuring the cDNA level of the biomarker. In other embodiments, the expression level of the biomarkers provided herein is determined by measuring the protein level of the biomarker.

[0153] Several methods for detecting or quantifying mRNA levels are known in the art. Exemplary methods include, but are not limited to, Northern blot, RNA sequencing, microarray, ribonuclease protection assay, PCR-based methods, and the like. Using the mRNA sequence of the biomarker, a probe that is at least partially complementary to the mRNA sequence can be prepared. This probe can then be used to detect mRNA in a sample by any suitable assay, such as a PCR-based method, Northern blotting, dipstick assay, and the like.

[0154] In other embodiments, a nucleic acid assay for detecting or quantifying a biomarker in a biological sample can be prepared. The assay can include a solid support and at least one nucleic acid in contact with the support, where the nucleic acid corresponds to at least a portion of the mRNA of the biomarker. The assay can also have means for detecting the expression (e.g., change in expression) of the mRNA in the sample.

[0155] The assay method can vary depending on the type of desired mRNA information. Exemplary methods include, but are not limited to, Northern blot and PCR-based methods (e.g., qRT-PCR). Methods such as qRT-PCR can also accurately quantify the amount of mRNA in a sample. Exemplary methods also include next-generation sequencing (NGS).

[0156] The presence of mRNA in a sample can be determined using any suitable assay platform. For example, the assay can be in the form of a dipstick, membrane, chip, disk, test strip, filter, microsphere, slide, multiwell plate, or optical fiber. The assay system can have a solid support, onto which nucleic acids corresponding to the mRNA are attached. The solid support can include, for example, plastic, silicon, metal, resin, glass, membrane, particle, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, plate, or slide. The assay components can be prepared and packaged together as a kit for mRNA detection.

[0157] If necessary, the nucleic acid can be labeled to create a population of labeled mRNA. Generally, the sample can be labeled using methods well known in the art (e.g., using DNA ligase, terminal transferase, or by labeling the RNA backbone, etc.). For example, Ausubel et al., Short Protocols in Molecular Biology (Wiley & Sons, 3 rd ed. 1995); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, N.Y., 3 rdSee (ed. 2001). In some embodiments, the sample is labeled with a fluorescent label. Exemplary fluorescent dyes include, but are not limited to, xanthene dyes, fluorescein dyes (e.g., fluorescein isothiocyanate (FITC), 6-carboxyfluorescein (FAM), 6-carboxy-2’,4’,7’,4,7-hexachlorofluorescein (HEX), 6-carboxy-4’,5’-dichloro-2’,7’-dimethoxyfluorescein (JOE)), rhodamine dyes (e.g., rhodamine 110 (R110), N,N,N’,N’-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX), 5-carboxyrhodamine 6G (R6G5 or G5), 6-carboxyrhodamine 6G (R6G6 or G6)), cyanine dyes (e.g., Cy3, Cy5, and Cy7), Alexa dyes (e.g., Alexa-fluor-555), coumarin, diethylaminocoumarin, umbelliferone, benzimidazole dyes (e.g., Hoechst 33258), phenanthridine dyes (e.g., Texas Red), ethidium dyes, acridine dyes, carbazole dyes, phenoxazine dyes, porphyrin dyes, polymethine dyes, BODIPY dyes, quinoline dyes, pyrene, fluorescein chlorotriazinyl, eosin dyes, tetramethylrhodamine, lysamine, napthofluorescein, etc.

[0158] The nucleic acids may be present at specific and assignable positions on a solid support, each position corresponding to at least a portion of an mRNA sequence that exhibits differential expression during treatment of a compound in a cell or patient.

[0159] For example, in certain embodiments, a method of detecting and quantifying the RNA (e.g., mRNA) level of a biomarker from a biological sample includes: (a) obtaining RNA from the sample; (b) creating a first DNA molecule having a sequence complementary to the RNA by contacting the RNA with a primer that specifically binds to a sequence in the RNA; (c) amplifying DNA corresponding to a segment of the gene encoding the biomarker; and (d) determining the RNA level of the biomarker based on the amount of the amplified DNA.

[0160] Typical mRNA assay methods can include: (1) obtaining a surface-bound target probe; (2) hybridizing a population of mRNA to the surface-bound probe under conditions sufficient to effect specific binding; (3) removing nucleic acids that did not specifically bind to the surface-bound probe by washing after hybridization; and (4) detecting the hybridized mRNA. The reagents used in each of these steps and their conditions of use can vary depending on the specific application.

[0161] Hybridization can be performed under suitable hybridization conditions, which can be of various stringencies as desired. Typical conditions are sufficient to result in a probe / target complex on a solid surface between complementary binding members, i.e., between the surface-bound target probe and the complementary mRNA in the sample. In certain embodiments, stringent hybridization conditions may be used.

[0162] Hybridization is typically carried out under stringent hybridization conditions. For standard hybridization techniques (e.g., under conditions sufficient to effect specific binding of a probe to target mRNA in a sample), see Kallioniemi et al., Science 1992, 258:818-821 and WO 93 / 18186. Guides to several common techniques, e.g., Tijssen, Hybridization with Nucleic Acid Probes, Parts I and II (Elsevier, Amsterdam 1993) are available. For descriptions of techniques suitable for in situ hybridization, see Gall et al., Meth. Enzymol. 1981, 21:470-480; Angerer et al., Genetic Engineering: Principles and Methods, Vol 7, pgs 43-65 (Plenum Press, New York, Setlow and Hollaender, eds. 1985). The selection of appropriate conditions will depend on experimental design including, for example, the source of the sample, the identity of the capture agent, the degree of complementarity expected, etc., including temperature, salt concentration, polynucleotide concentration, hybridization time, stringency of the wash conditions, etc., and can be determined by one of ordinary skill in the art as a routine matter of experimentation.

[0163] One of ordinary skill in the art will readily recognize that alternative, but equivalent, hybridization and wash conditions can be utilized to provide similar stringency conditions.

[0164] After the mRNA hybridization procedure, the surface-bound polynucleotide is typically washed to remove unbound nucleic acid. The wash may be carried out using any convenient wash protocol, where the wash conditions are typically stringent as described above. Next, hybridization of the probe to the target mRNA is detected using standard techniques.

[0165] Other methods, such as PCR-based methods, can also be used to detect or quantify the expression of biomarkers. For examples of PCR methods, reference can be made to U.S. Patent No. 6,927,024, which is hereby incorporated by reference in its entirety. For examples of RT-PCR methods, reference can be made to U.S. Patent No. 7,122,799, which is hereby incorporated by reference in its entirety. For fluorescence in situ PCR methods, reference is made to U.S. Patent No. 7,186,507, which is hereby incorporated by reference in its entirety.

[0166] In some embodiments, quantitative reverse transcription PCR (qRT-PCR) can be used for both the detection and quantification of RNA targets (Bustin et al., Clin. Sci. 2005, 109:365-379). The quantitative results obtained by qRT-PCR are generally more informative than qualitative data. Thus, in some embodiments, qRT-PCR-based assays can be useful for measuring mRNA levels in cell-based assays. The qRT-PCR method is also useful for monitoring patient therapies. For examples of qRT-PCR-based methods, reference can be made, for example, to U.S. Patent No. 7,101,663, which is hereby incorporated by reference in its entirety.

[0167] In contrast to conventional reverse transcriptase PCR and agarose gel analysis, qRT-PCR provides quantitative results. An additional advantage of qRT-PCR is that it is relatively easy and convenient to use. Instruments for qRT-PCR are commercially available, such as the Applied Biosystems 7500, and reagents are also commercially available, such as TaqMan® Sequence Detection Chemistry. For example, TaqMan® Gene Expression Assays can be used according to the manufacturer's instructions. These kits are pre-designed gene expression assays for fast and reliable detection and quantification of human, mouse, and rat mRNA transcripts. An exemplary qRT-PCR program is, for example, 2 min at 50°C, 10 min at 95°C, 40 cycles of 15 sec at 95°C, then 1 min at 60°C.

[0168] The cycle number at which the fluorescent signal associated with a particular amplicon accumulation crosses the threshold (C T To determine the sequence of the nucleotide sequence (referred to as nucleotide sequence), for example, use the 7500 Real-Time PCR System Sequence Detection Software or C T The data can be analyzed using a relative comparison quantification calculation method. With this method, the output is expressed as the fold change in expression level. In some embodiments, the threshold level can be selected to be automatically determined by the software. In some embodiments, the threshold level is set to be above the baseline but low enough within the exponential growth range of the amplification curve.

[0169] To measure the level of a biomarker, several protein detection and quantification methods can be used. Any suitable protein quantification method can be used. In some embodiments, antibody-based methods are used. In some embodiments, the level of a protein biomarker is measured using an immunoassay. In some embodiments, immunoassays include Western blot, enzyme-linked immunosorbent assay (ELISA), flow cytometry, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay (RIA), dot blotting, and flow cytometry. In some embodiments, ELISA is direct ELISA (enzyme-linked immunosorbent assay), indirect ELISA, sandwich ELISA, competitive ELISA, multiplex ELISA, ELISPOT technology, and other similar techniques known in the art.

[0170] In some embodiments, the level of the protein biomarker is determined by using mass spectrometry (MS). In some embodiments, the MS includes liquid chromatography-tandem mass spectrometry (LC MS / MS), liquid chromatography-mass spectrometry (LC-MS), multiple reaction monitoring (MRM), selected reaction monitoring (SRM), affinity capture MS (AC-MS), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS, MALDI-TOF post-source decay (PSD), MALDI-TOF / TOF, surface enhanced laser desorption / ionization time-of-flight mass spectrometry (SELDI-TOF) MS, electrospray ionization mass spectrometry (ESI-MS), ESI-MS / MS, ESI-MS / (MS)n (where n is an integer greater than zero), ESI 3D or linear (2D) ion trap MS, ESI triple quadrupole MS, ESI quadrupole orthogonal TOF (Q-TOF), ESI Fourier transform MS system, desorption / ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS / MS, APCI-(MS)n, ion mobility spectrometry (IMS), inductively coupled plasma mass spectrometry (ICP-MS), atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS / MS, and APPI-(MS)n.

[0171] Exemplary antibody-based assays for determining the level of a protein biomarker include contacting the protein in a sample with a primary antibody that immunospecifically binds to the biomarker protein. In some embodiments, the methods provided herein include (i) contacting the biomarker protein bound to the primary antibody with a secondary antibody having a detectable label, wherein the secondary antibody immunospecifically binds to the biomarker protein and the secondary antibody immunospecifically binds to an epitope different from the primary antibody on the biomarker protein; (ii) detecting the presence of the secondary antibody bound to the biomarker protein; and (iii) further determining the amount of the biomarker protein based on the amount of the detectable label in the secondary antibody. In other embodiments, the methods provided herein include (i) contacting the biomarker protein bound to the primary antibody with a secondary antibody having a detectable label, wherein the secondary antibody immunospecifically binds to the primary antibody; (ii) detecting the presence of the secondary antibody bound to the primary antibody; and (iii) further determining the amount of the biomarker protein based on the amount of the detectable label in the secondary antibody.

[0172] In certain embodiments of the various methods provided herein, two or more of these steps are performed sequentially. In other embodiments of the methods provided herein, two or more of the steps are performed in parallel (e.g., at the same time).

[0173] 5.4. Subjects, Samples, and Cell Types In certain embodiments, the various methods provided herein use a sample (e.g., a biological sample) from a subject or individual (e.g., a patient). The subject can be a patient, such as a patient having CFS, for example. The subject can be a mammal, such as a human, for example. The subject can be male or female, and can be an adult, pediatric, or infant. The sample can be analyzed at a point in time during the active phase of CFS or when CFS is in the inactive phase. In certain embodiments, two or more samples are obtained from the subject.

[0174] In some embodiments, a subject (e.g., a patient) has one or more symptoms of CFS. In some embodiments, the subject is a patient having fatigue, e.g., a bedridden patient having fatigue. In some embodiments, this fatigue is caused by an infection, e.g., a viral infection or a bacterial infection. In some embodiments, the patient has had or has EBV, CMV, HHV-6, HHV-7. In some embodiments, the patient is positive for EBV, CMV, HHV-6A, HHV-6B, or HHV-7 (e.g., based on, for example, the detection of viral DNA in a blood sample, e.g., a plasma sample). In some embodiments, the patient has had a coronavirus (e.g., SARS-CoV-2) infection. In some embodiments, the patient has had symptoms of COVID-19.

[0175] In some embodiments, the patient has been diagnosed with an autoimmune disease. In some embodiments, the patient has or has had an autoimmune disease. In some embodiments, the autoimmune disease is Hashimoto's thyroiditis, fibromyalgia, inflammatory bowel disease (IBD) (e.g., Crohn's disease or ulcerative colitis), postural orthostatic tachycardia syndrome (POTS), Graves' disease, psoriasis, rheumatoid arthritis or Sjogren's syndrome. In some embodiments, the patient has been positive or has had a positive test result for autoantibodies indicating an autoimmune disease.

[0176] In certain embodiments, the sample used in the methods provided herein comprises a body fluid from a subject. Non-limiting examples of body fluids include blood (e.g., whole blood), plasma, amniotic fluid, aqueous humor, bile, cerumen, Cowper's gland fluid, bulbourethral gland fluid, chyle, chymous porridge, female ejaculate, interstitial fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, tears, urine, vaginal lubricant, vomit, water, feces, internal body fluids (including cerebrospinal fluid that surrounds the brain and spinal cord), synovial fluid, intracellular fluid (the fluid inside cells), and vitreous humor (the fluid in the eye). In some embodiments, the sample is a blood sample. Blood samples can be obtained using conventional techniques as described, for example, in Innis et al, eds., PCR Protocols (Academic Press, 1990). White blood cells can be isolated from blood samples using conventional techniques or commercially available kits, such as the RosetteSep kit (Stem Cell Technologies, Vancouver, Canada). Subpopulations of white blood cells, such as monocytes, B cells, T cells, macrophages, granulocytes, or lymphocytes, can be further isolated using conventional techniques, such as magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, California) or fluorescence-activated cell sorting (FACS) (Becton Dickinson, San Jose, California).

[0177] In one embodiment, the blood sample is from about 0.1 mL to about 10.0 mL, from about 0.2 mL to about 7 mL, from about 0.3 mL to about 5 mL, from about 0.4 mL to about 3.5 mL, or from about 0.5 mL to about 3.0 mL. In another embodiment, the blood sample is about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1.0 mL, about 1.5 mL, about 2.0 mL, about 2.5 mL, about 3.0 mL, about 3.5 mL, about 4.0 mL, about 4.5 mL, about 5.0 mL, about 6.0 mL, about 7.0 mL, about 8.0 mL, about 9.0 mL, or about 10.0 mL.

[0178] In some embodiments, the sample used in the method includes a biopsy. The biopsy can be from any organ or tissue, such as skin, liver, lung, heart, colon, kidney, bone marrow, teeth, lymph nodes, hair, spleen, brain, breast, or other organs. For the isolation of a sample from a subject, any biopsy technique known to those skilled in the art can be used, such as open biopsy, closed biopsy, core biopsy, incisional biopsy, excisional biopsy, or fine needle aspiration biopsy.

[0179] In one embodiment, the sample used in the method provided herein is obtained from a subject before the subject receives treatment for a disease or disorder. In another embodiment, the sample is obtained from a subject while the subject is receiving treatment for a disease or disorder. In another embodiment, the sample is obtained from a subject after the subject has received treatment for a disease or disorder. In various embodiments, the treatment includes administering a compound to the subject.

[0180] In certain embodiments, the sample used in the method provided herein includes a plurality of cells. In certain embodiments, the number of cells used in the method provided herein can range from a single cell to about 10 9 cells. In some embodiments, the number of cells used in the method provided herein is about 1×10 4 cells, about 5×10 4 cells, about 1×10 5 cells, about 5×10 5 cells, about 1×10 6 cells, about 5×10 6 cells, about 1×10 7 cells, about 5×10 7 cells, about 1×10 8 cells, about 5×10 8 cells, or about 1×10 9 cells.

[0181] The number and type of cells collected from a subject can be monitored by measuring changes in cell surface markers using standard cell detection techniques such as, for example, flow cytometry, cell sorting, immunocytochemistry (e.g., staining with tissue-specific or cell marker-specific antibodies), fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), etc., or by examining cell morphology using optical or confocal microscopy, and / or by measuring changes in gene expression using techniques well known in the art such as PCR and gene expression profiling. These techniques can also be used to identify cells that are positive for one or more specific markers.

[0182] In certain embodiments, a subset of cells is used in the methods provided herein. Specific methods for the selection and isolation of cell populations are well known in the art and can be based on cell size, morphology, or intracellular or extracellular markers. Such methods include, but are not limited to, flow cytometry, flow sorting, FACS, bead-based separation methods such as magnetic cell sorting, size-based separation methods (e.g., sieves, obstacle arrays, or filters), sorting in microfluidic devices, antibody-based separation methods, sedimentation, affinity adsorption, affinity extraction, density gradient centrifugation, laser capture microdissection, etc. Fluorescence-activated cell sorting (FACS) is a well-known method for separating particles, including cells, based on the fluorescence characteristics of the particles (Kamarch, Methods Enzymol. 1987, 151:150-165). When the fluorescent moiety in an individual particle is laser-excited, a slight electrical change occurs, allowing for the electromagnetic separation of positive and negative particles from a mixture. In one embodiment, cell surface marker-specific antibodies or ligands are labeled with a characteristic fluorescent label. When the cells are processed through a cell sorter, separation of the cells based on their ability to bind the antibody used becomes possible. Particles that have undergone FACS sorting can be directly placed into individual wells of a 96-well or 384-well plate, which can facilitate separation and cloning.

[0183] In one embodiment, RNA (e.g., mRNA) or protein is purified, and the presence or absence of a biomarker is measured by gene or protein expression analysis. In certain embodiments, the presence or absence of a biomarker is measured by quantitative real-time PCR (qRT-PCR), microarray, flow cytometry, or immunofluorescence. In other embodiments, the presence or absence of a biomarker is measured by ELISA or other similar methods known in the art. For other exemplary methods, see Section 5.3 above.

[0184] 5.5. Kits In another aspect, provided herein are kits for performing the methods provided herein. In some embodiments, provided herein are kits for identifying a subject having CFS or for confirming CFS in a subject, the kit comprising agents for obtaining the expression level of a biomarker in a sample from the subject, wherein the biomarker is selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the kit further comprises instructions for identifying or confirming that the subject has CFS if the expression level of the biomarker is higher than the reference expression level of the biomarker. In some embodiments, the kit provides (e.g., in the instructions) the reference expression level of the biomarker. In some embodiments, the kit further comprises means for obtaining a sample from the subject.

[0185] In some embodiments, provided herein is a kit for determining the severity of CFS in a subject, the kit comprising an agent for obtaining the expression level of a biomarker in a sample from the subject, wherein the biomarker is selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the kit further comprises instructions regarding how to determine the severity of CFS. In some embodiments, the kit provides (e.g., in the instructions) a reference expression level of the biomarker. In some embodiments, the kit provides instructions regarding comparing the expression level of the biomarker to the reference level of the biomarker and determining the severity of CFS based on the comparison. In some embodiments, the kit further comprises an implement for obtaining a sample from the subject.

[0186] In some embodiments, provided herein is a kit for identifying a subject likely or unlikely to respond to treatment of CFS, or predicting whether a subject will respond to treatment of CFS, the kit comprising an agent for obtaining the expression level of a biomarker in a sample from the subject, wherein the biomarker is an agent selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the kit further comprises instructions on how to identify a subject likely or unlikely to respond to treatment of CFS, or how to predict whether a subject will respond to treatment of CFS. In some embodiments, the kit provides (e.g., in the instructions) a reference expression level of the biomarker. In some embodiments, the kit provides instructions for identifying or predicting that a subject is likely to respond to treatment of CFS if the expression level of the biomarker is higher than the reference expression level of the biomarker. In some embodiments, the kit further comprises an instrument for obtaining a sample from the subject. In some embodiments, the kit is for identifying a subject likely or unlikely to respond to an immunomodulatory drug (IMiD), or predicting whether a subject will respond to an IMiD. In other embodiments, the kit is for identifying a subject likely or unlikely to respond to a CRBN modulator or a compound having the ability to bind to CRBN and / or induce a conformational change thereof, or predicting whether a subject will respond to a CRBN modulator or a compound having the ability to bind to CRBN and / or induce a conformational change thereof. In some embodiments, the kit is for identifying a subject likely or unlikely to respond to an agent that depletes B cells (e.g., an anti-CD20 antibody, e.g., rituximab), or predicting whether a subject will respond to an agent that depletes B cells (e.g., an anti-CD20 antibody, e.g., rituximab).

[0187] In some embodiments of the various kits provided herein, the reference expression level of a biomarker provided in the kit is a predetermined expression level of the biomarker from a public database. In some embodiments of the various kits provided herein, the reference expression level of a biomarker provided in the kit is the expression level of the biomarker in a healthy subject or a subject without CFS. In some embodiments, the reference expression level of the biomarker is the expression level of the biomarker in a subject with moderate CFS. In some embodiments of the various kits provided herein, the reference expression level of a biomarker provided in the kit is the expression level of the biomarker determined based on a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without CFS, or a cohort of subjects with moderate CFS). In some embodiments, the reference expression level of the biomarker is the median or average value of the expression levels of the biomarker in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without CFS, or a cohort of subjects with moderate CFS). In some embodiments, the biomarker is HSPA8 or ABCE1, and the reference expression level of the biomarker is the expression level of the biomarker in a healthy subject or a subject without CFS, or a cohort of healthy subjects or subjects without CFS. In some embodiments, the biomarker is selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8, and the reference expression level of the biomarker is the expression level of the biomarker in a subject with mild CFS or a cohort of subjects with mild CFS.

[0188] In other embodiments, provided herein is a kit for determining or monitoring the effectiveness of treatment in a subject having CFS, the kit comprising an agent for obtaining a first expression level of a biomarker in a first sample from the subject, wherein the biomarker is selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8; and an agent for obtaining a second expression level of the biomarker in a second sample obtained from the subject after administration of treatment to the subject. In some embodiments, the kit further comprises instructions regarding how to determine or monitor the effectiveness of treatment in a subject having CFS. In some embodiments, the kit provides instructions for determining the effectiveness of treatment based on a comparison of the first expression level and the second expression level. In some embodiments, the kit provides instructions for determining that the treatment is effective if the second expression level is lower than the first expression level. In some embodiments, the kit further comprises an instrument for obtaining a sample from the subject. In some embodiments, the kit is for determining or monitoring the effectiveness of an immunomodulatory drug (IMiD) in a subject having CFS. In some embodiments, the kit is for determining or monitoring the effectiveness of a CRBN modulator or a compound having the ability to bind to CRBN and / or induce a conformational change thereof in a subject having CFS. In some embodiments, the kit is for determining or monitoring the effectiveness of an agent that depletes B cells (e.g., an anti-CD20 antibody, e.g., rituximab) in a subject having CFS. In some embodiments, the kit further comprises instructions regarding determining or adjusting the dosage of treatment for the subject.

[0189] In yet other embodiments, provided herein is a kit for screening the efficacy of a compound in treating CFS, the kit comprising an agent for obtaining a first level of a biomarker in a sample, wherein the biomarker is selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8; and an agent for obtaining a second level of the biomarker in the sample after administering the compound to the sample. In some embodiments, the kit further comprises instructions on how to select a compound for treating CFS. In some embodiments, the kit provides instructions for selecting the compound if the second level is lower than the first level. In some embodiments, the kit further comprises an instrument for obtaining the sample. In some embodiments, the kit is for screening the efficacy of an immunomodulatory drug (IMiD) in treating CFS. In some embodiments, the kit is for screening the efficacy of a compound having the ability to modulate CRBN or bind to CRBN and / or induce a conformational change thereof in treating CFS. In some embodiments, the kit is for screening the efficacy of an agent that depletes B cells (e.g., an anti-CD20 antibody, e.g., rituximab) in treating CFS.

[0190] In yet other embodiments, provided herein is a kit for identifying a subject having long COVID or for verifying long COVID in a subject, the kit comprising an agent for determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a CAP selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D. In some embodiments, the kit further comprises instructions for identifying or verifying that the subject has long COVID if the expression level of the biomarker is higher than the reference expression level of the biomarker. In some embodiments, the kit provides a reference expression level of the biomarker. In some embodiments, the kit further comprises an instrument for obtaining a sample from the subject.

[0191] In some embodiments, provided herein is a kit for identifying a subject likely or unlikely to respond to treatment for long COVID or for predicting whether a subject will respond to treatment for long COVID, the kit comprising an agent for determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a CAP selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D. In some embodiments, the kit further comprises instructions for identifying or predicting that the subject is likely to respond to treatment if the expression level of the biomarker is higher than the reference expression level of the biomarker. In certain embodiments, the kit further comprises instructions for administering treatment to a subject identified or predicted to be likely to respond to treatment. In some embodiments, the kit provides a reference expression level of the biomarker. In some embodiments, the kit further comprises an instrument for obtaining a sample from the subject.

[0192] In some embodiments, provided herein is a kit for selectively treating a subject having or suspected of having long COVID, the kit comprising an agent for determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a CAP selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D. In some embodiments, the kit further comprises instructions for identifying or predicting that the subject is likely to respond to treatment for long COVID if the expression level of the biomarker is higher than a reference expression level of the biomarker. In certain embodiments, the kit further comprises instructions for administering treatment to a subject identified or predicted to be likely to respond to treatment. In some embodiments, the kit provides a reference expression level of the biomarker. In some embodiments, the kit further comprises an implement for obtaining a sample from the subject.

[0193] In some embodiments of the various kits provided herein, the reference expression level of a biomarker is a predetermined expression level of the biomarker. In some embodiments, the reference expression level of a biomarker is a predetermined expression level of the biomarker obtained from a public database. In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker in a healthy subject or a subject without long COVID. In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker in a subject with acute COVID (e.g., severe acute COVID). In some embodiments, the reference expression level of a biomarker is the expression level of the biomarker determined based on a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without long COVID, or a cohort of subjects with acute COVID (e.g., severe acute COVID)). In some embodiments, the reference expression level of a biomarker is the median or average value of the expression levels of the biomarker in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without long COVID, a cohort of subjects with acute COVID (e.g., severe acute COVID)).

[0194] In some embodiments of the various kits provided herein, the biomarker is HSPA8 or IKZF3, and the reference expression level of the biomarker is the expression level of the biomarker in a healthy subject or a subject without long COVID, or in a cohort of healthy subjects or subjects without long COVID. In some embodiments, the biomarker is selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D, and the reference expression level of the biomarker is the expression level of the biomarker in a subject with acute COVID (e.g., severe acute COVID) or in a cohort of subjects with acute COVID (e.g., severe acute COVID).

[0195] In some embodiments of the various kits provided herein, the kit comprises identifying that a subject has Long COVID if the expression level of a biomarker is at least about 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% higher than the reference expression level of the biomarker. The expression level of the biomarker can be determined using any known method in the art, and exemplary methods are described in more detail in Section 5.3 below. In some embodiments, a level is determined to be higher than the reference level if it is higher (e.g., statistically significantly higher) than the reference level when observed by a measurement assay.

[0196] In some embodiments of the various kits provided herein, when multiple biomarkers are used, the expression level of each biomarker is compared to the reference expression level of that biomarker, and the kit includes instructions for identifying or confirming a subject having long COVID, or for identifying or predicting that a subject is likely to respond to long COVID treatment if the expression level of each biomarker is higher than the reference expression level of that biomarker. In other embodiments, when multiple biomarkers are used, an overall score is calculated based on the multiple biomarkers and compared to a reference overall score. In some embodiments, the kit includes instructions for identifying or confirming a subject having long COVID, or for identifying or predicting that a subject is likely to respond to long COVID treatment if the overall score is higher than the reference overall score. In some embodiments, the overall score is calculated using the median Z-score method. Briefly, the median Z-score is determined by first calculating the mean value of each gene from all samples within the gene expression matrix. Next, this mean value is subtracted from each corresponding gene for all samples, and then scaling is performed by dividing the value by its standard deviation. The median of the scaled values from the desired multiple genes forms the overall score. Another exemplary method for calculating the overall score is the single-sample gene set enrichment (ssGSEA) method. The single-sample gene score represents the degree to which genes within a particular gene set are coordinately upregulated or downregulated within a sample. This score is calculated by adjusting the cumulative sum statistic based on a decreasing walk through the ranked expression list. The enrichment score is the maximum deviation from zero encountered during the walk; this corresponds to a weighted Kolmogorov-Smirnov-like statistic (see, e.g., Subramanian et al., PNAS, 102(43):15545-15550 (2005); and Barbie et al., Nature, 462(7269):108-112).

[0197] In other embodiments, provided herein is a kit for determining or monitoring the effectiveness of treatment in a subject having long COVID, the kit comprising an agent for obtaining a first expression level of a biomarker in a first sample from the subject, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8; and an agent for obtaining a second expression level of the biomarker in a second sample obtained from the subject after administration of treatment to the subject. In some embodiments, the kit further comprises instructions regarding how to determine or monitor the effectiveness of treatment in a subject having long COVID. In some embodiments, the kit provides instructions for determining the effectiveness of treatment based on a comparison of the first expression level and the second expression level. In some embodiments, the kit provides instructions for determining that the treatment is effective if the second expression level is lower than the first expression level. In some embodiments, the kit further comprises an implement for obtaining a sample from the subject. In some embodiments, the kit is for determining or monitoring the effectiveness of an immunomodulatory drug (IMiD) in a subject having long COVID. In some embodiments, the kit is for determining or monitoring the effectiveness of a CRBN modulator or a compound having the ability to bind to CRBN and / or induce a conformational change thereof in a subject having long COVID. In some embodiments, the kit is for determining or monitoring the effectiveness of an agent that depletes B cells (e.g., an anti-CD20 antibody, e.g., rituximab) in a subject having long COVID.

[0198] In certain embodiments, the kit further comprises instructions for determining or adjusting (e.g., increasing) the dosage of the treatment or administering a different long COVID treatment to the subject if the second expression level is not lower than the first expression level.

[0199] In yet other embodiments, provided herein is a kit for screening the effectiveness of a compound in treating long COVID, the kit comprising an agent for obtaining a first level of a biomarker in a sample, wherein the biomarker is a CAP selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8; and an agent for obtaining a second level of the biomarker in the sample after administering the compound to the sample. In some embodiments, the kit further comprises instructions on how to select a compound for treating long COVID. In some embodiments, the kit provides instructions for selecting a compound if the second level is lower than the first level. In some embodiments, the kit further comprises an instrument for obtaining the sample. In some embodiments, the kit is for screening the effectiveness of an immunomodulatory drug (IMiD) in treating long COVID. In some embodiments, the kit is for screening the effectiveness of a compound having the ability to modulate CRBN or bind to CRBN and / or induce a conformational change thereof in treating long COVID. In some embodiments, the kit is for screening the effectiveness of an agent for depleting B cells (e.g., an anti-CD20 antibody, e.g., rituximab) in treating long COVID.

[0200] In some embodiments, the kit provided herein comprises instructions for determining that the treatment is effective if the second expression level is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower than the first expression level. Based on the comparison of the first expression level and the second expression level, in one or more subsequent treatment cycles, different treatments or different dosage regimens may be administered to the subject.

[0201] In some embodiments, the treatments disclosed herein include immunomodulatory drugs (IMiDs) as disclosed in Section 5.2. In some embodiments, the treatment includes a CRBN modulator or a compound having the ability to bind to CRBN and / or induce a conformational change thereof as disclosed in Section 5.2. In some embodiments, the treatment includes an agent that depletes B cells (e.g., an anti-CD20 antibody, e.g., rituximab) as disclosed in Section 5.2.

[0202] In some embodiments of the various kits provided herein, the kit includes an agent for determining / measuring the expression levels of two or more biomarkers, three or more biomarkers, four or more biomarkers, five or more biomarkers, or all of the biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8.

[0203] In some embodiments, the kit comprises an agent for determining / measuring the expression levels of IKZF2 and at least 1, 2, 3, or 4 of IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the kit comprises an agent for determining / measuring the expression levels of IKZF3 and at least 1, 2, 3, or 4 of IKZF2, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the kit comprises an agent for determining / measuring the expression levels of ABCE1 and at least 1, 2, 3, or 4 of IKZF2, IKZF3, BACH2, CD3D, and HSPA8. In some embodiments, the kit comprises an agent for determining / measuring the expression levels of BACH2 and at least 1, 2, 3, or 4 of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8. In some embodiments, the kit comprises an agent for determining / measuring the expression levels of CD3D and at least 1, 2, 3, or 4 of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8. In some embodiments, the kit comprises an agent for determining / measuring the expression levels of HSPA8 and at least 1, 2, 3, or 4 of IKZF2, IKZF3, ABCE1, BACH2, and CD3D. In some embodiments, the kit comprises an agent for determining / measuring the expression levels of all biomarkers in the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the kit comprises an agent for determining / measuring the expression level of IKZF2. In some embodiments, the kit comprises an agent for determining / measuring the expression level of IKZF3. In some embodiments, the kit comprises an agent for determining / measuring the expression level of ABCE1. In some embodiments, the kit comprises an agent for determining / measuring the expression level of BACH2. In some embodiments, the kit comprises an agent for determining / measuring the expression level of CD3D. In some embodiments, the kit comprises an agent for determining / measuring the expression level of HSPA8.In certain embodiments, the kit comprises an agent for determining / measuring the expression levels of two or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the kit comprises an agent for determining / measuring the expression levels of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the kit comprises an agent for determining / measuring the expression levels of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the kit comprises an agent for determining / measuring the expression levels of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the kit comprises an agent for determining / measuring the expression levels of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the kit comprises an agent for determining / measuring the expression levels of IKZF2 and IKZF3. In some embodiments, the kit comprises an agent for determining / measuring the expression levels of IKZF2 and ABCE1. In some embodiments, the kit comprises an agent for determining / measuring the expression levels of IKZF2 and BACH2. In some embodiments, the kit comprises an agent for determining / measuring the expression levels of IKZF2 and CD3D. In some embodiments, the kit comprises an agent for determining / measuring the expression levels of IKZF2 and HSPA8. In some embodiments, the kit comprises an agent for determining / measuring the expression levels of IKZF3 and ABCE1. In some embodiments, the kit comprises an agent for determining / measuring the expression levels of IKZF3 and BACH2. In some embodiments, the kit comprises an agent for determining / measuring the expression levels of IKZF3 and CD3D. In some embodiments, the kit comprises an agent for determining / measuring the expression levels of IKZF3 and HSPA8. In some embodiments, the kit comprises an agent for determining / measuring the expression levels of ABCE1 and BACH2. In some embodiments, the kit comprises an agent for determining / measuring the expression levels of ABCE1 and CD3D.In some embodiments, the kit includes agents for determining / measuring the expression levels of ABCE1 and HSPA8. In some embodiments, the kit includes agents for determining / measuring the expression levels of BACH2 and CD3D. In some embodiments, the kit includes agents for determining / measuring the expression levels of BACH2 and HSPA8. In some embodiments, the kit includes agents for determining / measuring the expression levels of CD3D and HSPA8. In some embodiments, the kit includes agents for determining / measuring the expression levels of IKZF2, IKZF3, and ABCE1. In some embodiments, the kit includes agents for determining / measuring the expression levels of IKZF2, IKZF3, and BACH2. In some embodiments, the kit includes agents for determining / measuring the expression levels of IKZF2, IKZF3, and CD3D. In some embodiments, the kit includes agents for determining / measuring the expression levels of IKZF2, IKZF3, and HSPA8. In some embodiments, the kit includes agents for determining / measuring the expression levels of IKZF2, ABCE1, and BACH2. In some embodiments, the kit includes agents for determining / measuring the expression levels of IKZF2, ABCE1, and CD3D. In some embodiments, the kit includes agents for determining / measuring the expression levels of IKZF2, ABCE1, and HSPA8. In some embodiments, the kit includes agents for determining / measuring the expression levels of IKZF2, BACH2, and CD3D. In some embodiments, the kit includes agents for determining / measuring the expression levels of IKZF2, BACH2, and HSPA8. In some embodiments, the kit includes agents for determining / measuring the expression levels of IKZF2, CD3D, and HSPA8. In some embodiments, the kit includes agents for determining / measuring the expression levels of IKZF3, ABCE1, and BACH2. In some embodiments, the kit includes agents for determining / measuring the expression levels of IKZF3, ABCE1, and CD3D. In some embodiments, the kit includes agents for determining / measuring the expression levels of IKZF3, ABCE1, and HSPA8.In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF3, BACH2, and CD3D. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF3, BACH2, and HSPA8. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF3, CD3D, and HSPA8. In some embodiments, the kit comprises agents for determining / measuring the expression levels of ABCE1, BACH2, and CD3D. In some embodiments, the kit comprises agents for determining / measuring the expression levels of ABCE1, BACH2, and HSPA8. In some embodiments, the kit comprises agents for determining / measuring the expression levels of ABCE1, CD3D, and HSPA8. In some embodiments, the kit comprises agents for determining / measuring the expression levels of BACH2, CD3D, and HSPA8. In some embodiments, the kit comprises agents for determining / measuring the expression levels of ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF3, BACH2, CD3D, and HSPA8. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF3, ABCE1, CD3D, and HSPA8. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF3, ABCE1, BACH2, and HSPA8. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF3, ABCE1, BACH2, and CD3D. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF2, BACH2, CD3D, and HSPA8. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF2, ABCE1, CD3D, and HSPA8. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF2, ABCE1, BACH2, and HSPA8. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF2, ABCE1, BACH2, and CD3D.In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF2, IKZF3, CD3D and HSPA8. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF2, IKZF3, BACH2 and HSPA8. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF2, IKZF3, BACH2 and CD3D. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF2, IKZF3, ABCE1 and HSPA8. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF2, IKZF3, ABCE1 and CD3D. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF2, IKZF3, ABCE1 and BACH2. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF3, ABCE1, BACH2, CD3D and HSPA8. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF2, ABCE1, BACH2, CD3D and HSPA8. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF2, IKZF3, BACH2, CD3D and HSPA8. In other embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF2, IKZF3, ABCE1, CD3D and HSPA8. In other embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF2, IKZF3, ABCE1, BACH2 and HSPA8. In other embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF2, IKZF3, ABCE1, BACH2 and CD3D. In some embodiments, the kit comprises agents for determining / measuring the expression levels of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8.

[0204] In some embodiments, when the kit contains agents for multiple biomarkers, the kit provides the reference expression levels of each biomarker. In other embodiments, the kit provides a reference comprehensive score for multiple biomarkers.

[0205] In some embodiments, the kit contains an agent for determining protein levels. In other embodiments, the kit contains an agent for determining mRNA levels. In still other embodiments, the kit contains an agent for determining cDNA levels.

[0206] In certain embodiments, provided herein is a kit for detecting the mRNA levels of one or more biomarkers. In certain embodiments, the kit contains one or more probes that specifically bind to the mRNA of one or more biomarkers. In certain embodiments, the kit further contains a washing solution. In certain embodiments, the kit further contains reagents for performing a hybridization assay, mRNA isolation or purification means, detection means, and positive and negative controls. In certain embodiments, the kit further contains instructions for using the kit. The kit can be adapted for home use, clinical use, or research use.

[0207] In certain embodiments, provided herein is a kit for detecting the protein levels of one or more biomarkers. In certain embodiments, the kit contains a dipstick coated with an antibody...

Claims

1. A composition comprising an agent for determining the expression level of at least one biomarker selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D, for use in a method for (i) identifying a subject having chronic fatigue syndrome (CFS) or confirming CFS in a subject, (ii) determining the severity of the CFS in a subject, (iii) identifying a subject that is likely or unlikely to respond to treatment for CFS or predicting whether a subject will respond to treatment for CFS, or (iv) selectively treating a subject that has or is suspected of having CFS, wherein the method is (a) Determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a cereblon (CRBN)-related protein (CAP) selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2 and CD3D; and (b) (i) If the expression level of the biomarker is higher than the reference expression level of the biomarker, identify or confirm that the subject has CFS, and optionally, the subject complains of chronic debilitating fatigue, unrecoverable sleep, mental and / or physical distress, neurological and cognitive impairment, and / or autoimmune or immunodeficiency, and optionally, the subject identified or confirmed to have CFS is socially withdrawn and / or bedridden; (ii) Determining the severity of the CFS in the subject based on a comparison of the expression level of the biomarker with the reference expression level of the biomarker, wherein, optionally, if the expression level of the biomarker is higher than the reference expression level of the biomarker, the severity of the CFS is determined to be severe, and optionally, the reference expression level of the biomarker is determined to be the expression level of the biomarker in a subject with mild CFS or in a cohort of subjects with mild CFS; or (iii) Identifying or predicting that a subject is likely to respond to the treatment if the expression level of the biomarker is higher than the reference expression level of the biomarker, wherein the method optionally further comprises administering the treatment to the subject identified or predicted to be likely to respond to the treatment. A composition containing the following:

2. The reference expression level of the biomarker is (i) A predetermined expression level of the biomarker; (ii) The expression level of the biomarker in subjects without CFS or in a cohort of subjects without CFS; (iii) The expression level of the biomarker in healthy subjects or a cohort of healthy subjects; or (iv) The expression level of the biomarker in subjects with mild CFS or in a cohort of subjects with mild CFS. The composition according to claim 1.

3. The method described above is (i) IKZF2, ABCE1, BACH2 and HSPA8; (ii) IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8; (iii) Two, three, four, five, or all of the biomarkers selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2, and CD3D; (iv) IKZF2 and at least one, two, three, or four of IKZF3, ABCE1, BACH2, CD3D, and HSPA8; (v) IKZF3 and at least one, two, three, or four of IKZF2, ABCE1, BACH2, CD3D, and HSPA8; (vi) ABCE1 and at least one, two, three, or four of IKZF2, IKZF3, BACH2, CD3D, and HSPA8; (vii) BACH2 and at least one, two, three, or four of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8; (viiii) CD3D and at least one, two, three, or four of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8; (ix) HSPA8 and at least one, two, three or four of IKZF2, IKZF3, ABCE1, BACH2 and CD3D; or (x) HSPA8 or ABCE1 The composition according to claim 1, comprising determining the expression level of the above.

4. The composition according to claim 3, wherein the method comprises (i) comparing the expression level of each of the biomarkers with its respective reference expression level, or (ii) obtaining a total score based on the expression levels of the biomarkers and comparing the total score with a reference score obtained from the reference expression levels of the biomarkers.

5. A composition comprising a drug for determining the expression level of at least one biomarker selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D, for use in a method for determining or monitoring the effectiveness of treatment in a subject having CFS, or (ii) a method for screening the effectiveness of treatment for CFS in a subject requiring treatment, wherein the method is (a) Determining a first expression level of a biomarker in a first sample obtained from the subject before administering the treatment to the subject, wherein the biomarker is a cereblon (CRBN)-related protein (CAP) selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2, and CD3D; (b) Performing the treatment on the subject; (c) Determining the second expression level of the biomarker in a second sample obtained from the subject after the subject has undergone the treatment; and (d) (i) Determining the effectiveness of the treatment based on a comparison of the first expression level and the second expression level, the method optionally includes determining that the treatment is effective if the second expression level is lower than the first expression level, and further optionally including determining or adjusting the dose of the treatment for the subject; or (ii) If the second expression level is lower than the first expression level, the treatment is selected. A composition containing the following:

6. The method described above is (i) IKZF2, ABCE1, BACH2 and HSPA8; (ii) IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8; (iii) Two, three, four, five, or all of the biomarkers selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2, and CD3D; (iv) IKZF2 and at least one, two, three, or four of IKZF3, ABCE1, BACH2, CD3D, and HSPA8; (v) IKZF3 and at least one, two, three, or four of IKZF2, ABCE1, BACH2, CD3D, and HSPA8; (vi) ABCE1 and at least one, two, three, or four of IKZF2, IKZF3, BACH2, CD3D, and HSPA8; (vii) BACH2 and at least one, two, three, or four of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8; (viiii) CD3D and at least one, two, three, or four of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8; (ix) HSPA8 and at least one, two, three or four of IKZF2, IKZF3, ABCE1, BACH2 and CD3D; or (x) HSPA8 or ABCE1 The composition according to claim 5, comprising determining the first and second expression levels of the first.

7. The composition according to claim 6, wherein the method comprises (i) comparing the first expression level of each of the biomarkers with its respective second expression level, or (ii) obtaining a first overall score based on the first expression level of the biomarkers and a second overall score based on the second expression level of the biomarkers, and comparing the first overall score with the second overall score.

8. The composition according to any one of claims 1 to 7, wherein the treatment comprises (i) an immunomodulator (IMiD), (ii) a cereblon (CRBN) modifier or a compound having the ability to bind to and / or induce a conformational change thereof, or (iii) a B cell depleting agent.

9. The composition according to any one of claims 1 to 7, wherein the CFS is related to an autoimmune disease or viral infection.

10. A composition comprising a drug for determining the expression level of at least one biomarker selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D, for use in a method for (i) identifying a subject having long COVID or confirming long COVID in a subject, (ii) identifying a subject that is likely or unlikely to respond to treatment for long COVID or predicting whether a subject will respond to treatment for long COVID, or (iii) selectively treating a subject having or suspected of having long COVID, wherein the method is (a) Determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a cereblon (CRBN)-related protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2 and CD3D; and (b) (i) Identifying or confirming that the subject has long COVID if the expression level of the biomarker is higher than the reference expression level of the biomarker; or (ii) Identifying or predicting that a subject is likely to respond to treatment for long COVID if the expression level of the biomarker is higher than the reference expression level of the biomarker, the method optionally further comprising administering the treatment to the subject identified or predicted to be likely to respond to the treatment. A composition comprising, optionally, the subject having previously contracted COVID-19.

11. The reference expression level of the biomarker is (i) A predetermined expression level of the biomarker; (ii) The expression level of the biomarker in subjects who do not have long COVID or in a cohort of subjects who do not have long COVID; (iii) The expression level of the biomarker in healthy subjects or a cohort of healthy subjects; or (iv) The expression level of the biomarker in subjects with acute COVID-19 or in a cohort of subjects with acute COVID-19 The composition according to claim 10.

12. The method described above is (i) Two, three, four, five, or all of the biomarkers selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; (ii) IKZF2 and at least one, two, three, or four of IKZF3, ABCE1, BACH2, CD3D, and HSPA8; (iii) IKZF3 and at least one, two, three, or four of IKZF2, ABCE1, BACH2, CD3D, and HSPA8; (iv) ABCE1 and at least one, two, three, or four of IKZF2, IKZF3, BACH2, CD3D, and HSPA8; (v) BACH2 and at least one, two, three, or four of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8; (vi) CD3D and at least one, two, three, or four of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8; (vii) HSPA8 and at least one, two, three, or four of IKZF2, IKZF3, ABCE1, BACH2, and CD3D; (viiii) HSPA8, ABCE1, IKZF2, BACH2 and CD3D; or (ix) HSPA8 or ABCE1 The composition according to claim 10, comprising determining the expression level of the above.

13. The composition according to claim 12, comprising comparing the expression level of each of the biomarkers with their respective reference expression levels, or obtaining a total score based on the expression levels of the biomarkers and comparing the total score with a reference score obtained from the reference expression levels of the biomarkers.

14. (i) a method for determining or monitoring the effectiveness of treatment in subjects with long COVID, or (ii) a method for screening the effectiveness of treatment for long COVID in subjects requiring treatment, comprising a composition for determining the expression level of at least one biomarker selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2 and CD3D, wherein the method (a) Determining a first expression level of a biomarker in a first sample obtained from the subject before administering the treatment to the subject, wherein the biomarker is a cereblon (CRBN)-related protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; (b) Performing the treatment on the subject; (c) Determining the second expression level of the biomarker in a second sample obtained from the subject after the subject has undergone the treatment; and (d) (i) Determining the effectiveness of the treatment based on a comparison of the first expression level and the second expression level, wherein the method optionally determines that the treatment is effective if the second expression level is lower than the first expression level, and further optionally the method further determines or adjusts the dose of the treatment for the subject; or (ii) Comparing the first expression level with the second expression level, and selecting the treatment if the second expression level is lower than the first expression level. A composition comprising, optionally, the subject having previously contracted COVID-19.

15. The method described above is (i) Two, three, four, five, or all of the biomarkers selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; (ii) IKZF2 and at least one, two, three, or four of IKZF3, ABCE1, BACH2, CD3D, and HSPA8; (iii) IKZF3 and at least one, two, three, or four of IKZF2, ABCE1, BACH2, CD3D, and HSPA8; (iv) ABCE1 and at least one, two, three, or four of IKZF2, IKZF3, BACH2, CD3D, and HSPA8; (v) BACH2 and at least one, two, three, or four of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8; (vi) CD3D and at least one, two, three, or four of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8; (vii) HSPA8 and at least one, two, three, or four of IKZF2, IKZF3, ABCE1, BACH2, and CD3D; (viiii) HSPA8, ABCE1, IKZF2, IKZF3, BACH2 and CD3D; or (ix) HSPA8 or ABCE1 The composition according to claim 14, comprising determining the first and second expression levels of the first.

16. The composition according to claim 15, wherein the method comprises (i) comparing the first expression level of each of the biomarkers with the second expression level thereof; or (ii) obtaining a first overall score based on the first expression level of the biomarkers and a second overall score based on the second expression level of the biomarkers, and comparing the first overall score with the second overall score.

17. The composition according to any one of claims 10 to 16, wherein the treatment comprises (i) an immunomodulator (IMiD), (ii) a CRBN modifier or a compound having the ability to bind to and / or induce a conformational change thereof, or (iii) a B cell depleting agent.

18. The expression level of the biomarker is (i) Measuring the mRNA level of the biomarker, wherein the mRNA level is optionally determined by quantitative reverse transcriptase PCR (RT-qPCR), microarray, Northern blotting, or RNA sequencing; or (ii) Measuring the protein level of the biomarker, wherein the protein level of the biomarker is determined by any choice of mass spectrometry (MS), liquid chromatography-tandem mass spectrometry (LC-MS / MS), immunoassay, flow cytometry, immunohistochemistry, Western blotting, or enzyme-linked immunosorbent assay (ELISA). The composition according to claim 1, 5, 10, or 14, as determined by...

19. A kit comprising the composition according to claim 1, 5, 10, or 14, wherein the kit optionally further comprises equipment for obtaining the sample, instructions for interpreting the determined expression level, and / or the reference expression level of the biomarker.

20. The composition according to claim 1, 5, 10, or 14, wherein the subject determined to have severe CFS is confined and / or bedridden, and the subject or cohort of the subject having mild CFS is not confined and / or bedridden, and optionally, the subject determined to have severe CFS has an autoimmune disease, or the cohort of the subject having mild CFS has an autoimmune disease.

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