Biomarkers for chronic fatigue syndrome and long COVID and uses thereof

Abstract

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

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 346,698, filed on May 27, 2022, the contents of which are incorporated herein by reference in their entirety. 1. Technical Field

[0002] The present disclosure provides methods and kits for predicting and monitoring post-viral syndromes (e.g., chronic fatigue syndrome (CFS) and / or long COVID) using certain biomarkers, selectively treating such syndromes, and assessing clinical sensitivity to treatment and therapeutic response. 2. Background technology

[0003] Post-viral syndromes, which may include chronic fatigue syndrome (CFS) and / or long COVID, are complex conditions that can involve physical, cognitive, emotional, and neurological difficulties. These conditions can cause symptoms including fatigue, post-exertional malaise, cognitive dysfunction, sensory-motor symptoms, headaches, memory problems, insomnia, muscle pain, palpitations, shortness of breath, dizziness and balance problems, speech and language problems, joint pain, and chest tightness. Symptoms may persist for weeks, months, or longer after the viral infection has cleared. In some cases, affected patients become housebound and / or bedridden.

[0004] CFS, also known as myalgic encephalomyelitis (ME), is a debilitating condition that can lead to severe impairment of physical, psychological, cognitive, social, and occupational functioning. Although the causes of CFS remain controversial, complex, and not fully understood, CFS can develop following a viral infection. To date, there is no clinical diagnostic test for CFS. Although some therapies have been used off-label to treat the symptoms of CFS, there are limited, if any, specific therapies for CFS.

[0005] Some people who have been infected with the virus that causes COVID-19 may experience long-term effects from their infection, called long COVID. Long COVID is also called post-COVID illness, long-haul COVID, post-acute COVID-19, long-term effects of COVID, and chronic COVID. People with long COVID can have a wide range of symptoms that may last weeks, months, or even years after infection.

[0006] There remains a need in the art for methods to identify, characterize, and treat CFS, long COVID, and post-viral syndromes associated with fatigue. 3. Summary of the invention

[0007] In one aspect, provided herein is a method for identifying a subject having chronic fatigue syndrome (CFS) or validating 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 cereblon (CRBN)-related protein (CAP) selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2, and CD3D; and (b) if the expression level of the biomarker is higher than a reference expression level of the biomarker, identifying or validating the subject as having CFS.

[0008] In some embodiments, the subject has reported chronic debilitating fatigue, unrefreshing sleep, mental and / or physical pain, neurological and cognitive impairment, and / or autoimmunity or immune deficiency.

[0009] On the other hand, provided herein is a method for determining the severity of 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 cerebellin (CRBN)-related protein (CAP) selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2 and CD3D; (b) comparing the expression level of the biomarker with a reference expression level of the biomarker; and (c) determining the severity of CFS in the subject based on the comparison in step (b).

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

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

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

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

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

[0015] 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 the expression level of the biomarker in a subject not suffering from CFS or a cohort of subjects not suffering from CFS. In some embodiments, the reference expression level of a 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 a biomarker is the expression level of the biomarker in a subject suffering from mild CFS or a cohort of subjects suffering from 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 not suffering from CFS, or a cohort of healthy subjects or subjects not suffering from CFS.

[0017] In some embodiments, the method comprises determining the expression levels of two, three, four, five or all 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 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 of IKZF2, IKZF3, BACH2, CD3D and HSPA8. In some embodiments, the method comprises determining the expression level of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8.

[0018] In some embodiments, the method includes comparing the expression level of each of the biomarkers to their respective reference expression levels. In some embodiments, the method includes obtaining a comprehensive score based on the expression level of the biomarkers and comparing the comprehensive score to a reference score derived from the reference expression level of the biomarkers.

[0019] In another aspect, provided herein is a method for determining or monitoring the effectiveness of a 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 the treatment to the subject, wherein the biomarker is cerebellin (CRBN)-related protein (CAP) selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, 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 to the second expression level.

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

[0021] In another aspect, provided herein is a method for screening a treatment for effectiveness in treating CFS, 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 cerebellin (CRBN)-related protein (CAP) selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, 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.

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

[0023] In some embodiments, the method comprises determining a first expression level and a second expression level of two, three, four, five or all biomarkers selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2 and CD3D. In some embodiments, the method comprises determining a first expression level and a second expression level of each of: (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 IKZF2, IKZF3, BACH2, CD3D and HSPA 8; (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 a first expression level and a second expression level of all biomarkers in the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2 and CD3D.

[0024] In some embodiments, the method includes comparing a first expression level of each of the biomarkers to their respective second expression levels. In some embodiments, the method includes obtaining a first comprehensive score based on the first expression level of the biomarker and a second comprehensive score based on the second expression level of the biomarker, and comparing the first comprehensive score to the second comprehensive score.

[0025] In some embodiments, treatment includes an immunomodulatory drug (IMiD). In some embodiments, treatment includes a cerebellar protein (CRBN) modulator or a compound capable of binding to CRBN and / or inducing a conformational change thereof. In some embodiments, 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] On the other hand, provided herein is a method for identifying a subject having long COVID or validating 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 cerebellar protein (CRBN)-related protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2 and CD3D; and (b) if the expression level of the biomarker is higher than a reference expression level of the biomarker, identifying or validating the subject as having long COVID.

[0028] On the other hand, the present invention provides a method for identifying a subject who is likely or unlikely to respond to long COVID treatment or predicting the responsiveness of a subject to long COVID treatment, the method comprising: (a) determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a cerebellum protein (CRBN)-related protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2 and CD3D; and (b) if the expression level of the biomarker is higher than a reference expression level of the biomarker, identifying or predicting the subject as likely to respond to the treatment.

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

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

[0031] 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 the expression level of the biomarker in a subject without long COVID or a cohort of subjects without long COVID. In some embodiments, the reference expression level of a 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 a biomarker is the expression level of the biomarker in a subject with acute COVID or a cohort of subjects with 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 healthy subjects or subjects without long COVID, or in a cohort of healthy subjects or subjects without long COVID.

[0033] In some embodiments, the method comprises determining the expression levels of two, three, four, five or all 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 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 of IKZF2, IKZF3, BACH2, CD3D and HSPA8. In some embodiments, the method comprises determining the expression level of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D.

[0034] In some embodiments, the method includes comparing the expression level of each of the biomarkers to their respective reference expression levels. In some embodiments, the method includes obtaining a comprehensive score based on the expression level of the biomarkers and comparing the comprehensive score to a reference score derived from the reference expression level of the biomarkers.

[0035] On the other hand, provided herein is a method for determining or monitoring the effectiveness of a treatment in a subject with 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 cerebellar leukocyte antigen (CRBN)-related 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 to the second expression level.

[0036] In some embodiments, the method comprises: if the second expression level is lower than the first expression level, determining that the treatment is effective. 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 a treatment for effectiveness in 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 cerebellum protein (CRBN)-related 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 comprises determining a first expression level and a second expression level of two, three, four, five or all biomarkers selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2 and CD3D. In some embodiments, the method comprises determining a first expression level and a second expression level of each of: (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 IKZF2, IKZF3, BACH2, CD3D and HSPA 8; (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 a first expression level and a second expression level of all biomarkers in the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2 and CD3D.

[0040] In some embodiments, the method includes comparing a first expression level of each of the biomarkers to their respective second expression levels. In some embodiments, the method includes obtaining a first comprehensive score based on the first expression level of the biomarker and a second comprehensive score based on the second expression level of the biomarker, and comparing the first comprehensive score to the second comprehensive score.

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

[0042] In some embodiments, the subject has had 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 a 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 aforementioned methods, the kit 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.

[0045] In some embodiments, the kit further comprises a means for obtaining a sample.

[0046] In some embodiments, the kit further comprises instructions for interpreting the determined expression levels.

[0047] In some embodiments, the kit further comprises a reference expression level of a biomarker. 4. Description of the drawings

[0048] Figure 1 Depicted is the data analysis workflow for the Autoimmune Profile (AIP) gene expression data.

[0049] Figure 2A-2B Plots the correlation of gene expression measurements between samples collected at two time points ( Figure 2A ) and the correlation of gene expression measurements of housekeeping genes between the two batches ( Figure 2B ). The raw and normalized relative fluorescence units (RFU) of three housekeeping genes (ACTB, GAPDH, TFRC) were compared between replicates using a Wilcoxon signed-rank test. To assess potential batch effects, the two batches were combined and principal component analysis (PCA) was performed to visualize the clustering of normalized genes and immune module genes, respectively.

[0050] Figure 3 Depicted is an unsupervised cluster analysis (heat map) of the AIP data, which shows the relationship between genes that are significantly upregulated or downregulated in a subset of participants with certain demographic or clinical attributes. A single column represents a patient, and the column annotation is consistent with its associated clinical or demographic category. A separate row represents the 51 AIP set of genes, which are then separated by the differentially expressed subgroups of the gene on the left portion of the heat map.

[0051] Figures 4A-4H Forest plots and bootstrapped t-tests with coefficient weights comparing bedridden and non-bedridden CFS patients are depicted ( Figure 4A and Figure 4B ) and single gene maps ( Figures 4C-4H ). Figure 4A Depicted is a univariate bootstrap analysis that identified six genes of interest (in boxes) that were differentially expressed by CFS bedridden status. A forest plot visually displays the mean coefficient value for each gene and the lower and upper 95% confidence interval limits. Coefficients that do not overlap zero are indicated in boxes. Figure 4B Depicted are multivariate bootstrap analyses of six key genes differentially expressed by CFS bedridden status. Forest plots visually display the mean coefficient values ​​for each gene as well as the lower and upper limits of the 95% confidence interval. CFS bedridden patients were set as baseline variables for comparison with CFS non-bedridden patients (line with triangles) and control patients (line with circles).

[0052] Figure 5 bedridden CFS patients with other autoimmune diseases were profiled. These patients showed large differential expression in the same six genes (IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8).

[0053] Fig. 6AUnsupervised clustering of urinary organic acids based on metabolic class is depicted. Heatmap based on scaled and centered mmol values ​​divided by CFS bedridden status (top annotations) and clustered by metabolic class (right annotations). Figure 6B A summary of p-values ​​and fold changes of organic acids in normal versus CFS is depicted (time point -1 only). Figure 6C-6F One-way analysis of raw values ​​by cohort (CFS or normal) for various organic acids (xanthuric acid, glycolic acid, pyruvic acid, and hippuric acid) is depicted. Figure 6G Heat map depicting CFS bedridden versus non-bedridden. Figure 6H A summary of p values ​​and fold changes in CFS bedridden versus CFS ambulatory is depicted (time point -1 only). Vanillylmandelic acid had p<0.05 and a fold change of 1.28. Fig.6I One-way analysis of raw values ​​by severity (bedridden or not) for vanillylmandelic acid is depicted.

[0054] Figure 7 A summary of the key biological functions of the six genes identified by the AIP is depicted.

[0055] Figure 8 Similarities between ME / CFS and long-haul COVID are drawn.

[0056] Figures 9A-9F Describes the comparison of ABCE1 ( Fig.9A )、BACH2( Fig. 9B )、CD3D( Fig. 9C )、HSPA8( Fig.9D )、IKZF2( Fig.9E ) and IKZF3( Fig.9F ) Individual box plots of gene expression for . Wilcoxon signed-rank p-values ​​are shown.

[0057] Figures 10A-10F Describes the comparison of ABCE1 ( Fig. 10A )、BACH2( Fig. 10B )、CD3D( Fig. 10C )、HSPA8( Fig. 10D )、IKZF2( Fig.10E ) and IKZF3( Fig.10F ) Individual box plots of gene expression for . Wilcoxon signed-rank p-values ​​are shown. 5. Specific implementation methods

[0058] This disclosure is based in part on the findings that there are significant similarities in symptoms between long COVID and CFS and that certain cerebellar leukocyte nuclease (CRBN)-related protein (CAP) biomarkers (e.g., Figure 7 The levels of those biomarkers (mRNA, cDNA or protein) in the study were associated with CFS and long COVID and their severity. 5.1 Definitions

[0059] Unless otherwise defined herein, the technical and scientific terms used in this specification have the meanings commonly understood by those of ordinary skill in the art. Where appropriate, terms used in the singular also include the plural, and vice versa. If any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall prevail.

[0060] As used herein, the term "biomarker" is a substance whose detection, amount, change, or any other characteristic thereof indicates a particular biological state, such as a disease condition or its progression. In some embodiments, a biomarker can be determined individually. In other embodiments, several biomarkers can be measured simultaneously.

[0061] As used herein, the term "expression" refers to transcription from a gene to produce 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. As used herein, the term "expression" also refers to translation from an RNA molecule to produce a protein, a polypeptide or a portion thereof.

[0062] As used herein, the term "level" related to a biomarker refers to the amount, accumulation or rate of a biomarker molecule (e.g., mRNA or protein expression of a gene or organic acid). The level can be represented by, for example, the amount or rate of synthesis of a 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 accumulated in a cell or biological fluid. As used herein, a level can be the absolute amount of a molecule in a sample determined under steady-state or non-steady-state conditions or the relative amount of a molecule. In some embodiments, the expression level of a biomarker is a normalized expression level. In some embodiments, the normalized expression level 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 for the expression level of the housekeeping gene (e.g., ACTB, GAPDH or TFRC) to determine the normalized expression level of the biomarker.

[0063] Any method known in the art for normalization can be used herein. Generally speaking, the normalization method for gene expression data can be divided into three categories, i.e., data-driven reference, external reference, and whole gene set reference. For data-driven programs, first, a subset of genes with no change or minimal change between samples is identified as a data-driven housekeeping gene to normalize the data set. For external controls, external controls, such as spike-in controls, have been designed in many experiments. These natural controls can be used as exogenous references for normalization of gene expression data. For the whole gene set, all genes in the experiment are used to derive a value or several values ​​for data normalization. Several algorithms use the whole genome as a reference for data normalization. Another exemplary method of normalization is "global" normalization, which means using the whole gene set (e.g., the whole transcriptome), and the RFU value of the target gene is divided by the median or average RFU value of the whole set. In certain embodiments, the normalization method for gene expression profiling data assumes that most genes in the genome are equally expressed in each experimental unit and that genes are symmetrically distributed between overexpression and underexpression. Denormalizing the data provides an alternative when the above normalization assumptions do not apply, such as when the data contain large partitions of differentially expressed genes.

[0064] Normalized values ​​can be scaled. An exemplary scaling program for gene expression profiling data is provided below: the relationship between the gene expression data observed in different technology platforms cannot be assumed to be the same in terms of their raw data. In order to compare the data between platforms, it is assumed that the observed data range is similar. For example, in scaling, it can be assumed that the observed data of the device are the same, and the simplest function of scaling the data range of one platform relative to the data range of another platform can be completed based on logarithmic scaling. Scaling allows the measurement range between the mean data point as the center and calibration of each platform, so that the gene expression profiling data from different platforms can be compared. In a specific exemplary method, the original RFU is used, and divided by the geometric mean of a group of housekeeping genes. In one embodiment, the group of genes includes 3 housekeeping genes ACTB, GAPDH and TFRC or is composed of these housekeeping genes. The Log2 of the normalized value can be calculated, and it scales the value in the range of -1 to 1. This allows all genes to be drawn together in a figure and compared.

[0065] The term "reference level" refers to the level of a biomarker for comparison purposes (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 of the methods provided herein. In some embodiments, the reference level of a biomarker is a predetermined level of the biomarker. In some embodiments, a predetermined expression level can be found in a public database. In some embodiments, the reference level is the expression level of a gene in 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 of the biomarker (e.g., the expression level of a gene (e.g., mRNA or protein expression level) or the level of an organic acid) in the same subject or from different samples measured at different time points. In some embodiments, the reference level of a biomarker is the level of the biomarker in the corresponding tissue measured in 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) (e.g., the expression level of a gene (e.g., mRNA or protein expression level) or the level of an organic acid). In some embodiments, the reference level of a biomarker is the level of the biomarker in the corresponding tissue measured in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects without CFS, a cohort of subjects with mild CFS (interchangeably used herein as moderate CFS), a cohort of subjects without long COVID, a cohort of subjects with acute COVID) (e.g., the median or mean expression level of a gene (e.g., mRNA or protein expression level) or the median or mean level of an organic acid). 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 the biomarker in a sample obtained from a subject before the treatment and / or compound is administered to the subject.

[0066] As used herein, the terms "treat" or "treatment" or "treating" or "to treat" or "alleviate" or "alleviation" or "alleviating" or "to alleviate" refer to therapeutic measures intended to cure, slow down, alleviate the symptoms of and / or stop the progression of a pathological condition or disorder.

[0067] As used herein and unless otherwise indicated, the term "therapeutically effective amount" of a compound is an amount sufficient to provide a therapeutic benefit for treating or managing a disease or disorder (e.g., CFS or long COVID) or to delay or minimize one or more symptoms associated with the presence of a disease or disorder (e.g., CFS or long COVID). A therapeutically effective amount of a compound means the amount of a therapeutic agent alone or in combination with other therapies that provides a therapeutic benefit for treating or managing a disease or disorder (e.g., CFS or long COVID). The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or disorder (e.g., CFS or long COVID), or increases the therapeutic effect of another therapeutic agent. The term also refers to an amount of a compound sufficient to elicit a biological or medical response of a biomolecule (e.g., protein, enzyme, RNA, DNA or organic acid), cell, tissue, system, animal or human that is being sought by a researcher, veterinarian, doctor or clinician.

[0068] As used herein, the term "prevent" or "prevention" or "preventing" refers to partially or completely inhibiting the development, recurrence, onset or spread of a disease, disorder or condition, or symptoms thereof, in a subject.

[0069] The terms "response" or "responsiveness" when used with respect to any of the methods provided herein refer to the degree of effectiveness of the treatment in alleviating or reducing the symptoms of a disease (e.g., CFS or long COVID).

[0070] The term "predict" or "predicting" generally means to determine or tell in advance. The term "predicting" when used with respect to any of the methods provided herein means to determine the likelihood of a method outcome at the outset, prior to a certain action (eg, performing a treatment).

[0071] As used herein, the term "obtaining" in relation to biomarker levels refers to actions or steps related to obtaining information about biomarker levels. In some embodiments, the term "obtaining" includes the steps of "determining", "measuring", "evaluating", "assessing" and / or "determining". In some embodiments, "obtaining" involves organizing a determination, evaluation, assessment and / or determination performed by a third party, or using the results of such determination, evaluation, assessment and / or determination. 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 the steps necessary to prepare a sample under conditions where an assay for nucleic acids or proteins for measuring biomarkers in a sample is to be performed.

[0072] The terms "determine," "measure," "evaluate," "assess," and "assay" are used interchangeably herein to refer to a form of measurement, including determining whether an element is present or not. Measurements can be quantitative and / or qualitative determinations. "Determining the expression level" can include measuring the amount of something present, as well as determining whether it is present or not.

[0073] As used herein, the term "monitoring" generally refers to overseeing, regulating, regulating, watching, tracking, or monitoring activities. For example, the term "monitoring the effectiveness of a compound" refers to tracking the effectiveness of treating a patient's disease or disorder (e.g., CFS or long COVID). The term "monitoring the effectiveness of a compound" also refers to tracking the effectiveness of treating a patient's CFS or long COVID (i.e., reduction in fatigue). Similarly, the term "monitoring," when used in connection with patient compliance, whether alone or in a clinical trial, refers to tracking or confirming that a patient is actually taking the drug being tested as prescribed. For example, monitoring can be performed by tracking the expression of mRNA or protein biomarkers or the levels of urinary organic acids.

[0074] The terms "likely" or "likelihood" generally refer to an increase in the probability of an event occurring. The term "likelihood" when used with respect to the effectiveness of a treatment for a subject generally contemplates an increased likelihood that the progression or extent of a disease will be reduced. The term "likelihood" when used with respect to the effectiveness of treating CFS or long COVID generally can mean a reduction in symptoms of CFS or long COVID (e.g., severe fatigue).

[0075] As used herein, the term "sample" refers to a material or mixture of materials, e.g., in fluid or solid form, containing one or more components of interest. A sample can be a biological sample obtained from a biological subject, including a sample of biological tissue or fluid origin obtained, obtained 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, and the like.

[0076] The terms "polypeptide" and "protein" as used interchangeably herein refer to a polymer of three or more amino acids connected by peptide bonds and arranged in series. The term "polypeptide" includes proteins, protein fragments, protein analogs, oligopeptides, etc. As used herein, the term "polypeptide" may also refer to peptides. The amino acids constituting the polypeptide may be of natural origin or synthetic. The polypeptide may be purified from a biological sample. Polypeptides, proteins or peptides also include modified polypeptides, proteins and peptides, for example, glycopolypeptides, glycoproteins or glycopeptides; or lipopolypeptides, lipoproteins or lipopeptides.

[0077] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to describe polymers of any length consisting of nucleotides (e.g., deoxyribonucleotides or ribonucleotides), or synthetically produced compounds that can hybridize with naturally occurring nucleic acids in a sequence-specific manner similar to two naturally occurring nucleic acids, for example, can participate in Watson-Crick base pairing interactions. As used herein in the context of polynucleotide sequences, the terms "bases" (or "bases") and "nucleotides" (or "nucleotides") are synonymous, i.e., monomeric subunits of polynucleotides. The terms "nucleoside" and "nucleotide" are intended to include those containing not only known purine and pyrimidine bases, but also parts of other heterocyclic bases that have been modified. These modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated ribose or other heterocycles. In addition, the terms "nucleoside" and "nucleotide" include those containing not only conventional ribose and deoxyribose but also parts of other sugars. Modified nucleosides or nucleotides also include modifications to the sugar moiety, for example, where one or more hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, etc. "Analogs" refer to molecules with structural features that are considered in the literature to be mimetics, derivatives, having similar structures or other similar terms, and include, for example, polynucleotides incorporating non-natural nucleotides, nucleotide mimetics such as 2'-modified nucleosides, peptide nucleic acids, oligonucleoside phosphonates, and any polynucleotides to which substituents (such as protecting groups or linking moieties) have been added.

[0078] The term "about" or "approximately" means an acceptable error for a particular value determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" means 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.

[0079] As used herein, the term "and / or" as used in phrases such as "A and / or B" is intended to include: both A and B; A or B; A (alone); and B (alone). Likewise, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass 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).

[0080] As used in the disclosure and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0081] It should be understood that when the term "comprising" is used anywhere herein to describe an embodiment, otherwise similar embodiments described according to "consisting of" and / or "consisting essentially of" are also provided. It should also be understood that when the phrase "consisting essentially of" is used anywhere herein to describe an embodiment, otherwise similar embodiments described according to "consisting of" are also provided. 5.2 Biomarkers and their use

[0082] In one aspect, provided herein are methods of using the level of at least one cerebellum protein-associated protein (CAP) biomarker (e.g., expression level of a gene (e.g., mRNA or protein expression level)) identified herein and associated with a post-viral syndrome characterized by fatigue. In some embodiments, fatigue is fatigue that persists for at least 3 months after infection with a virus. In some embodiments, fatigue is fatigue that persists for at least 6 months after infection with a virus. In some embodiments, fatigue is debilitating. In some embodiments, fatigue interferes with daily living activities. In some embodiments, patients with post-viral syndrome are unable to leave their homes, are bedridden, or both. In some embodiments, post-viral syndrome includes one or more of the following: discomfort after exertion, cognitive dysfunction, sensory motor symptoms, headache, memory problems, insomnia, muscle pain, palpitations, shortness of breath, dizziness and balance problems, speech and language problems, joint pain, chest tightness. In certain embodiments, post-viral syndrome is CFS. In certain embodiments, post-viral syndrome is long COVID.

[0083] "Cerebrin-associated protein" or "CAP" refers to a protein that interacts with or binds to CRBN directly or indirectly. In certain embodiments, a CAP is any protein that directly binds to cerebellin, as well as any protein that is an indirect downstream effector of the cerebellin pathway. In certain embodiments, a "cerebellin-associated protein" or "CAP" is a substrate of CRBN, such as a protein substrate of an E3 ubiquitin ligase complex involving CRBN, or a downstream substrate thereof. In certain embodiments, a CAP is an IKAROS family zinc finger (IKZF2), an IKAROS family zinc finger 3 (IKZF3), an ATP-binding cassette subfamily E member 1 (ABCE1), a BTB domain and CNC homolog 2 (BACH2), a CD3δ subunit of the T cell receptor (CD3D), or a heat shock protein family A member 8 (HSPA8).

[0084] In some embodiments, provided herein is a method for identifying a subject 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, and identifying the subject as having post-viral syndrome if the expression level of the biomarker is higher than a reference expression level of the biomarker.

[0085] In some embodiments, provided herein is a method for verifying post-viral 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 if the expression level of the biomarker is higher than a reference expression level of the biomarker, verifying the subject as having post-viral syndrome.

[0086] In some embodiments, a method for determining the severity of post-viral syndrome in a subject is provided herein, 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 severity of post-viral syndrome in the subject based on the expression level. In some embodiments, the method further comprises comparing the expression level of the biomarker with a reference expression level of the biomarker. In some embodiments, a higher expression level of a biomarker indicates a more severe post-viral syndrome.

[0087] In certain embodiments, a method for monitoring the progress of post-viral syndrome in a subject is provided herein, 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 evaluating the progress of post-viral syndrome based on the expression level of the biomarker. In certain embodiments, compared with the reference expression level of the biomarker (e.g., the expression level of the biomarker at an earlier time point), the higher expression level of the biomarker indicates that the post-viral syndrome progresses. In certain embodiments, compared with the reference expression level of the biomarker (e.g., the expression level of the biomarker at an earlier time point), the lower expression level of the biomarker indicates that the post-viral syndrome disappears.

[0088] In some embodiments, provided herein is a method for identifying a subject who is likely or unlikely to respond to a post-viral syndrome treatment or predicting a subject's responsiveness to a post-viral syndrome treatment, 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 if the expression level of the biomarker is higher than a reference expression level of the biomarker, identifying or predicting the subject as likely to respond to a post-viral syndrome treatment.

[0089] In some embodiments, provided herein is a method for selectively treating a subject having or suspected of having post-viral syndrome with a therapy, 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 the subject as likely to respond to the post-viral syndrome therapy if the expression level of the biomarker is higher than a reference expression level of the biomarker; and administering the therapy to the subject identified or predicted as likely to respond to the therapy.

[0090] In some embodiments, the reference expression level of a biomarker is a predetermined expression level of a biomarker from a public database. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker in a subject without post-virus syndrome or a healthy subject. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker in a subject with mild post-virus syndrome or an acute viral infection. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker determined based on a cohort of a subject (e.g., a cohort of a healthy subject, a cohort of a subject without post-virus syndrome, a cohort of a subject with mild post-virus syndrome, or a cohort of a subject with an acute viral infection). In some embodiments, the reference expression level of a biomarker is the median or average expression level of a biomarker of the expression level of a biomarker in a cohort of a subject (e.g., a cohort of a healthy subject, a cohort of a subject without post-virus syndrome, a cohort of a subject with mild post-virus syndrome, or a cohort of a subject with an acute viral infection).

[0091] In certain embodiments, the method includes 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 with the reference expression level of the biomarker. In other embodiments, when multiple biomarkers are used, a comprehensive score based on multiple biomarkers is calculated and compared with a reference comprehensive score. In some embodiments, the comprehensive score is calculated using the median Z score method. In other embodiments, the comprehensive score is calculated using a single sample gene set enrichment (ssGSEA) method.

[0092] In some embodiments, a method for determining or monitoring the effectiveness of a treatment in a subject with post-viral syndrome is provided herein, 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 the treatment to the subject; obtaining a second expression level of the biomarker in a second sample obtained from the subject after the treatment is administered to the subject; and determining the effectiveness of the treatment based on a comparison of the first expression level with the second expression level. In some embodiments, the method comprises: if the second expression level is lower than the first expression level, determining that the treatment is effective. In some embodiments, the method comprises: if the second expression level is not lower than the first expression level, determining that the treatment is ineffective. In some embodiments, the method comprises: if the second expression level is not lower than the first expression level, determining or adjusting (e.g., increasing) the dosage of the treatment or administering a different treatment to the subject.

[0093] In some embodiments, provided herein is a method for screening a treatment for effectiveness in treating post-viral syndrome, the method comprising: (a) determining a first expression level of a biomarker in a sample before administering a compound to the sample, wherein the biomarker is cerebellum protein (CRBN)-related 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.

[0094] In certain embodiments, the method includes using two or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In some embodiments, when using multiple biomarkers, the second expression level of each biomarker is compared with the first expression level of the biomarker, and the method includes: if the second expression level of each biomarker is lower than the first expression level of the biomarker, it is determined that the treatment is effective. In other embodiments, when using multiple biomarkers, a comprehensive score based on multiple biomarkers is calculated and compared with a reference comprehensive score. In some embodiments, the comprehensive score is calculated using the median Z score method. In other embodiments, the comprehensive score is calculated using a single sample gene set enrichment (ssGSEA) method.

[0095] In some embodiments, the treatment of post-viral syndrome disclosed herein comprises an immunomodulatory drug (IMiD) as disclosed in Section 5.2. In some embodiments, the treatment of post-viral syndrome comprises a cerebellum protein (CRBN) modulator as disclosed in Section 5.2 or a compound capable of binding to CRBN and / or inducing a conformational change thereof. In some embodiments, the treatment of post-viral syndrome comprises an agent that depletes B cells as disclosed in Section 5.2 (e.g., an anti-CD20 antibody, e.g., rituximab). 5.2.1 Biomarkers for CFS

[0096] As used herein, "chronic fatigue syndrome" or "CFS" refers to a disease with fatigue symptoms that last for a period of time (e.g., for 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 continuous or intermittent during the period. In some embodiments, the fatigue symptoms are persistent during the period. In some embodiments, the fatigue symptoms cause the patient to be bedridden. In some embodiments, the disease includes a combination of symptoms, which may include one or more of the following: debilitating fatigue, discomfort after exertion, restless sleep or sleep disorders (or both) and / or cognitive difficulties. In some embodiments, the combination of symptoms includes debilitating fatigue, discomfort after exertion, restless sleep or sleep disorders (or both) and cognitive difficulties. In some embodiments, this combination of symptoms exists within a period of time disclosed herein. In some embodiments, the period is at least six weeks in adults and at least four weeks in children. Most commonly, fatigue symptoms last for more than six months. In some cases, the disease may have a variety of other symptoms, such as restless sleep, mental and physical pain, neurological and cognitive impairment, and autoimmunity or immune deficiency.

[0097] In some embodiments, CFS is post-viral syndrome. In some embodiments, CFS occurs in the absence of a viral infection and is therefore not a post-viral syndrome.

[0098] In some embodiments, provided herein is a method for identifying a subject with 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 if the expression level of the biomarker is higher than a reference expression level of the biomarker, the subject is identified as having CFS. 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 a biomarker. In some embodiments, the reference expression level of a biomarker is a predetermined expression level of a biomarker obtained from a public database. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker in a healthy subject or a subject not suffering from CFS. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker in a subject suffering from moderate CFS. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker determined based on a cohort of a subject (e.g., a cohort of a healthy subject, a cohort of a subject not suffering from CFS, or a cohort of a subject suffering from moderate CFS). In some embodiments, the reference expression level of a biomarker is the median or average expression level of a biomarker of the expression level of a biomarker in a cohort of a subject (e.g., a cohort of a healthy subject, a cohort of a subject not suffering from CFS, or a cohort of a subject suffering from moderate CFS). In some embodiments, a biomarker is HSPA8 or ABCE1, and the reference expression level of a biomarker is a healthy subject or a subject not suffering from CFS, or a healthy subject or a cohort of a subject not suffering from CFS.

[0100] In some embodiments, the method comprises identifying the subject as having 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 comprises identifying the subject as having 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 comprises identifying the subject as having 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 comprises identifying the subject as having 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 comprises identifying the subject as having 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 comprises identifying the subject as having 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 comprises identifying the subject as having 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 comprises: if the expression level of the biomarker is at least 70% higher than the reference expression level of the biomarker, then the subject is identified as having CFS. In some embodiments, the method comprises: if the expression level of the biomarker is at least 80% higher than the reference expression level of the biomarker, then the subject is identified as having CFS. In some embodiments, the method comprises: if the expression level of the biomarker is at least 90% higher than the reference expression level of the biomarker, then the subject is identified as having CFS. In some embodiments, the method comprises: if the expression level of the biomarker is at least 2 times the reference expression level of the biomarker, then the subject is identified as having CFS. In some embodiments, the method comprises: if the expression level of the biomarker is at least 3 times the reference expression level of the biomarker, then the subject is identified as having CFS. In some embodiments, the method comprises: if the expression level of the biomarker is at least 4 times the reference expression level of the biomarker, then the subject is identified as having CFS. In some embodiments, the method comprises: if the expression level of the biomarker is at least 5 times the reference expression level of the biomarker, then the subject is identified as having CFS. In some embodiments, the method comprises: if the expression level of the biomarker is at least 6 times the reference expression level of the biomarker, then the subject is identified as having CFS. In some embodiments, the method comprises: if the expression level of the biomarker is at least 7 times the reference expression level of the biomarker, then the subject is identified as having CFS.In some embodiments, the method includes: if the expression level of the biomarker is at least 8 times the reference expression level of the biomarker, the subject is identified as having CFS. In some embodiments, the method includes: if the expression level of the biomarker is at least 9 times the reference expression level of the biomarker, the subject is identified as having CFS. In some embodiments, the method includes: if the expression level of the biomarker is at least 10 times the reference expression level of the biomarker, the subject is identified as having CFS. The expression level of the biomarker can be determined using any method known in the art, and exemplary methods are described in more detail in Section 5.3 below. In some embodiments, if the expression level of the biomarker is higher (e.g., statistically significantly higher) than a reference level, as observed according to a measurement assay, the expression level of the biomarker is determined to be higher than the reference level.

[0101] In certain embodiments, the method includes using two or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8 to identify a subject with CFS. In certain embodiments, the method includes using three or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8 to identify a subject with CFS. In certain embodiments, the method includes using four or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8 to identify a subject with CFS. In certain embodiments, the method includes using five or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8 to identify a subject with CFS. In certain embodiments, the method comprises using all biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8 to identify a subject with CFS. 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 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 with the reference expression level of the biomarker, and the method includes: if the expression level of each biomarker is higher than the reference expression level of the biomarker, the subject is identified as suffering from CFS. In other embodiments, when multiple biomarkers are used, a comprehensive score based on multiple biomarkers is calculated and compared with a reference comprehensive score. In some embodiments, the method includes: if the comprehensive score is higher than the reference comprehensive score, the subject is identified as suffering from CFS. In some embodiments, the comprehensive score is calculated using the median Z score method. In brief, the median Z score is derived by first calculating the mean value of each gene from all samples in the gene expression matrix. The mean value is then subtracted from each corresponding gene of all samples, and then scaled by dividing these values ​​by their standard deviation. The median scaling value from multiple target genes includes a comprehensive score. Another exemplary method for calculating the comprehensive score is the single sample gene set enrichment (ssGSEA) method. The single sample gene score represents the degree to which the genes in a specific gene set are synergistically upregulated or downregulated in the sample. The score is calculated by adjusting the running sum statistic based on the decreasing trend in the ordered expression list. The enrichment score is the maximum zero deviation encountered in the trend; it corresponds to a weighted Kolmogorov-Smirnov-like statistic (see, e.g., Subramanian et al., PNAS [Proceedings of the National Academy of Sciences of the United States of America], 102(43): 15545-15550 (2005); and Barbie et al., Nature [Nature], 462(7269): 108-112).

[0102] In other embodiments, provided herein is a method of verifying 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 if the expression level of the biomarker is higher than the reference expression level of the biomarker, the subject is verified as having CFS. In some embodiments, the subject has reported symptoms associated with CFS. Therefore, in some embodiments, provided herein is a method of verifying CFS in a subject, the method comprising selecting a subject who has reported symptoms associated with CFS; 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 if the expression level of the biomarker is higher than the reference expression level of the biomarker, the subject is verified as having CFS. Exemplary symptoms associated with CFS include, but are not limited to, chronic debilitating fatigue, restless sleep, mental and / or physical pain, neurological and cognitive impairment, and / or autoimmunity or immunodeficiency. In a certain embodiment, the biomarker is IKZF2. In a certain embodiment, the biomarker is IKZF3. In a certain embodiment, the biomarker is ABCE1. In a certain embodiment, the biomarker is BACH2. In a certain embodiment, the biomarker is CD3D. In a certain embodiment, the biomarker is HSPA8.

[0103] In some embodiments, the reference expression level of a biomarker is a predetermined expression level of a biomarker from a public database. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker in a subject who does not suffer from CFS. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker in a subject who suffers from moderate CFS. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker determined based on a cohort of a subject (e.g., a cohort of healthy subjects, a cohort of a subject who does not suffer from CFS, or a cohort of a subject who suffers from moderate CFS). In some embodiments, the reference expression level of a biomarker is the median or average expression level of a biomarker of the expression level of a biomarker in a cohort of a subject (e.g., a cohort of healthy subjects, a cohort of a subject who does not suffer from CFS, or a cohort of a subject who suffers from moderate CFS). In some embodiments, a biomarker is HSPA8 or ABCE1, and the reference expression level of a biomarker is a healthy subject or a subject who does not suffer from CFS, or a healthy subject or a cohort of a subject who does not suffer from 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 not suffering from CFS, or in a cohort of healthy subjects or subjects not suffering from CFS.

[0104] In some embodiments, the method includes: if the expression level of the biomarker is at least 5% higher than the reference expression level of the biomarker, then the subject is verified as having CFS. In some embodiments, the method includes: if the expression level of the biomarker is at least 10% higher than the reference expression level of the biomarker, then the subject is verified as having CFS. In some embodiments, the method includes: if the expression level of the biomarker is at least 20% higher than the reference expression level of the biomarker, then the subject is verified as having CFS. In some embodiments, the method includes: if the expression level of the biomarker is at least 30% higher than the reference expression level of the biomarker, then the subject is verified as having CFS. In some embodiments, the method includes: if the expression level of the biomarker is at least 40% higher than the reference expression level of the biomarker, then the subject is verified as having CFS. In some embodiments, the method includes: if the expression level of the biomarker is at least 50% higher than the reference expression level of the biomarker, then the subject is verified as having CFS. In some embodiments, the method includes: if the expression level of the biomarker is at least 60% higher than the reference expression level of the biomarker, then the subject is verified as having CFS. In some embodiments, the method comprises: if the expression level of the biomarker is at least 70% higher than the reference expression level of the biomarker, then the subject is verified as having CFS. In some embodiments, the method comprises: if the expression level of the biomarker is at least 80% higher than the reference expression level of the biomarker, then the subject is verified as having CFS. In some embodiments, the method comprises: if the expression level of the biomarker is at least 90% higher than the reference expression level of the biomarker, then the subject is verified as having CFS. In some embodiments, the method comprises: if the expression level of the biomarker is at least 2 times the reference expression level of the biomarker, then the subject is verified as having CFS. In some embodiments, the method comprises: if the expression level of the biomarker is at least 3 times the reference expression level of the biomarker, then the subject is verified as having CFS. In some embodiments, the method comprises: if the expression level of the biomarker is at least 4 times the reference expression level of the biomarker, then the subject is verified as having CFS. In some embodiments, the method comprises: if the expression level of the biomarker is at least 5 times the reference expression level of the biomarker, then the subject is verified as having CFS. In some embodiments, the method includes verifying that the 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 verifying that the 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: if the expression level of the biomarker is at least 8 times the reference expression level of the biomarker, the subject is verified as having CFS. In some embodiments, the method includes: if the expression level of the biomarker is at least 9 times the reference expression level of the biomarker, the subject is verified as having CFS. In some embodiments, the method includes: if the expression level of the biomarker is at least 10 times the reference expression level of the biomarker, the subject is verified as having CFS. The expression level of the biomarker can be determined using any method known 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 a reference level, as observed according to a measurement assay, the level is determined to be higher than the reference level.

[0105] In certain embodiments, the method includes using two or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8 to verify the subject with CFS. In certain embodiments, the method includes using three or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8 to verify the subject with CFS. In certain embodiments, the method includes using four or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8 to verify the subject with CFS. In certain embodiments, the method includes using five or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8 to verify the subject with CFS. In certain embodiments, the method comprises using all biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8 to verify a subject with CFS. 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 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 a reference expression level of the biomarker, and the method includes: if the expression level of each biomarker is higher than the reference expression level of the biomarker, the subject is verified as having CFS. In other embodiments, when multiple biomarkers are used, a comprehensive score based on multiple biomarkers is calculated and compared with a reference comprehensive score. In some embodiments, the method includes: if the comprehensive score is higher than the reference comprehensive score, the subject is verified as having CFS. In some embodiments, the comprehensive score is calculated using the median Z score method. In other embodiments, the comprehensive score is calculated using the single sample gene set enrichment (ssGSEA) method.

[0106] The present disclosure is also based in part on the following discovery: the expression level of certain CAP biomarkers is associated with the severity of CFS. Therefore, in other embodiments, a method for determining the severity of CFS in a subject is provided herein, 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 with the reference expression level of the biomarker. In some embodiments, a higher expression level of the biomarker indicates a more severe CFS. As used herein, "severe CFS" refers to a type of CFS in which the patient is severely affected and cannot go out and / or is bedridden. In certain embodiments, severe CFS patients are unable to go out and / or bedridden most of the time. In certain embodiments, severe CFS patients (e.g., very severe CFS patients) are completely bedridden and need help 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 and / or bedridden.

[0107] 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.

[0108] In some embodiments, the reference expression level of a biomarker is a predetermined expression level of a biomarker from a public database. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker in a healthy subject or a subject not suffering from CFS. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker determined based on a cohort of healthy subjects. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker in a subject whose severity of CFS has been determined and known. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker in a cohort of a subject suffering from moderate CFS or a subject suffering from moderate CFS. In some embodiments, the reference expression level of a biomarker is the median or average expression level of a biomarker of the expression level of a biomarker in a cohort of a subject (e.g., a cohort of a healthy subject, a cohort of a subject suffering from moderate CFS, or a cohort of a subject whose severity of CFS has been determined and known).

[0109] 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 healthy subjects or subjects 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 subjects with mild CFS or a cohort of subjects with mild CFS.

[0110] In other embodiments, there is provided herein a method for monitoring the progress 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 assessing the progress 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, regularly. 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 in the same subject at an earlier time point. In certain embodiments, the higher expression level of the biomarker indicates CFS progression compared to the reference expression level of the biomarker (e.g., the expression level of the biomarker at an earlier time point). In certain embodiments, a lower expression level of a biomarker compared to a reference expression level of the biomarker (eg, the expression level of the biomarker at an earlier time point) indicates resolution of CFS.

[0111] The expression level of a biomarker can be determined using any method known 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 a reference level as observed according to a measurement assay, the level is determined to be higher than the reference level. In some embodiments, if the level is (e.g., statistically significantly) lower than a reference level as observed according to a measurement assay, the level is determined to be lower than the reference level.

[0112] In certain embodiments, the method includes using two or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the method includes using three or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the method includes using four or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the method includes using five or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the method includes using all biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the expression level of IKZF2 and IKZF3 is 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 with the reference level of the biomarker. In other embodiments, when multiple biomarkers are used, a composite score based on the multiple biomarkers is calculated and compared to a reference composite score. In some embodiments, the composite score is calculated using the median Z score method. In other embodiments, the composite score is calculated using a single sample gene set enrichment (ssGSEA) method.

[0113] In another aspect, the biomarkers provided herein are used to predict the responsiveness of a subject to a CFS treatment. In some embodiments, provided herein is a method of identifying a subject who may or may not respond to a CFS treatment or predicting the responsiveness of a subject to a CFS treatment, 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 if the expression level of the biomarker is higher than the reference expression level of the biomarker, the subject is identified or predicted as likely to respond to a CFS treatment. 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.

[0114] In some embodiments, the reference expression level of a biomarker is a predetermined expression level of a biomarker obtained from a public database. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker in a healthy subject or a subject not suffering from CFS. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker in a subject suffering from moderate CFS. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker determined based on a cohort of a subject (e.g., a cohort of a healthy subject, a cohort of a subject not suffering from CFS, or a cohort of a subject suffering from moderate CFS). In some embodiments, the reference expression level of a biomarker is the median or average expression level of a biomarker of the expression level of a biomarker in a cohort of a subject (e.g., a cohort of a healthy subject, a cohort of a subject not suffering from CFS, or a cohort of a subject suffering from moderate CFS). In some embodiments, a biomarker is HSPA8 or ABCE1, and the reference expression level of a biomarker is a healthy subject or a subject not suffering from CFS, or a healthy subject or a cohort of a subject not suffering from CFS.

[0115] In some embodiments, the method comprises: if the expression level of the biomarker is at least 5% higher than the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the expression level of the biomarker is at least 10% higher than the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the expression level of the biomarker is at least 20% higher than the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the expression level of the biomarker is at least 30% higher than the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the expression level of the biomarker is at least 40% higher than the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the expression level of the biomarker is at least 50% higher than the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the expression level of the biomarker is at least 60% higher than the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the expression level of the biomarker is at least 70% higher than the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the expression level of the biomarker is at least 80% higher than the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the expression level of the biomarker is at least 90% higher than the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the expression level of the biomarker is at least 2 times the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the expression level of the biomarker is at least 3 times the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises identifying or predicting the subject as likely to respond to a CFS treatment if the expression level of the biomarker is at least 4-fold greater than a reference expression level of the biomarker.In some embodiments, the method comprises: if the expression level of the biomarker is at least 5 times the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the expression level of the biomarker is at least 6 times the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the expression level of the biomarker is at least 7 times the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the expression level of the biomarker is at least 8 times the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the expression level of the biomarker is at least 9 times the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the expression level of the biomarker is at least 10 times the reference expression level of the biomarker, then the subject is identified or predicted as likely to respond to CFS treatment. The expression level of a biomarker can be determined using any method known 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 a reference level as observed according to a measurement assay, the level is determined to be higher than the reference level.

[0116] In certain embodiments, the method includes using two or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the method includes using three or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the method includes using four or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the method includes using five or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the method includes using all biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the expression level of IKZF2 and IKZF3 is 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 the biomarker, and the method includes: if the level of each biomarker is higher than the reference level of the biomarker, the subject is identified or predicted as likely to respond to CFS treatment. In other embodiments, when multiple biomarkers are used, a comprehensive score based on multiple biomarkers is calculated and compared to a reference comprehensive score, and the method includes: if the comprehensive score is higher than the reference comprehensive score, the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the comprehensive score is calculated using the median Z score method. In other embodiments, the comprehensive score is calculated using the single sample gene set enrichment (ssGSEA) method.

[0117] In another aspect, a selective treatment method is provided herein, comprising administering a CFS treatment to a subject identified or predicted as likely to respond to the treatment according to the methods provided herein. Specifically, in some embodiments, a method of selectively treating a subject suffering from or suspected of suffering from CFS with a treatment is provided herein, 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; if the expression level of the biomarker is higher than a reference expression level of the biomarker, the subject is identified or predicted as likely to respond to the CFS treatment; and administering the treatment to a subject identified or predicted as likely to respond to the treatment.

[0118] Treatment of CFS in the above methods includes any treatment for alleviating any symptom associated with CFS. In some embodiments, the treatment includes an immunomodulatory compound.

[0119] In some specific embodiments, the treatment for CFS includes immunomodulatory drugs (IMiD). IMiD includes a group of compounds that can be used to treat several types of human diseases (including certain cancers). Unless otherwise specified, the term "immunomodulatory compound" as used herein may cover 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 synthesized or commercially available. The inflammatory cytokine TNF-α produced by macrophages and monocytes during acute inflammation causes a series of different signaling events in the cell. Without being limited by a particular theory, one of the biological effects exerted by the immunomodulatory compounds disclosed herein is to reduce the production of myeloid cell TNF-α. The immunomodulatory compounds disclosed herein can enhance the degradation of TNF-αmRNA. In addition, without being limited by theory, the immunomodulatory compounds disclosed herein may also be effective T cell co-stimulators and significantly increase cell proliferation in a dose-dependent manner. The immunomodulatory compounds disclosed herein may also have a greater costimulatory effect on CD8+T cell subsets than on CD4+T cell subsets. In addition, the compound may have anti-inflammatory properties against myeloid cell responses, but effectively costimulates T cells to produce more IL-2, IFN-γ, and enhances T cell proliferation and CD8+T cell cytotoxic activity. In addition, without being limited by a specific theory, the immunomodulatory compounds disclosed herein may be able to act on natural killer ("NK") cells and natural killer T ("NKT") cells indirectly and directly through cytokine activation, and increase the ability of NK cells to produce beneficial cytokines (such as but not limited to IFN-γ), and enhance the cytotoxic activity of NK and NKT cells. Various immunomodulatory compounds disclosed herein contain one or more chiral centers and may exist as a racemic mixture of enantiomers or a mixture of diastereomers. Therefore, the use of stereoisomerically pure forms of these compounds is also provided herein, as well as the use of mixtures of these forms. For example, a mixture of enantiomers of a specific immunomodulatory compound containing equal or unequal amounts can be used. These isomers can be synthesized asymmetrically or resolved using standard techniques such as chiral columns or chiral resolving agents.See, e.g., Jacques, J. et al., Enantiomers, Racemates and Resolutions (Wiley-Interscience, New York, 1981); Wilen, SH et al., Tetrahedron 33:2725 (1977); Eliel, EL, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, SH, Tables of Resolving Agents and Optical Resolutions, p. 268 (ELiel, ed., Univ. of Notre Dame Press, Notre Dame, Indiana, 1972).

[0120] In other embodiments, the CFS treatments provided herein include CRBN modulators. In some embodiments, CRBN modulators are agents that can directly or indirectly modulate at least one biological activity of CRBN. In some embodiments, CRBN modulators are agents that can physically bind to CRBN. In other embodiments, CRBN modulators do not directly bind to CRBN, but can act in other ways via CRBN-mediated pathways. 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 26, 2326-2335 (2012); Bjorklund et al., Leukemia. 2020; 34(4): 1197-1201). It is believed that the interaction of CRBN with certain immunomodulatory compounds mediates their antiproliferative effects in multiple myeloma (MM) cells (Lopez-Girona et al., Leukemia 2012; Zhu et al., Blood 2011, 118, Abstract 127). CRBN is encoded by a 25kb gene on chromosome 6, which consists of 11 exons and 10 introns. Therefore, the alternative splicing process can potentially generate multiple functional proteins and variants of a protein from a single gene with different structural organizations and functional activities. Truncated proteins that have lost the interaction domain or key functional amino acid residues can produce non-functional or abnormal CRBN proteins, which can interfere with the function of the full-length CRBN protein and reduce or change the therapeutic activity of the therapeutic compound, which exerts its activity via its interaction with the full-length CRBN protein. There are at least two isoforms of the protein cerebellum protein (CRBN), which are 442 and 441 amino acids long, respectively, and CRBN is conserved from plants to humans. In humans, the CRBN gene has been identified as a candidate gene for autosomal recessive non-syndromic mental retardation (ARNSMR). See Higgins, JJ et al., Neurology [Neurology], 2004, 63: 1927-1931. CRBN was initially characterized as a novel RGS-containing protein that interacts with a calcium-activated potassium channel protein (SLO1) in the rat brain and was subsequently shown to interact with a voltage-gated chloride channel (CIC-2) in the retina with 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 originally identified as a nucleotide excision repair protein associated with damaged DNA binding protein 2 (DDB2). Its defective activity leads to repair defects in patients with xeroderma pigmentosum complementation group E (XPE). DDB1 also appears as a component of many different DCX (DDB1-CUL4-X-box) E3 ubiquitin-protein ligase complexes that mediate ubiquitination and subsequent proteasomal degradation of target proteins. CRBN has also been identified as a target for the development of therapeutic agents for cerebral cortical diseases. See WO 2010 / 137547 A1. In some embodiments, the beneficial effects of certain therapeutic compounds provided herein require binding to CRBN or one or more substrates of CRBN. In some embodiments, the compounds provided herein for treating CFS can induce CRBN to undergo conformational changes. In some embodiments, the use of the therapeutic compounds provided herein causes different conformational changes or other changes in the properties of the CRBN surface, thereby producing different phenotypic responses. In certain embodiments, the CRBN modulator is an immunomodulatory compound. In other embodiments, CRBN is not an immunomodulatory compound.

[0121] In some embodiments, the CFS treatment 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 a B cell. In some embodiments, the antigen of a B cell is CD20, CD19, CD22, CD38, or B cell activation 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.

[0122] In some embodiments, the therapeutic compound of the selective treatment methods of the present invention is formulated in a pharmaceutical composition comprising a therapeutic compound provided herein and a pharmaceutically acceptable excipient. By combining the compounds provided herein with an optional physiologically acceptable excipient (see, e.g., Remington, Remington's Pharmaceutical Sciences (18th edition 1980)) in aqueous solution or lyophilized or other dry form to prepare pharmaceutical compositions containing therapeutic compounds provided herein for storage. The therapeutic compounds disclosed herein can be formulated in any suitable form for delivery to target cells / tissues, for example, as microcapsules or coarse emulsions (Remington, supra; Park et al., 2005, Molecules [molecules] 10: 146-61; Malik et al., 2007, Curr. Drug. Deliv. [Contemporary Drug Delivery] 4: 141-51), as sustained-release formulations (Putney and Burke, 1998, Nature Biotechnol. [Natural Biotechnology] 16: 153-57) or in liposomes (Maclean et al., 1997, Int. J. Oncol. [International Journal of Oncology] 11: 325-32; Kontermann, 2006, Curr. Opin. Mol. Ther. [New Progress in Molecular Therapy] 8: 39-45). The therapeutic compounds provided herein can also be encapsulated in prepared microcapsules, such as, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in coarse emulsions, respectively. Such techniques are disclosed in, for example, Remington, supra. Various compositions and delivery systems are known and can be used with compounds as described herein. In certain embodiments, the composition can be provided as a controlled release or 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. [Critical Reviews in Biomedical Engineering] 14:201-40; Buchwald et al., 1980, Surgery [Surgery] 88:507-16; and Saudek et al., 1989, N. Engl. J. Med. [New England Journal of Medicine] 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 a composition provided herein (see, e.g., Medical Applications of Controlled Release [Medical Applications of Controlled Release] (Langer and Wise, eds., 1974); Controlled Drug Bioavailability,Drug Product Design and PerformanceControlled 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. Pat. Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463 and 5,128,326; PCT Publication Nos. WO 99 / 15154 and WO 99 / 20253). Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactic acid (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained-release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In another embodiment, a controlled-release or sustained-release system can be placed near a specific target tissue, such as the nasal passages or lungs, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release[Medical Applications of Controlled Release] Vol. 2, 115-38 (1984). Controlled release systems are discussed, for example, by Langer, 1990, Science 249: 1527-33. Any technique known to those skilled in the art can be used to produce sustained-release formulations containing the therapeutic compounds described herein (see, e.g., U.S. Pat. No. 4,526,938, PCT 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 to administer the therapeutic compounds provided herein.

[0123] In another aspect according to the present disclosure, the responsiveness of the subject to the therapeutic compound can be determined by the change in the expression level of the biomarker provided herein. For example, a decrease in the expression level of the biomarker provided herein after treatment indicates that the subject is responsive to the treatment; and the absence of a change in the expression level of the biomarker indicates that the subject is not responsive. The extent of the biomarker change can also be used to indicate the extent of responsiveness. The treatment and dosage adjustment can then be designed accordingly. Therefore, in some embodiments, there is provided herein a method for determining or monitoring the effectiveness of treatment in a subject with 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 the treatment to the subject; obtaining a second expression level of the biomarker in a second sample obtained from the subject after the treatment is administered to the subject; and determining the effectiveness of the treatment based on a comparison of the first expression level with the second expression level. In some embodiments, the method includes: if the second expression level is lower than the first expression level, determining that the treatment is effective. In some embodiments, the method includes: if the second expression level is not lower than the first expression level, determining that the treatment is ineffective. In some embodiments, the method includes: if the second expression level is not lower than the first expression level, determining or adjusting (e.g., increasing) the dosage of the treatment or administering a different treatment to the subject.

[0124] 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 comprises an immunomodulatory drug (IMiD). In other embodiments, the treatment comprises a CRBN modulator. In some embodiments, the treatment comprises an agent that depletes B cells (e.g., an anti-CD20 antibody, e.g., rituximab).

[0125] Based on the comparison of the first expression level with the second expression level, different treatments or different dosing regimens can be applied to the subject in subsequent treatment cycles. 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 method known in the art, and exemplary methods are described in more detail in Section 5.3 below. In some embodiments, as observed according to the measurement assay, if the level is higher than (e.g., statistically significantly higher than) the second level, it is determined that the level is higher than the second level. In some embodiments, as observed according to the measurement assay, if the level is (e.g., statistically significantly) lower than the second level, it is determined that the level is lower than the second level.

[0126] In certain embodiments, the method includes using two or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the method includes using three or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the method includes using four or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the method includes using five or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the method includes using all biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the expression level of IKZF2 and IKZF3 is 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 with the first expression level of the biomarker, and the method includes: if the second expression level of each biomarker is lower than the first expression level of the biomarker, it is determined that the treatment is effective. In other embodiments, when multiple biomarkers are used, a comprehensive score based on multiple biomarkers is calculated and compared with a reference comprehensive score. In some embodiments, the comprehensive score is calculated using the median Z score method. In other embodiments, the comprehensive score is calculated using the single sample gene set enrichment (ssGSEA) method.

[0127] The present disclosure also includes the use of the biomarkers provided herein for screening compounds for effectiveness in treating CFS. Thus, in some embodiments, provided herein is a method for screening compounds for effectiveness 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; applying the compound to the sample; obtaining a second expression level of the biomarker in the sample after the compound is applied to the sample; comparing the first expression level to 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 comprises: 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 20% lower than the first expression level. In some embodiments, the method comprises: selecting the compound if the second expression level is at least 30% lower than the first expression level. In some embodiments, the method comprises: selecting the compound if the second expression level is at least 40% lower than the first expression level. In some embodiments, the method includes: if the second expression level is at least 50% lower than the first expression level, then the compound is selected. In some embodiments, the method includes: if the second expression level is at least 60% lower than the first expression level, then the compound is selected. In some embodiments, the method includes: if the second expression level is at least 70% lower than the first expression level, then the compound is selected. In some embodiments, the method includes: if the second expression level is at least 80% lower than the first expression level, then the compound is selected. In some embodiments, the method includes: if the second expression level is at least 10% lower than the first expression level, then the compound is selected. In some embodiments, the method includes: if the second expression level is at least 90% lower than the first expression level, then the compound is selected. In some embodiments, the therapeutic compound is an immunomodulatory drug (IMiD). In other embodiments, the therapeutic compound is a CRBN modulator or a compound capable of binding to CRBN and / or inducing 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 includes using three or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the method includes using four or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the method includes using five or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the method includes using all biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the expression levels of IKZF2 and IKZF3 are determined. In certain embodiments, the expression levels of IKZF2 and ABCE1 are determined. In certain embodiments, the expression levels of IKZF2 and BACH2 are determined. In certain 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 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 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 with the first expression level of the biomarker, and the method includes: if the second expression level of each biomarker is lower than the first expression level of the biomarker, the compound is selected. In other embodiments, when multiple biomarkers are used, a composite score based on multiple biomarkers is calculated and compared with a reference composite score, and if the composite score is higher than the reference composite score, the compound is selected. In some embodiments, the method includes obtaining a first composite score based on a first expression level of a biomarker and a second composite score based on a second expression level of a biomarker, and comparing the first composite score with the second composite score. In some embodiments, the composite score is calculated using the median Z score method. In other embodiments, the composite score is calculated using a single sample gene set enrichment (ssGSEA) method.

[0128] On the other hand, the present disclosure also provides methods and kits based on the surprising discovery that there is a correlation between CSF and certain organic acids. As shown in the example section, urine organic acid spectrum analysis revealed the differences between the CFS cohort and the normal cohort. More specifically, among 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, xanthocyanic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanillylmandelic acid, methylmalonic acid, glyceric acid, urine organic acid spectrum showed statistically significant (p < 0.05) differences between CFS patients and normal subjects (see Figures 6A-6F). Among them, 12 organic acids had a median fold change of >1.4 (CFS reduction) and p<0.05, and they were xanthocyanic 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 CFS bedridden and non-bedridden subjects. Therefore, one or more of these organic acids can be used in combination with IKZF2, IKZF3, ABCE1, BACH2, CD3D, and / or HSPA8 in the various methods described above. In other embodiments, one or more of these organic acids can be used independently of IKZF2, IKZF3, ABCE1, BACH2, CD3D and / or HSPA8 in various methods, for example, for identifying a subject having CFS or verifying CFS in a subject, determining the severity of CFS in a subject, identifying a subject who is likely or unlikely to respond to a CFS treatment or predicting the responsiveness of a subject to a CFS treatment, selectively treating a subject having or suspected of having CFS with a treatment, and / or determining or monitoring the effectiveness of a treatment in a subject having CFS, and screening compounds for effectiveness in treating CFS, as described in Examples 56-94 below.

[0129] In some embodiments, provided herein is a method for identifying a subject with chronic fatigue syndrome (CFS) or verifying CFS in a subject, the method comprising obtaining the level of a urine organic acid in a sample from the subject, wherein the urine 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, xanthocyanic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanillylmandelic acid, methylmalonic acid, and glyceric acid; and if the level of the urine organic acid in the sample is different from a reference level of the urine organic acid, the subject is identified or verified as having CFS. In some embodiments, the urine organic acid is selected from the group consisting of xanthuric 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 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 xanthuric 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 vanillylmandelic 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 not suffering from CFS. In some embodiments, the reference level of the organic acid is the level of the organic acid in a subject suffering from 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 not suffering from CFS, or a cohort of subjects suffering from moderate CFS).In some embodiments, the reference level of an organic acid is the median or average level of the organic acid in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects not suffering from CFS, or a cohort of subjects suffering from moderate CFS). In some embodiments, the method comprises: if the level of urinary organic acid is below the reference level of urinary organic acid, the subject is identified or verified as suffering from CFS. In some embodiments, the method comprises: if the level of organic acid is at least 5% lower than the reference level of organic acid, the subject is identified as suffering from CFS. In some embodiments, the method comprises: if the level of organic acid is at least 10% lower than the reference level of organic acid, the subject is identified as suffering from CFS. In some embodiments, the method comprises: if the level of organic acid is at least 20% lower than the reference level of organic acid, the subject is identified as suffering from CFS. In some embodiments, the method comprises: if the level of organic acid is at least 30% lower than the reference level of organic acid, the subject is identified as suffering from CFS. In some embodiments, the method comprises: if the level of organic acid is at least 40% lower than the reference level of organic acid, the subject is identified as suffering from CFS. In some embodiments, the method comprises identifying the subject as having 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 comprises identifying the subject as having 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 comprises identifying the subject as having 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 comprises identifying the subject as having 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 comprises identifying the subject as having 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 comprises identifying the subject as having CFS if the level of the organic acid is at most 1 / 2 of the reference level of the organic acid. In some embodiments, the method comprises identifying the subject as having CFS if the level of the organic acid is at most 1 / 3 of the reference level of the organic acid. In some embodiments, the method comprises identifying the subject as having CFS if the level of the organic acid is at most 1 / 4 of the reference level of the organic acid. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 5 of the reference level of the organic acid, then the subject is identified as having CFS. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 6 of the reference level of the organic acid, then the subject is identified as having CFS. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 7 of the reference level of the organic acid, then the subject is identified as having CFS.In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 8 of the reference level of the organic acid, the subject is identified as having CFS. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 9 of the reference level of the organic acid, the subject is identified as having CFS. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 10 of the reference level of the organic acid, the subject is identified as having CFS. The level of the organic acid can be determined using any method known in the art, and exemplary methods are described in more detail in Section 5.3 below. In some embodiments, if the level of the organic acid is (e.g., statistically significantly) below the reference level, as observed according to the measurement assay, the level of the organic acid is determined to be below the reference level. In certain embodiments, the method includes 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, xanthocyanic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanillylmandelic acid, methylmalonic acid and glyceric acid to identify a subject with CFS. In some embodiments, the method includes: if the level of urine organic acid in the sample is lower than the reference level of urine organic acid, the subject is identified or verified as having CFS. In some embodiments, CFS is associated with autoimmune disease or infection.

[0130] In some embodiments, provided herein is a method for determining the severity of CFS in a subject, the method comprising obtaining the level of a urine organic acid in a sample from the subject, wherein the urine 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, xanthocyanic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanillylmandelic acid, methylmalonic acid, and glyceric acid; comparing the level of the urine organic acid in the sample with a reference level of the urine organic acid; and determining the severity of CFS in the subject based on the comparison of the level of the urine organic acid in the sample with the reference level of the urine organic acid. In some embodiments, the urine organic acid is selected from the group consisting of xanthocyanic acid, glycolic acid, pyruvic acid, hippuric acid, isovalerylglycine, kynurenic acid, 3-hydroxyisovaleric acid, vanillylmandelic acid, pyroglutamic acid, 3-hydroxypropionic acid, glyceric acid and alpha-ketoadipic acid. In some embodiments, the method further comprises comparing the level of the organic acid to a reference level of the organic acid. In some embodiments, a lower level of the organic acid indicates more severe CFS. In other embodiments, a method for monitoring the progress of CFS in a subject is provided herein, 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, xanthocyanic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanillylmandelic acid, methylmalonic acid and glyceric acid, and the progress of CFS is assessed based on the level of biomarkers. 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 alpha-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 xanthuric 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 vanillylmandelic acid. In some embodiments, the organic acid is methylmalonic acid. In some embodiments, the organic acid is glyceric acid. In certain embodiments, the method includes 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, xanthocyanic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanillylmandelic acid, methylmalonic acid and glyceric acid to identify a subject with CFS. In some embodiments, the reference level of an organic acid is a predetermined level of an organic acid obtained from a public database. In some embodiments, the reference level of an organic acid is the level of an organic acid in a healthy subject or a subject not suffering from CFS. In some embodiments, the reference level of an organic acid is the level of an organic acid in a subject suffering from moderate CFS. In some embodiments, the reference level of organic acid is the level of organic acid determined based on a cohort of healthy subjects. In some embodiments, the reference level of organic acid is the level of organic acid in a subject whose severity of CFS has been determined and known. In some embodiments, the reference level of organic acid is the median or average level of the level of 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 subjects whose severity of CFS has been determined and known). In other embodiments, the reference level of organic acid is the level of organic acid measured in the same subject but at different times. The level of 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, as observed according to the measurement assay, if the level is higher than (e.g., statistically significantly higher than) the reference level, it is determined that the level is higher than the reference level. In some embodiments, as observed according to the measurement assay, if the level is (e.g., statistically significantly) lower than the reference level, it is determined that the level is lower than the reference level. In certain embodiments, the method comprises 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, xanthocyanic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanillylmandelic acid, methylmalonic acid and glyceric acid to identify a subject suffering from CFS. In certain embodiments, CFS is associated with an autoimmune disease or infection.

[0131] In some embodiments, provided herein is a method of identifying a subject who is likely or unlikely to respond to a CFS treatment or predicting a subject's responsiveness to a CFS treatment, the method comprising obtaining a level of a urine organic acid in a sample from the subject, wherein the urine 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, xanthocyanic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanillylmandelic acid, methylmalonic acid, and glyceric acid; and if the level of the urine organic acid in the sample is different from a reference level of the urine organic acid, identifying or predicting the subject as likely to respond to a CFS treatment. In some embodiments, the urine organic acid is selected from the group consisting of xanthuric 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 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 xanthuric 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 vanillylmandelic 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 not suffering from CFS. In some embodiments, the reference level of the organic acid is the level of the organic acid in a subject suffering from 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 not suffering from CFS, or a cohort of subjects suffering from moderate CFS).In some embodiments, the reference level of an organic acid is the median or average level of the organic acid in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects not suffering from CFS, or a cohort of subjects suffering from moderate CFS). In some embodiments, the method comprises: if the level of the organic acid is below the reference level of the organic acid, then the subject is identified or predicted as likely to respond to a CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at least 5% lower than the reference level of the organic acid, then the subject is identified or predicted as likely to respond to a CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at least 10% lower than the reference level of the organic acid, then the subject is identified or predicted as likely to respond to a CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at least 20% lower than the reference level of the organic acid, then the subject is identified or predicted as likely to respond to a CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at least 30% lower than the reference level of the organic acid, then the subject is identified or predicted as likely to respond to a CFS treatment. In some embodiments, the method comprises identifying or predicting the subject as likely to respond to a CFS treatment if the level of the organic acid is at least 40% lower than a reference level of the organic acid. In some embodiments, the method comprises identifying or predicting the subject as likely to respond to a CFS treatment if the level of the organic acid is at least 50% lower than a reference level of the organic acid. In some embodiments, the method comprises identifying or predicting the subject as likely to respond to a CFS treatment if the level of the organic acid is at least 60% lower than a reference level of the organic acid. In some embodiments, the method comprises identifying or predicting the subject as likely to respond to a CFS treatment if the level of the organic acid is at least 70% lower than a reference level of the organic acid. In some embodiments, the method comprises identifying or predicting the subject as likely to respond to a CFS treatment if the level of the organic acid is at least 80% lower than a reference level of the organic acid. In some embodiments, the method comprises identifying or predicting the subject as likely to respond to a CFS treatment if the level of the organic acid is at least 90% lower than a reference level of the organic acid. In some embodiments, the method comprises identifying or predicting the subject as likely to respond to a CFS treatment if the level of the organic acid is at most 1 / 2 of the reference level of the organic acid. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 3 of the reference level of the organic acid, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 4 of the reference level of the organic acid, then the subject is identified or predicted as likely to respond to CFS treatment.In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 5 of the reference level of the organic acid, then the subject is identified or predicted as likely to respond to the CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 6 of the reference level of the organic acid, then the subject is identified or predicted as likely to respond to the CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 7 of the reference level of the organic acid, then the subject is identified or predicted as likely to respond to the CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 8 of the reference level of the organic acid, then the subject is identified or predicted as likely to respond to the CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 9 of the reference level of the organic acid, then the subject is identified or predicted as likely to respond to the CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 10 of the reference level of the organic acid, then the subject is identified or predicted as likely to respond to the CFS treatment. In some embodiments, the method includes: if the level of urine organic acid is lower than the reference level of urine organic acid, the subject is identified or predicted as possibly responding to CFS treatment. In some embodiments, the method includes applying the treatment to a subject identified or predicted as possibly responding to the treatment. In certain embodiments, the method includes 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, xanthocyanic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanillylmandelic acid, methylmalonic acid and glyceric acid to identify a subject suffering from CFS. In some embodiments, CFS is associated with autoimmune disease or infection.

[0132] In some embodiments, provided herein is a method of selectively treating a subject having or suspected of having CFS with a treatment, the method comprising obtaining a level of a urine organic acid in a sample from the subject, wherein the urine 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, xanthocyanic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanillylmandelic acid, methylmalonic acid, and glyceric acid; identifying or predicting the subject as likely to respond to the CFS treatment if the level of the urine organic acid in the sample is different from a reference level of the urine organic acid; and administering the treatment to the subject identified or predicted as likely to respond to the treatment. In some embodiments, the urine organic acid is selected from the group consisting of xanthuric 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 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 xanthuric 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 vanillylmandelic 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 not suffering from CFS. In some embodiments, the reference level of the organic acid is the level of the organic acid in a subject suffering from 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 not suffering from CFS, or a cohort of subjects suffering from moderate CFS).In some embodiments, the reference level of an organic acid is the median or average level of the organic acid in a cohort of subjects (e.g., a cohort of healthy subjects, a cohort of subjects not suffering from CFS, or a cohort of subjects suffering from moderate CFS). In some embodiments, the method comprises: if the level of the organic acid is below the reference level of the organic acid, then the subject is identified or predicted as likely to respond to a CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at least 5% lower than the reference level of the organic acid, then the subject is identified or predicted as likely to respond to a CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at least 10% lower than the reference level of the organic acid, then the subject is identified or predicted as likely to respond to a CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at least 20% lower than the reference level of the organic acid, then the subject is identified or predicted as likely to respond to a CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at least 30% lower than the reference level of the organic acid, then the subject is identified or predicted as likely to respond to a CFS treatment. In some embodiments, the method comprises identifying or predicting the subject as likely to respond to a CFS treatment if the level of the organic acid is at least 40% lower than a reference level of the organic acid. In some embodiments, the method comprises identifying or predicting the subject as likely to respond to a CFS treatment if the level of the organic acid is at least 50% lower than a reference level of the organic acid. In some embodiments, the method comprises identifying or predicting the subject as likely to respond to a CFS treatment if the level of the organic acid is at least 60% lower than a reference level of the organic acid. In some embodiments, the method comprises identifying or predicting the subject as likely to respond to a CFS treatment if the level of the organic acid is at least 70% lower than a reference level of the organic acid. In some embodiments, the method comprises identifying or predicting the subject as likely to respond to a CFS treatment if the level of the organic acid is at least 80% lower than a reference level of the organic acid. In some embodiments, the method comprises identifying or predicting the subject as likely to respond to a CFS treatment if the level of the organic acid is at least 90% lower than a reference level of the organic acid. In some embodiments, the method comprises identifying or predicting the subject as likely to respond to a CFS treatment if the level of the organic acid is at most 1 / 2 of the reference level of the organic acid. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 3 of the reference level of the organic acid, then the subject is identified or predicted as likely to respond to CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 4 of the reference level of the organic acid, then the subject is identified or predicted as likely to respond to CFS treatment.In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 5 of the reference level of the organic acid, then the subject is identified or predicted as likely to respond to the CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 6 of the reference level of the organic acid, then the subject is identified or predicted as likely to respond to the CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 7 of the reference level of the organic acid, then the subject is identified or predicted as likely to respond to the CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 8 of the reference level of the organic acid, then the subject is identified or predicted as likely to respond to the CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 9 of the reference level of the organic acid, then the subject is identified or predicted as likely to respond to the CFS treatment. In some embodiments, the method comprises: if the level of the organic acid is at most 1 / 10 of the reference level of the organic acid, then the subject is identified or predicted as likely to respond to the CFS treatment. In certain embodiments, the method comprises 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, xanthocyanic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanillylmandelic acid, methylmalonic acid and glyceric acid to identify a subject suffering from CFS. In certain embodiments, CFS is associated with an autoimmune disease or infection.

[0133] In some embodiments, provided herein is a method of determining or monitoring the effectiveness of a treatment in a subject having CFS, the method comprising obtaining a first level of urine organic acids in a first sample from the subject before administering the treatment to the subject, wherein the urine 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, xanthocyanic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanillylmandelic acid, methylmalonic acid, and glyceric acid; administering the treatment to the subject, and obtaining a second level of urine organic acids 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 level to the second level. In some embodiments, the urine organic acid is selected from the group consisting of xanthuric 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 some embodiments, the urine organic acid is selected from the group consisting of xanthuric 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 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 alpha-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 xanthocyanic 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 vanillylmandelic acid. In some embodiments, the organic acid is methylmalonic acid. In some embodiments, the organic acid is glyceric acid. In some embodiments, the method comprises: if the second level is higher than the first level, determining that the treatment is effective. In some embodiments, the method comprises: if the second level is at least 10% higher than the first level, determining that the treatment is effective.In some embodiments, the method includes determining that the treatment is effective if the second level is at least 20% higher than the first level. In some embodiments, the method includes determining that the treatment is effective if the second level is at least 30% higher than the first level. In some embodiments, the method includes determining that the treatment is effective if the second level is at least 40% higher than the first level. In some embodiments, the method includes determining that the treatment is effective if the second level is at least 50% higher than the first level. In some embodiments, the method includes determining that the treatment is effective if the second level is at least 60% higher than the first level. In some embodiments, the method includes determining that the treatment is effective if the second level is at least 70% higher than the first level. In some embodiments, the method includes determining that the treatment is effective if 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 the dose of the treatment for the subject. In some embodiments, the method includes determining the levels of two or more urinary organic acids and comparing the levels of each urinary organic acid with their respective reference levels. 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 organic acid in healthy subjects or subjects not suffering from CFS. In some embodiments, the reference level of organic acid is the level of organic acid in subjects suffering from moderate CFS. In some embodiments, the reference level of organic acid is the level of organic acid determined based on a queue of healthy subjects. In some embodiments, the reference level of organic acid is the level of organic acid in the first sample obtained from a subject. In some embodiments, the reference level of organic acid is the median or average level of the level of organic acid in a queue of a subject (e.g., a queue of healthy subjects, a queue of subjects suffering from moderate CFS, or a queue of the first sample obtained from a subject). In some embodiments, CFS is associated with an autoimmune disease or infection.

[0134] In some embodiments, provided herein is a method for screening a compound for effectiveness in treating CFS, the method comprising obtaining a first level of a urine organic acid in a sample, wherein (i) the urine 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, xanthocyanic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanillylmandelic acid, methylmalonic acid, and glyceric acid; (ii) xanthocyanic 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; and / or (iii) the urine organic acid is vanillylmandelic acid; applying the compound to the sample; obtaining a second level of urine organic acids in the sample after applying the compound to the sample; comparing the first level with the second level; and if the second level is different from the first level, selecting the compound, wherein if the second level is higher than the first level, selecting the compound. 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 alpha-ketoadipate. 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 xanthocyanic 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 vanillylmandelic acid. In some embodiments, the organic acid is methylmalonic acid. In some embodiments, the organic acid is glyceric acid. In some embodiments, the method comprises: if the second level is higher than the first level, then selecting the treatment. In some embodiments, the method comprises: if the second level is 10% higher than the first level, then selecting the treatment. In some embodiments, the method comprises: if the second level is 20% higher than the first level, then selecting the treatment. In some embodiments, the method comprises: if the second level is 30% higher than the first level, then selecting the treatment. In some embodiments, the method comprises: if the second level is 40% higher than the first level, then selecting the treatment.In some embodiments, the method includes: if the second level is 50% higher than the first level, then the treatment is selected. In some embodiments, the method includes: if the second level is 60% higher than the first level, then the treatment is selected. In some embodiments, the method includes: if the second level is 70% higher than the first level, then the treatment is selected. In some embodiments, the method includes: if the second level is 80% higher than the first level, then the treatment is selected. In some embodiments, the method includes: if the second level is 90% higher than the first level, then the treatment is selected. In some embodiments, the method includes determining the levels of two or more urinary organic acids and comparing the level of each urinary organic acid with their respective reference levels. 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 not suffering from CFS. In some embodiments, the reference level of the organic acid is the level of the organic acid in a subject suffering from 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 an organic acid is a median or average level of an 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 a first sample obtained from a subject). In some embodiments, CFS is associated with an autoimmune disease or infection.

[0135] In some embodiments, CFS treatment includes any treatment for alleviating any symptoms associated with CFS. In some embodiments, treatment includes immunomodulatory compounds. In some embodiments, treatment includes immunomodulatory drugs (IMiDs). IMiDs include a group of compounds that can be used to treat several types of human diseases (including certain cancers). Unless otherwise indicated, the term "immunomodulatory compound" as used herein may encompass 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 synthesized or commercially available. In other embodiments, CFS treatments provided herein include CRBN regulators. In some embodiments, CRBN regulators are agents that can directly or indirectly regulate at least one biological activity of CRBN. In some embodiments, CRBN regulators are agents that can be physically combined with CRBN. In other embodiments, CRBN regulators do not directly bind to CRBN, but can otherwise play a role via CRBN-mediated pathways. In some embodiments, the CFS treatment 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 a B cell. In some embodiments, the antigen of a B cell is CD20, CD19, CD22, CD38, or B cell activation 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 inelizumab. In some embodiments, the agent that depletes B cells is an anti-BAFF antibody. In some embodiments, the anti-BAFF antibody is belimumab.

[0136] In some embodiments, the therapeutic compound is formulated in a pharmaceutical composition comprising a therapeutic compound provided herein and a pharmaceutically acceptable excipient. In some embodiments, CFS is associated with an autoimmune disease or infection.

[0137] In some embodiments, when multiple organic acids are used, the second level of each organic acid is compared to the first level of the organic acid, and the method includes: if the second level of each organic acid is lower than the first level of the organic acid, the subject is identified as having CFS. In some embodiments, when two or more organic acids are used, a comprehensive score based on multiple organic acids is calculated and compared with a reference comprehensive score. In some embodiments, the method includes determining a first level and a second level of two or more urine organic acids, and obtaining a first comprehensive score based on the first level of urine organic acids and a second comprehensive score based on the second level of urine organic acids, and comparing the first comprehensive score with the second comprehensive score. In some embodiments, the comprehensive score is calculated using the median Z score method. In other embodiments, the comprehensive score is calculated using the single sample gene set enrichment (ssGSEA) method.

[0138] In some embodiments, a kit is provided herein, comprising a kit 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, xanthocyanic acid, isovalerylglycine, 3-hydroxyisovaleric acid, isocitric acid, cis-aconitic acid, pyroglutamic acid, vanillylmandelic 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 alpha-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 xanthocyanic 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 vanillylmandelic 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 a tool for obtaining a sample. In some embodiments, the kit includes instructions for interpreting the determined levels. In some embodiments, CFS is associated with an autoimmune disease or infection. 5.2.2 Biomarkers of Long COVID

[0139] Long COVID is also called post-COVID illness, long COVID, post-acute COVID-19, long-term effects of COVID, and chronic COVID. Long COVID is a wide range of new, recurring, or persistent health problems that people experience after being infected with the virus that causes COVID-19 (see, e.g., Thaweethai et al., JAMA [Journal of the American Medical Association] (published online May 25, 2023) "Development of a Definition of Postacute Sequelae of SARS-CoV-2 Infection"). Most people with COVID-19 get better within a few days to a few weeks after infection, so at least 3 months after infection is the first time that the start of long COVID can be identified. Symptoms of long COVID may include tiredness or fatigue that interferes with daily life, shortness of breath, cough, chest pain, palpitations, difficulty thinking or concentrating (sometimes called "brain fog"), headaches, sleep problems, dizziness (lightheadedness) when standing up, pins and needles, changes in smell or taste, depression or anxiety, diarrhea, stomach pain, joint or muscle pain, rash, and menstrual cycle changes. In some embodiments, the 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 some embodiments, the 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 symptom is selected from fatigue, post-exertional malaise, cognitive dysfunction, sensory motor symptoms, headaches, memory problems, insomnia, muscle pain, palpitations, shortness of breath, dizziness and balance problems, speech and language problems, joint pain, and chest tightness.

[0140] In some embodiments, provided herein is a method for identifying a subject having long COVID or validating 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 cerebellar leukocyte antigen (CRBN)-related protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; and (b) identifying or validating the subject as having long COVID if the expression level of the biomarker is higher than a reference expression level of the biomarker.

[0141] In some embodiments, a method for identifying a subject who may or may not respond to long COVID treatment or predicting the responsiveness of a subject to long COVID treatment is provided herein, the method comprising: (a) determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a cerebellum protein (CRBN)-related protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; and (b) if the expression level of the biomarker is higher than the reference expression level of the biomarker, the subject is identified or predicted as likely to respond to the treatment. In certain embodiments, the method further comprises administering the treatment to a subject identified or predicted as likely to respond to the treatment.

[0142] In some embodiments, provided herein is a method for selectively treating a subject having or suspected of having long COVID with a therapy, the method comprising: (a) determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is a cerebellar leukocyte antigen (CRBN)-related protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; (b) identifying or predicting the subject as likely to respond to the long COVID therapy if the expression level of the biomarker is higher than a reference expression level of the biomarker; and (c) administering the therapy to the subject identified or predicted as likely to respond to the therapy.

[0143] 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.

[0144] In some embodiments, the reference expression level of a biomarker is a predetermined expression level of a biomarker. In some embodiments, the reference expression level of a biomarker is a predetermined expression level of a biomarker obtained from a public database. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker in a healthy subject or a subject not suffering from long COVID. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker in a subject suffering from acute COVID (e.g., severe acute COVID). In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker determined based on a cohort of a subject (e.g., a cohort of healthy subjects, a cohort of subjects not suffering from long COVID, or a cohort of subjects suffering from acute COVID (e.g., severe acute COVID)). In some embodiments, the reference expression level of a biomarker is the median or average expression level of a biomarker of the expression level of a biomarker in a cohort of a subject (e.g., a cohort of healthy subjects, a cohort of subjects not suffering from long COVID, a cohort of subjects suffering from acute COVID (e.g., severe acute COVID)).

[0145] 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 healthy subjects or subjects without long COVID, or 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 subjects with acute COVID (e.g., severe acute COVID) or a cohort of subjects with acute COVID (e.g., severe acute COVID).

[0146] As used herein, a subject with acute COVID refers to a subject who is confirmed to be infected with SARS-CoV-2 and is experiencing the acute phase of the disease, which begins with the onset of the first symptoms associated with SARS-CoV-2 infection and ends after 15 days. A subject with severe acute COVID refers to a subject with acute COVID and respiratory compromise defined by the need for oxygen supplementation without the need for mechanical ventilation. In certain embodiments, a subject with severe acute COVID is hospitalized (or awaiting hospitalization in the ED).

[0147] In some embodiments, the method includes identifying the subject as having long COVID if the expression level of the 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 a reference expression level of the biomarker. The expression level of the biomarker can be determined using any method known 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 a reference level as observed according to a measurement assay, the level is determined to be higher than the reference level.

[0148] 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 to identify or validate a subject with long COVID, or to identify or predict a subject as likely to respond to long COVID treatment. 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, 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.

[0149] In some embodiments, when multiple biomarkers are used, the expression level of each biomarker is compared with the reference expression level of the biomarker, and the method includes identifying or verifying a subject with long COVID, or if the expression level of each biomarker is higher than the reference expression level of the biomarker, the subject is identified or predicted as being likely to respond to long COVID treatment. In other embodiments, when multiple biomarkers are used, a comprehensive score based on multiple biomarkers is calculated and compared with a reference comprehensive score. In some embodiments, the method includes identifying or verifying a subject with long COVID, or if the comprehensive score is higher than the reference comprehensive score, the subject is identified or predicted as being likely to respond to long COVID treatment. In some embodiments, the comprehensive score is calculated using the median Z score method. In short, the median Z score is derived by first calculating the mean value of each gene from all samples in the gene expression matrix. The mean value is then subtracted from each corresponding gene of all samples, and then scaled by dividing these values ​​by their standard deviations. The median scaling value from multiple target genes includes a comprehensive score. Another exemplary method for calculating a comprehensive score is a single sample gene set enrichment (ssGSEA) method. Single sample gene score represents the extent to which genes in a particular gene set are co-regulated up or down within a sample. The score is calculated by adjusting the running sum statistic based on the decreasing trend in the sorted expression list. The enrichment score is the maximum zero deviation encountered in the trend; it corresponds to a weighted Kolmogorov-Smirnov statistic (see, e.g., Subramanian et al., PNAS [Proceedings of the National Academy of Sciences of the United States], 102(43): 15545-15550 (2005); and Barbie et al., Nature [Natural], 462(7269): 108-112).

[0150] In some embodiments, a method for determining or monitoring the effectiveness of a treatment in a subject with long COVID is provided herein, the method comprising: (a) determining a first expression level of a biomarker in a first sample obtained from the subject before the treatment is administered to the subject, wherein the biomarker is a cerebellum protein (CRBN)-related 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 a biomarker in a second sample obtained from the subject after the treatment is administered to the subject; and (d) determining the effectiveness of the treatment based on a comparison of the first expression level with the second expression level. In certain embodiments, the method comprises: if the second expression level is lower than the first expression level, determining that the treatment is effective. In certain embodiments, the method comprises: if the second expression level is not lower than the first expression level, determining that the treatment is ineffective. In certain embodiments, the method further comprises: if the second expression level is not lower than the first expression level, determining or adjusting (e.g., increasing) the dosage of the treatment or administering a different long COVID treatment to the subject.

[0151] In some embodiments, provided herein is a method for screening a treatment for effectiveness in treating long COVID, the method comprising: (a) determining a first expression level of a biomarker in a sample before administering a compound to the sample, wherein the biomarker is cerebellum protein (CRBN)-related 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.

[0152] In some embodiments, the method includes: 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, then determining that the treatment is effective. Based on the comparison of the first expression level with the second expression level, different treatments or different dosing regimens can be administered to the subject in subsequent treatment cycles.

[0153] 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.

[0154] The expression level of a biomarker can be determined using any method known 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 a second level as observed according to 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 lower) than a second level as observed according to a measurement assay, the level is determined to be lower than the second level.

[0155] 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 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 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.

[0156] In some embodiments, when multiple biomarkers are used, the second expression level of each biomarker is compared with the first expression level of the biomarker, and the method includes: if the second expression level of each biomarker is lower than the first expression level of the biomarker, it is determined that the treatment is effective. In other embodiments, when multiple biomarkers are used, a comprehensive score based on multiple biomarkers is calculated and compared with a reference comprehensive score. In some embodiments, the comprehensive score is calculated using the median Z score method. In other embodiments, the comprehensive score is calculated using the single sample gene set enrichment (ssGSEA) method.

[0157] In certain embodiments, the treatment for long COVID disclosed herein includes immunomodulatory drugs (IMiDs). IMiDs include a group of compounds that can be used to treat several types of human diseases, including certain cancers. Unless otherwise specified, the term "immunomodulatory compound" as used herein may cover 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 synthesized or commercially available. The inflammatory cytokine TNF-α produced by macrophages and monocytes during acute inflammation causes a series of different signaling events in cells. Without being limited by a particular theory, one of the biological effects exerted by the immunomodulatory compounds disclosed herein is to reduce the production of myeloid cell TNF-α. The immunomodulatory compounds disclosed herein can enhance the degradation of TNF-αmRNA. In addition, without being limited by theory, the immunomodulatory compounds disclosed herein may also be effective T cell co-stimulators and significantly increase cell proliferation in a dose-dependent manner. The immunomodulatory compounds disclosed herein may also have a greater costimulatory effect on CD8+T cell subsets than on CD4+T cell subsets. In addition, the compound may have anti-inflammatory properties against myeloid cell responses, but effectively costimulates T cells to produce more IL-2, IFN-γ, and enhances T cell proliferation and CD8+T cell cytotoxic activity. In addition, without being limited by a specific theory, the immunomodulatory compounds disclosed herein may be able to act on natural killer ("NK") cells and natural killer T ("NKT") cells indirectly and directly through cytokine activation, and increase the ability of NK cells to produce beneficial cytokines (such as but not limited to IFN-γ), and enhance the cytotoxic activity of NK and NKT cells. Various immunomodulatory compounds disclosed herein contain one or more chiral centers and may exist as a racemic mixture of enantiomers or a mixture of diastereomers. Therefore, the use of stereoisomerically pure forms of these compounds is also provided herein, as well as the use of mixtures of these forms. For example, a mixture of enantiomers of a specific immunomodulatory compound containing equal or unequal amounts can be used. These isomers can be synthesized asymmetrically or resolved using standard techniques such as chiral columns or chiral resolving agents.See, e.g., Jacques, J. et al., Enantiomers, Racemates and Resolutions (Wiley-Interscience, New York, 1981); Wilen, SH et al., Tetrahedron 33:2725 (1977); Eliel, EL, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, SH, Tables of Resolving Agents and Optical Resolutions, p. 268 (ELiel, ed., Univ. of Notre Dame Press, Notre Dame, Indiana, 1972).

[0158] In certain embodiments, the treatment for long COVID disclosed herein includes a CRBN modulator. In some embodiments, a CRBN modulator is an agent that can directly or indirectly modulate at least one biological activity of CRBN. In some embodiments, a CRBN modulator is an agent that can be physically bound to CRBN. In other embodiments, a CRBN modulator does not directly bind to CRBN, but can otherwise act via a CRBN-mediated pathway. Cerebellin (CRBN), a component of a DDB1-CUL4a-Roc1 ubiquitin ligase complex, has been identified as a target for certain immunomodulatory compounds, such as thalidomide, lenalidomide, pomalidomide, and ibedomide (Lopez-Girona et al., Leukemia [Leukemia] Vol. 26, pp. 2326-2335 (2012); Bjorklund et al., Leukemia. [Leukemia] 2020; 34(4): 1197-1201). It is believed that the interaction of CRBN with certain immunomodulatory compounds mediates their antiproliferative effects 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, which consists of 11 exons and 10 introns. Therefore, the process of alternative splicing can potentially generate multiple functional proteins and variants of a protein from a single gene with different structural organizations and functional activities. Truncated proteins that have lost the interaction domain or key functional amino acid residues can produce non-functional or abnormal CRBN proteins that can interfere with the function of the full-length CRBN protein and reduce or alter the therapeutic activity of therapeutic compounds that exert their activity via their interaction with the full-length CRBN protein. There are at least two isoforms of the protein cerebellin (CRBN), which are 442 and 441 amino acids long, 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, JJ et al., Neurology, 2004, 63: 1927-1931. CRBN was initially characterized as a novel protein containing RGS that interacts with calcium-activated potassium channel protein (SLO1) in rat brain and was subsequently shown to interact with voltage-gated chloride channels (CIC-2) in the retina with 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 originally identified as a nucleotide excision repair protein associated with damaged DNA binding protein 2 (DDB2). Its defective activity leads to repair defects in patients with xeroderma pigmentosum complementation group E (XPE). DDB1 also appears as a component of many different DCX (DDB1-CUL4-X-box) E3 ubiquitin-protein ligase complexes that mediate ubiquitination and subsequent proteasomal degradation of target proteins. CRBN has also been identified as a target for the development of therapeutic agents for cerebral cortical diseases. See WO 2010 / 137547 A1. In some embodiments, the beneficial effects of certain therapeutic compounds provided herein require binding to CRBN or one or more substrates of CRBN. In some embodiments, the therapeutic compounds provided herein can induce CRBN to undergo conformational changes. In some embodiments, the use of the therapeutic compounds provided herein causes different conformational changes or other changes in the properties of the CRBN surface, thereby producing different phenotypic responses. In certain embodiments, the CRBN modulator is an immunomodulatory compound. In other embodiments, CRBN is not an immunomodulatory compound.

[0159] In some embodiments, the treatment for 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 a B cell. In some embodiments, the antigen of a B cell is CD20, CD19, CD22, CD38, or B cell activation 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 inelizumab. In some embodiments, the agent that depletes B cells is an anti-BAFF antibody. In some embodiments, the anti-BAFF antibody is belimumab.

[0160] In some embodiments, the therapeutic compounds provided herein are formulated in a pharmaceutical composition comprising a therapeutic compound provided herein and a pharmaceutically acceptable excipient. Remington's Pharmaceutical Sciences(18th edition 1980)) in aqueous solution or lyophilized or other dry form to prepare pharmaceutical compositions containing therapeutic compounds provided herein for storage. The therapeutic compounds disclosed herein can be formulated in any suitable form for delivery to target cells / tissues, for example, as microcapsules or coarse emulsions (Remington, supra; Park et al., 2005, Molecules [molecules] 10: 146-61; Malik et al., 2007, Curr. Drug. Deliv. [Contemporary Drug Delivery] 4: 141-51), as sustained-release formulations (Putney and Burke, 1998, Nature Biotechnol. [Natural Biotechnology] 16: 153-57) or in liposomes (Maclean et al., 1997, Int. J. Oncol. [International Journal of Oncology] 11: 325-32; Kontermann, 2006, Curr. Opin. Mol. Ther. [New Progress in Molecular Therapy] 8: 39-45). The therapeutic compounds provided herein can also be encapsulated in prepared microcapsules, such as, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in coarse emulsions, respectively. Such techniques are disclosed in, for example, Remington, supra. Various compositions and delivery systems are known and can be used with compounds as described herein. In certain embodiments, the composition can be provided as a controlled release or 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. [Critical Reviews in Biomedical Engineering] 14:201-40; Buchwald et al., 1980, Surgery [Surgery] 88:507-16; and Saudek et al., 1989, N. Engl. J. Med. [New England Journal of Medicine] 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 a composition provided herein (see, e.g., Medical Applications of Controlled Release [Medical Applications of Controlled Release] (Langer and Wise, eds., 1974); Controlled Drug Bioavailability,Drug Product Design and PerformanceControlled 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. Pat. Nos. 5,679,377, 5,916,597, 5,912,015, 5,989,463 and 5,128,326; PCT Publication Nos. WO 99 / 15154 and WO 99 / 20253). Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactic acid (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one embodiment, the polymer used in the sustained-release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In another embodiment, a controlled-release or sustained-release system can be placed near a specific target tissue, such as the nasal passages or lungs, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, Medical Applications of Controlled Release[Medical Applications of Controlled Release] Vol. 2, 115-38 (1984). Controlled release systems are discussed, for example, by Langer, 1990, Science 249: 1527-33. Any technique known to those skilled in the art can be used to produce sustained-release formulations containing the therapeutic compounds described herein (see, e.g., U.S. Pat. No. 4,526,938, PCT 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 to administer the therapeutic compounds provided herein. 5.3. Methods for detecting and quantifying biomarkers

[0161] Any method and technique known in the art for detecting or quantifying biomarkers can be used for the present method. 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 a biomarker provided herein is determined by measuring the mRNA level of the biomarker. In other embodiments, the expression level of a biomarker provided herein is determined by measuring the cDNA level of the biomarker. In other embodiments, the expression level of a biomarker provided herein is determined by measuring the protein level of the biomarker.

[0162] Several methods for detecting or quantifying mRNA levels are known in the art. Exemplary methods include, but are not limited to, Northern blots, RNA sequencing, microarrays, ribonuclease protection assays, PCR-based methods, and the like. The mRNA sequence of a biomarker can be used to prepare a probe that is at least partially complementary to the mRNA sequence. The probe can then be used to detect the mRNA in a sample using any suitable assay, such as a PCR-based method, Northern blot, test strip assay, and the like.

[0163] 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, wherein the nucleic acid corresponds to at least a portion of the mRNA of the biomarker. The assay can also have a means for detecting expression (e.g., expression changes) of mRNA in a sample.

[0164] The assay method can vary depending on the type of mRNA information desired. Exemplary methods include, but are not limited to, Northern blots 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).

[0165] Any suitable assay platform can be used to determine the presence of mRNA in a sample. For example, the assay can be in the form of a test strip, a membrane, a chip, a disc, a test paper, a filter, a microsphere, a slide, a multi-well plate, or an optical fiber. The assay system can have a solid support to which the nucleic acid corresponding to the mRNA is attached. The solid support can include, for example, plastic, silicon, metal, resin, glass, film, particle, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, disc, or slide. The assay components can be prepared and packaged together as a kit for detecting mRNA.

[0166] If desired, the nucleic acid can be labeled to prepare a population of labeled mRNA. Typically, 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.). See, e.g., Ausubel et al., Short Protocols in Molecular Biology [Short Protocols in Molecular Biology] (Wiley & Sons, 3rd edition 1995); Sambrook et al., Molecular Cloning:A Laboratory Manual[Molecular Cloning: A Laboratory Manual] (Cold Spring Harbor, NY [Cold Spring Harbor Laboratory Press], New York, 3rd edition 2001). In some embodiments, the sample is labeled with a fluorescent marker. 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), coumarins, diethylaminocoumarins, umbelliferone, benzalkonium 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, lissamine, naphthol fluorescein, and the like.

[0167] The nucleic acids may be present at specific, addressable locations on the solid support, each corresponding to at least a portion of an mRNA sequence that is differentially expressed in a cell or patient following treatment with the compound.

[0168] For example, in certain embodiments, a method for detecting and quantifying the level of RNA (e.g., mRNA) of a biomarker from a biological sample comprises: (a) obtaining RNA from the sample; (b) contacting the RNA with a primer that specifically binds to a sequence in the RNA to produce a first DNA molecule having a sequence complementary to the RNA; (c) amplifying DNA corresponding to a gene segment encoding the biomarker; and (d) determining the RNA level of the biomarker based on the amount of amplified DNA.

[0169] A typical mRNA assay method may include the following steps: 1) obtaining surface-bound target probes; 2) hybridizing a population of mRNAs to these surface-bound probes under conditions sufficient to provide specific binding; (3) post-hybridization washing to remove nucleic acids that are not specifically bound to these surface-bound probes; and (4) detecting hybridized mRNAs. The reagents used in each of these steps and the conditions under which they are used may vary depending on the specific application.

[0170] Hybridization can be carried out under suitable hybridization conditions, and its stringency can be varied as required. Typical conditions are sufficient to produce probe / target complexes between complementary binding members (i.e., between the target probe bound to the surface and the complementary mRNA in the sample) on a solid surface. In certain embodiments, stringent hybridization conditions can be used.

[0171] Hybridization is usually performed under stringent hybridization conditions. Kallioniemi et al., Science 1992, 258:818-821 and International Patent Application Publication No. WO 93 / 18186 describe standard hybridization techniques (e.g., under conditions sufficient to provide specific binding of the target mRNA in the sample to the probe). Several general technical guidelines are available, for example, Tijssen, Hybridization with Nucleic Acid Probes [Hybridization with Nucleic Acid Probes], Parts I and II (Elsevier, Amsterdam 1993). For a description 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 [Genetic Engineering: Principles and Methods], Volume 7, Pages 43-65 (Plenum Press, New York, Setlow and Hollaender, eds., 1985). The selection of appropriate conditions, including temperature, salt concentration, polynucleotide concentration, hybridization time, stringency of washing conditions, etc. will depend on the experimental design, including the source of the sample, the identity of the capture agent, the expected degree of complementarity, etc., and can be determined as a routine experiment by one of ordinary skill in the art.

[0172] One of ordinary skill will readily recognize that alternative but comparable hybridization and wash conditions can be used to provide conditions of similar stringency.

[0173] After the mRNA hybridization procedure, the surface-bound polynucleotides are usually washed to remove unbound nucleic acids. Any convenient washing scheme can be used for washing, wherein the washing conditions are usually stringent, as described above. Standard techniques are then used to detect hybridization of the target mRNA with the probe.

[0174] Other methods, such as PCR-based methods, can also be used to detect or quantify the expression of biomarkers. Examples of PCR methods can be found in U.S. Patent No. 6,927,024, which is incorporated herein by reference in its entirety. Examples of RT-PCR methods can be found in U.S. Patent No. 7,122,799, which is incorporated herein by reference in its entirety. U.S. Patent No. 7,186,507 (which is incorporated herein by reference in its entirety) describes methods for fluorescent in situ PCR.

[0175] In some embodiments, quantitative reverse transcription-PCR (qRT-PCR) can be used for detection and quantification of RNA targets (Bustin et al., Clin. Sci. [Clinical Science] 2005, 109: 365-379). Quantitative results obtained by qRT-PCR generally provide more information than qualitative data. Therefore, in some embodiments, qRT-PCR-based assays can be used to measure mRNA levels during cell-based assays. qRT-PCR methods can also be used to monitor patient therapy. Examples based on qRT-PCR methods can be found in, for example, U.S. Patent No. 7,101,663, which is incorporated herein by reference in its entirety.

[0176] Compared with conventional reverse transcriptase-PCR and agarose gel analysis, qRT-PCR gives quantitative results. Another advantage of qRT-PCR is that it is relatively simple and convenient to use. Instruments for qRT-PCR (such as Applied Biosystems 7500) are commercially available, as are reagents such as Sequence detection chemistry. For example, one can use Gene Expression Assays. These kits are pre-formulated gene expression assay kits for rapid and reliable detection and quantification of human, mouse, and rat mRNA transcripts. For example, an exemplary qRT-PCR program is 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds, then 60°C for 1 minute.

[0177] To determine when the fluorescence signal associated with the accumulation of a specific amplicon exceeds a threshold value (called C T ) cycle number, the data can be analyzed, for example, using the 7500 Real-Time PCR System Sequence Detection Software to compare the results using the comparative C T Relative quantification calculation method. Using this method, the output is expressed as a fold change in expression level. In some embodiments, a threshold level can be selected to be automatically determined by the software. In some embodiments, the threshold level is set above the baseline but low enough to be within the exponential growth region of the amplification curve.

[0178] Several protein detection and quantitative methods can be used to measure the level of biomarkers. Any suitable protein quantitative method can be used. In some embodiments, an antibody-based method is used. In some embodiments, the protein level of a biomarker is measured using an immunoassay. In some embodiments, immunoassays include Western blots, enzyme-linked immunosorbent assays (ELISA), flow cytometry, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassays (RIA), dot blots, and flow cytometry. In some embodiments, ELISA is direct ELISA (enzyme-linked immunosorbent assay), indirect ELISA, sandwich ELISA, competitive ELISA, multiple ELISA, ELISPOT technology, and other similar techniques known in the art.

[0179] In some embodiments, the protein level of the biomarker is determined by using mass spectrometry (MS). In some embodiments, 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 (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 systems, 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 mass spectrometry (IMS), inductively coupled plasma mass spectrometry (ICP-MS), atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS / MS, and APPI-(MS)n.

[0180] Exemplary antibody-based assays for determining the protein level of a biomarker include contacting the protein in the sample with a first antibody that immunospecifically binds to a biomarker protein. In some embodiments, the method provided herein further includes (i) contacting the biomarker protein bound to the first antibody with a second antibody having a detectable label, wherein the second antibody immunospecifically binds to the biomarker protein, and wherein the second antibody immunospecifically binds to an epitope different from the first antibody on the biomarker protein; (ii) detecting the presence of the second antibody bound to the biomarker protein; and (iii) determining the amount of the biomarker protein based on the amount of the detectable label in the second antibody. In other embodiments, the method provided herein further includes (i) contacting the biomarker protein bound to the first antibody with a second antibody having a detectable label, wherein the second antibody immunospecifically binds to the first antibody; (ii) detecting the presence of the second antibody bound to the first antibody; and (iii) determining the amount of the biomarker protein based on the amount of the detectable label in the second antibody.

[0181] In certain embodiments of the various methods provided herein, two or more steps are performed sequentially. In other embodiments of the methods provided herein, two or more steps are performed in parallel (e.g., simultaneously). 5.4. Subjects, samples, and cell types

[0182] In certain embodiments, the various methods provided herein use samples (e.g., biological samples) from a subject or individual (e.g., a patient). The subject can be a patient, such as, for example, a patient suffering from CFS. The subject can be a mammal, such as a human. The subject can be male or female, and can be an adult, a child, or an infant. The sample can be analyzed during the CFS active phase or the CFS inactive phase. In certain embodiments, more than one sample is obtained from a subject.

[0183] In some embodiments, the subject (e.g., patient) has one or more symptoms of CFS. In some embodiments, the subject is a patient suffering from fatigue, such as a bedridden patient suffering from fatigue. In some embodiments, fatigue is caused by infection (e.g., viral or bacterial infection). In some embodiments, the patient suffers from or has suffered from EBV, CMV, HHV-6, HHV-7. In some embodiments, the patient's EBV, CMV, HHV-6A, HHV-6B or HHV-7 is positive (e.g., based on detection of viral DNA, such as in a blood sample, such as in a plasma sample). In some embodiments, the patient has been infected with a coronavirus (e.g., SARS-CoV-2). In some embodiments, the patient had symptoms of COVID-19.

[0184] In some embodiments, the patient has been diagnosed with an autoimmune disease. In some embodiments, the patient suffers from or has suffered from 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 is positive or tests positive for autoantibodies indicating an autoimmune disease.

[0185] 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, Cooper's fluid (cowper'sfluid), pre-ejaculatory fluid, chyle, chyme, female ejaculation fluid, interstitial fluid, lymphatic fluid, menstruation, breast milk, mucus, pleural effusion, pus, saliva, sebum, semen, serum, sweat, tears, urine, vaginal lubrication, vomit, water, feces, internal body fluids (including cerebrospinal fluid around the brain and spinal cord), synovial fluid, intracellular fluid (fluid in cells), and vitreous humor (liquid in the eyeball). In certain embodiments, the sample is a blood sample. A blood sample can be used, for example, Innis et al., ed., PCR Protocols [PCR Protocol] (Academic Press, 1990) is obtained by conventional techniques described in. Leukocytes can be separated from blood samples using conventional techniques or commercially available kits, for example, RosetteSep kit (Stein Cell Technologies, Vancouver, Canada). Leukocyte (e.g., monocytes, B cells, T cells, mononuclear leukocytes, granulocytes or lymphocytes) subsets can be further separated using conventional techniques, for example, magnetic activated cell sorting (MACS) (Miltenyi Biotec, Auburn, California) or fluorescence activated cell sorting (FACS) (Becton Dickinson, San Jose, California).

[0186] In one embodiment, the blood sample is about 0.1 mL to about 10.0 mL, about 0.2 mL to about 7 mL, about 0.3 mL to about 5 mL, about 0.4 mL to about 3.5 mL, or 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.

[0187] In certain embodiments, the sample used in the present method comprises 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. Any biopsy technique known to those skilled in the art can be used to separate the sample from the subject, such as open biopsy, closed biopsy, core biopsy, incisional biopsy, excisional biopsy or fine needle aspiration biopsy.

[0188] In one embodiment, the sample used in the methods provided herein is obtained from the subject before the subject receives treatment for the disease or disorder. In another embodiment, the sample is obtained from the subject during the period when the subject receives treatment for the disease or disorder. In another embodiment, the sample is obtained from the subject after the subject receives treatment for the disease or disorder. In various embodiments, treatment includes administering a compound to the subject.

[0189] In certain embodiments, the sample used in the methods provided herein comprises a plurality of cells. In certain embodiments, the number of cells used in the methods provided herein can range from a single cell to about 10 9 In some embodiments, the number of cells used in the methods 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.

[0190] The number and type of cells collected from a subject can be monitored, for example, by measuring changes in cell surface markers using standard cell detection techniques (such as 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)), by examining the morphology of cells 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.

[0191] In certain embodiments, cell subsets are used in the methods provided herein. The methods of sorting and separating specific 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, separation based on beads (such as magnetic cell sorting), separation based on size (e.g., sieves, barrier arrays, or filters), sorting in microfluidic devices, separation based on antibodies, 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 particle fluorescence properties (Kamarch, Methods Enzymol. [Enzymology Methods], 1987, 151: 150-165). Laser excitation of the fluorescent part in a single particle will produce a small charge, thereby allowing electromagnetic separation of positive particles and negative particles from a mixture. In one embodiment, cell surface marker-specific antibodies or ligands are labeled with different fluorescent labels. The cells are processed through a cell sorter, allowing the cells to be separated based on their ability to bind to the antibodies used. FACS sorted particles can be deposited directly into individual wells of a 96-well or 384-well plate to facilitate separation and cloning.

[0192] 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. Other exemplary methods are described in Section 5.3 above. 5.5. Kit

[0193] On the other hand, a kit for performing the methods provided herein is provided herein. In certain embodiments, a kit for identifying a subject suffering from CFS or verifying the CFS of a subject is provided herein, 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 certain embodiments, the kit further comprises instructions for: if the expression level of the biomarker is higher than the reference expression level of the biomarker, the subject is identified or verified as suffering from CFS. In certain embodiments, the kit (e.g., in the instructions) provides the reference expression level of the biomarker. In certain embodiments, the kit further comprises a tool for obtaining a sample from a subject.

[0194] In some embodiments, a kit for determining the severity of CFS of a subject is provided herein, 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 for determining the severity of CFS. In some embodiments, the kit (e.g., in the instructions) provides a reference expression level of the biomarker. In some embodiments, the kit provides instructions for comparing the expression level of the biomarker with the reference level of the biomarker and determining the severity of CFS based on the comparison. In some embodiments, the kit further comprises an instrument for obtaining a sample from a subject.

[0195] In some embodiments, a kit for identifying a subject who may or may not respond to CFS treatment or predicting the responsiveness of a subject to CFS treatment is provided herein, 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 for identifying a subject who may or may not respond to CFS treatment or predicting the responsiveness of a subject to CFS treatment. In some embodiments, the kit (e.g., in the instructions) provides a reference expression level of the biomarker. In some embodiments, the kit provides instructions for identifying or predicting the subject as being likely to respond to CFS treatment if the expression level of the biomarker is higher than the reference expression level of the biomarker. In some embodiments, the kit further comprises a tool for obtaining a sample from a subject. In some embodiments, the kit is used to identify a subject who may or may not respond to an immunomodulatory drug (IMiD) or predict the responsiveness of a subject to IMiD. In other embodiments, the kit is used to identify subjects who are likely or unlikely to respond to CRBN modulators or compounds that can bind to CRBN and / or induce conformational changes, or predict the responsiveness of subjects to CRBN modulators or compounds that can bind to CRBN and / or induce conformational changes. In some embodiments, the kit is used to identify subjects who are likely or unlikely to respond to agents that deplete B cells (e.g., anti-CD20 antibodies, e.g., rituximab), or predict the responsiveness of subjects to agents that deplete B cells (e.g., anti-CD20 antibodies, e.g., rituximab).

[0196] In some embodiments of various kits provided herein, the reference expression level of the biomarker provided in the kit is the predetermined expression level of the biomarker from a public database. In some embodiments of various kits provided herein, the reference expression level of the biomarker provided in the kit is the expression level of the biomarker in healthy subjects or in subjects not suffering from CFS. In certain embodiments, the reference expression level of the biomarker is the expression level of the biomarker in subjects suffering from moderate CFS. In some embodiments of various kits provided herein, the reference expression level of the biomarker provided in the kit is the expression level of the biomarker determined based on the cohort of the subject (e.g., the cohort of healthy subjects, the cohort of subjects not suffering from CFS, or the cohort of subjects suffering from moderate CFS). In certain embodiments, the reference expression level of the biomarker is the median or average expression level of the biomarker of the expression level of the biomarker in the cohort of the subject (e.g., the cohort of healthy subjects, the cohort of subjects not suffering from CFS, or the cohort of subjects suffering from 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 healthy subjects or subjects 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 subjects with mild CFS or a cohort of subjects with mild CFS.

[0197] In other embodiments, a kit for determining or monitoring the effectiveness of treatment in a subject with CFS is provided herein, 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 a biomarker in a second sample obtained from the subject after the treatment is administered to the subject. In some embodiments, the kit further includes instructions on how to determine or monitor the effectiveness of treatment in a subject with CFS. In some embodiments, the kit provides instructions for determining the effectiveness of the treatment based on a comparison of the first expression level with 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 includes a tool for obtaining a sample from a subject. In some embodiments, the kit is used to determine or monitor the effectiveness of an immunomodulatory drug (IMiD) in a subject with CFS. In some embodiments, the kit is used to determine or monitor the effectiveness of a CRBN modulator or a compound capable of binding to CRBN and / or inducing a conformational change in a subject with CFS. In some embodiments, the kit is used to determine or monitor the effectiveness of an agent (e.g., an anti-CD20 antibody, e.g., rituximab) that depletes B cells in a subject with CFS. In some embodiments, the kit further includes instructions for determining or adjusting the dosage for treatment of the subject.

[0198] In other embodiments, a kit for screening compounds for effectiveness in treating CFS is provided herein, 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 a biomarker in the sample after the compound is applied to the sample. In some embodiments, the kit further includes 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 includes a tool for obtaining a sample. In some embodiments, the kit is used to screen immunomodulatory drugs (IMiDs) for effectiveness in treating CFS. In some embodiments, the kit is used to screen CRBN regulators or compounds capable of binding to CRBN and / or inducing conformational changes thereof for effectiveness in treating CFS. In some embodiments, the kit is used to screen agents (e.g., anti-CD20 antibodies, e.g., rituximab) that deplete B cells for effectiveness in treating CFS.

[0199] In other embodiments, a kit for identifying a subject with long COVID or verifying long COVID in a subject is provided herein, 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 the subject as having long COVID if the expression level of the biomarker is higher than a reference expression level of the biomarker. In some embodiments, the kit provides a reference expression level of a biomarker. In some embodiments, the kit further comprises a tool for obtaining a sample from a subject.

[0200] In some embodiments, a kit for identifying a subject who may or may not respond to long COVID treatment or predicting the responsiveness of a subject to long COVID treatment is provided herein, 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 includes instructions for: if the expression level of the biomarker is higher than the reference expression level of the biomarker, the subject is identified or predicted as being likely to respond to the treatment. In certain embodiments, the kit further includes instructions for applying the treatment to a subject identified or predicted as being likely to respond to the treatment. In some embodiments, the kit provides a reference expression level of a biomarker. In some embodiments, the kit further includes a tool for obtaining a sample from a subject.

[0201] In some embodiments, a kit for selectively treating a subject with or suspected of having long COVID with a treatment is provided herein, 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 includes instructions for identifying or predicting the subject as likely to respond to long COVID treatment if the expression level of the biomarker is higher than the reference expression level of the biomarker. In certain embodiments, the kit further includes instructions for administering the treatment to a subject identified or predicted as likely to respond to the treatment. In some embodiments, the kit provides a reference expression level of a biomarker. In some embodiments, the kit further includes a tool for obtaining a sample from a subject.

[0202] In some embodiments of various kits provided herein, the reference expression level of a biomarker is a predetermined expression level of a biomarker. In some embodiments, the reference expression level of a biomarker is a predetermined expression level of a biomarker obtained from a public database. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker in a healthy subject or a subject not suffering from long COVID. In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker in a subject suffering from acute COVID (e.g., severe acute COVID). In some embodiments, the reference expression level of a biomarker is the expression level of a biomarker determined based on a cohort of a subject (e.g., a cohort of healthy subjects, a cohort of a subject not suffering from long COVID, or a cohort of a subject suffering from acute COVID (e.g., severe acute COVID)). In some embodiments, the reference expression level of a biomarker is the median or average expression level of a biomarker of the expression level of a biomarker in a cohort of a subject (e.g., a cohort of healthy subjects, a cohort of a subject not suffering from long COVID, a cohort of a subject suffering from acute COVID (e.g., severe acute COVID)).

[0203] 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 not having long COVID, or a cohort of healthy subjects or subjects not having 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 a cohort of subjects with acute COVID (e.g., severe acute COVID).

[0204] In some embodiments of the various kits provided herein, the kit includes: if the expression level of the 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 subject is identified as having long COVID. The expression level of the biomarker can be determined using any method known 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 a reference level as observed according to a measurement assay, the level is determined to be higher than the reference level.

[0205] In some embodiments of various kits provided herein, when multiple biomarkers are used, the expression level of each biomarker is compared with the reference expression level of the biomarker, and the kit includes instructions for identifying or verifying subjects with long COVID, or if the expression level of each biomarker is higher than the reference expression level of the biomarker, the subject is identified or predicted as likely to respond to long COVID treatment. In other embodiments, when multiple biomarkers are used, a comprehensive score based on multiple biomarkers is calculated and compared with a reference comprehensive score. In some embodiments, the kit includes instructions for identifying or verifying subjects with long COVID, or if the comprehensive score is higher than the reference comprehensive score, the subject is identified or predicted as likely to respond to long COVID treatment. In some embodiments, the comprehensive score is calculated using the median Z score method. In short, the median Z score is derived by first calculating the mean value of each gene from all samples in the gene expression matrix. The mean value is then subtracted from each corresponding gene of all samples, and then scaled by dividing these values ​​by their standard deviations. The median scaled values ​​from multiple target genes include a comprehensive score. Another exemplary method for calculating the comprehensive score is the single sample gene set enrichment (ssGSEA) method. The single sample gene score represents the degree to which the genes in a specific gene set are synergistically upregulated or downregulated within the sample. The score is calculated by adjusting the running sum statistic based on the decreasing trend in the sorted expression list. The enrichment score is the maximum zero deviation encountered in the trend; it corresponds to a weighted Kolmogorov-Smirnov statistic (see, for example, Subramanian et al., PNAS [Proceedings of the National Academy of Sciences of the United States], 102 (43): 15545-15550 (2005); and Barbie et al., Nature [Natural], 462 (7269): 108-112).

[0206] In other embodiments, a kit for determining or monitoring the effectiveness of a treatment in a subject with long COVID is provided herein, 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 a biomarker in a second sample obtained from the subject after the treatment is administered to the subject. In some embodiments, the kit further includes instructions on how to determine or monitor the effectiveness of a treatment in a subject with long COVID. In some embodiments, the kit provides instructions for determining the effectiveness of the treatment based on a comparison of the first expression level with 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 includes a tool for obtaining a sample from a subject. In some embodiments, the kit is used to determine or monitor the effectiveness of an immunomodulatory drug (IMiD) in a subject with long COVID. In some embodiments, the kit is used to determine or monitor the effectiveness of a CRBN modulator or a compound capable of binding to CRBN and / or inducing a conformational change in a subject with long COVID. In some embodiments, the kit is used to determine or monitor the effectiveness of an agent that depletes B cells (e.g., an anti-CD20 antibody, e.g., rituximab) in a subject with long COVID.

[0207] In certain embodiments, the kit further includes instructions for determining or adjusting (e.g., increasing) the dose of the treatment or administering a different long COVID treatment to the subject if the second expression level is not less than the first expression level.

[0208] In other embodiments, a kit for screening compounds for effectiveness in treating long COVID is provided herein, 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 a biomarker in the sample after the compound is applied to the sample. In some embodiments, the kit further includes instructions on how to select a compound for treating long COVID. 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 includes a tool for obtaining a sample. In some embodiments, the kit is used to screen immunomodulatory drugs (IMiDs) for effectiveness in treating long COVID. In some embodiments, the kit is used to screen CRBN regulators or compounds capable of binding to CRBN and / or inducing conformational changes thereof for effectiveness in treating long COVID. In some embodiments, the kit is used to screen agents (e.g., anti-CD20 antibodies, e.g., rituximab) that deplete B cells for effectiveness in treating long COVID.

[0209] In some embodiments, the kits provided herein include 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 a comparison of the first expression level with the second expression level, different treatments or different dosing regimens may be administered to the subject in subsequent treatment cycles.

[0210] In some embodiments, the treatment disclosed herein comprises an immunomodulatory drug (IMiD) as disclosed in Section 5.2. In some embodiments, the treatment comprises a CRBN modulator as disclosed in Section 5.2 or a compound capable of binding to CRBN and / or inducing a conformational change thereof. In some embodiments, the treatment comprises an agent that depletes B cells as disclosed in Section 5.2 (e.g., an anti-CD20 antibody, e.g., rituximab).

[0211] In some embodiments of the various kits provided herein, the kit includes an agent for determining / measuring the expression level of two or more biomarkers, three or more biomarkers, four or more biomarkers, five or more biomarkers, or all biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8.

[0212] In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2 and at least one, two, three or four of IKZF3, ABCE1, BACH2, CD3D and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF3 and at least one, two, three or four of IKZF2, ABCE1, BACH2, CD3D and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of ABCE1 and at least one, two, three or four of IKZF2, IKZF3, BACH2, CD3D and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of BACH2 and at least one, two, three or four of IKZF2, IKZF3, ABCE1, CD3D and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of CD3D and at least one, two, three or four of IKZF2, IKZF3, ABCE1, BACH2 and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of HSPA8 and at least one, two, three or four of IKZF2, IKZF3, ABCE1, BACH2 and CD3D. In some embodiments, the kit includes an agent for determining / measuring the expression level of all biomarkers in the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF3. In some embodiments, the kit includes an agent for determining / measuring the expression level of ABCE1. In some embodiments, the kit includes an agent for determining / measuring the expression level of BACH2. In some embodiments, the kit includes an agent for determining / measuring the expression level of CD3D. In some embodiments, the kit includes an agent for determining / measuring the expression level of HSPA8. In certain embodiments, the kit includes an agent for determining / measuring the expression level of two or more biomarkers selected from the group consisting of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8. In certain embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, IKZF3, ABCE1, BACH2, CD3D and HSPA8.In certain embodiments, the kit includes an agent for determining / measuring the expression levels of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the kit includes an agent for determining / measuring the expression levels of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In certain embodiments, the kit includes an agent for determining / measuring the expression levels of IKZF2 and IKZF3. In certain embodiments, the kit includes an agent for determining / measuring the expression levels of IKZF2 and ABCE1. In certain embodiments, the kit includes an agent for determining / measuring the expression levels of IKZF2 and BACH2. In certain embodiments, the kit includes an agent for determining / measuring the expression levels of IKZF2 and CD3D. In certain embodiments, the kit includes an agent for determining / measuring the expression levels of IKZF2 and HSPA8. In certain embodiments, the kit includes an agent for determining / measuring the expression levels of IKZF3 and ABCE1. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF3 and BACH2. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF3 and CD3D. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF3 and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of ABCE1 and BACH2. In some embodiments, the kit includes an agent for determining / measuring the expression level of ABCE1 and CD3D. In some embodiments, the kit includes an agent for determining / measuring the expression level of ABCE1 and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of BACH2 and CD3D. In some embodiments, the kit includes an agent for determining / measuring the expression level of BACH2 and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of CD3D and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, IKZF3 and ABCE1. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, IKZF3, and BACH2. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, IKZF3, and CD3D. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, IKZF3, and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, ABCE1, and BACH2.In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, ABCE1 and CD3D. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, ABCE1 and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, BACH2 and CD3D. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, BACH2 and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, CD3D and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF3, ABCE1 and BACH2. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF3, ABCE1 and CD3D. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF3, ABCE1 and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF3, BACH2, and CD3D. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF3, BACH2, and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF3, CD3D, and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of ABCE1, BACH2, and CD3D. In some embodiments, the kit includes an agent for determining / measuring the expression level of ABCE1, BACH2, and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of ABCE1, CD3D, and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of BACH2, CD3D, and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF3, BACH2, CD3D, and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF3, ABCE1, CD3D, and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF3, ABCE1, BACH2, and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF3, ABCE1, BACH2, and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF3, ABCE1, BACH2, and CD3D.In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, BACH2, CD3D and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, ABCE1, CD3D and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, ABCE1, BACH2 and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, ABCE1, BACH2 and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, ABCE1, BACH2 and CD3D. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, IKZF3, CD3D and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, IKZF3, BACH2 and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, IKZF3, BACH2 and CD3D. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, IKZF3, ABCE1, and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, IKZF3, ABCE1, and CD3D. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, IKZF3, ABCE1, and BACH2. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF3, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, ABCE1, BACH2, CD3D, and HSPA8. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, IKZF3, BACH2, CD3D, and HSPA8. In other embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, IKZF3, ABCE1, CD3D, and HSPA8. In other embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, IKZF3, ABCE1, BACH2, and HSPA8. In other embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, IKZF3, ABCE1, BACH2, and CD3D. In some embodiments, the kit includes an agent for determining / measuring the expression level of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8.

[0213] In some embodiments, when the kit includes agents for multiple biomarkers, the kit provides a reference expression level for each biomarker. In other embodiments, the kit provides a reference comprehensive score for multiple biomarkers.

[0214] In some embodiments, the kit comprises an agent for determining protein levels. In other embodiments, the kit comprises an agent for determining mRNA levels. In other embodiments, the kit comprises an agent for determining cDNA levels.

[0215] In certain embodiments, provided herein is a kit for detecting the mRNA level of one or more biomarkers. In certain embodiments, the kit comprises one or more probes that specifically bind to the mRNA of one or more biomarkers. In certain embodiments, the kit further comprises a washing solution. In certain embodiments, the kit further comprises reagents for performing hybridization assays, mRNA separation or purification means, detection means, and positive and negative controls. In certain embodiments, the kit further comprises instructions for using the kit. The kit can be customized for home use, clinical use, or research use.

[0216] In certain embodiments, provided herein are kits for detecting protein levels of one or more biomarkers. In certain embodiments, the kit comprises a test strip coated with an antibody that recognizes a protein biomarker, a washing solution, a reagent for performing the assay, a protein separation or purification means, a detection means, and positive and negative controls. In certain embodiments, the kit further comprises instructions for using the kit. The kit can be customized for home use, clinical use, or research use.

[0217] Such a test kit can be used in the form of, for example, a test strip, a membrane, a chip, a disc, a test paper, a filter, a microsphere, a slide, a multi-well plate or an optical fiber. The solid support of the test kit can be, for example, plastic, silicon, metal, resin, glass, membrane, particle, precipitate, gel, polymer, sheet, sphere, polysaccharide, capillary, film, disk or slide. The biological sample can be, for example, a cell culture, a cell line, a tissue, an organ, an organelle, a biological fluid, a blood sample, a urine sample or a skin sample.

[0218] In another embodiment, the kit includes: a solid support; a nucleic acid attached to the support, wherein the nucleic acid is complementary to at least 1, 2, 3, 4, 5, 6, 10, 20, 50, 100, 200, 350 or more bases of an mRNA; and a means for detecting mRNA expression in a biological sample.

[0219] In a specific embodiment, the kit includes a compound or a pharmaceutical composition thereof in a container, and further includes a component for isolating RNA in one or more containers. In another specific embodiment, the kit includes a compound or a pharmaceutical composition in a container, and further includes a component for performing RT-PCR, qRT-PCR, deep sequencing or microarray in one or more containers.

[0220] In certain embodiments, the kit provided herein uses a means of detecting the expression of a biomarker by quantitative real-time PCR (qRT-PCR), microarray, flow cytometry, immunofluorescence, sequencing (e.g., next generation sequencing or NGS). In other embodiments, the expression of a biomarker is measured by an ELISA-based method or other similar methods known in the art.

[0221] In another specific embodiment, the kit includes a compound or pharmaceutical composition thereof in a container and further includes components for isolating proteins in one or more containers. In another specific embodiment, a pharmaceutical or assay kit includes a compound or pharmaceutical composition in a container and further includes components for performing flow cytometry or ELISA in one or more containers.

[0222] On the other hand, kits for measuring biomarkers are provided herein, which provide materials necessary for measuring the abundance of one or more gene products of a biomarker or a subset of a biomarker (e.g., one, two, three, four, five or more biomarkers) provided herein. Such a kit may include materials and reagents required for measuring RNA or protein. In some embodiments, such a kit includes a microarray, wherein the microarray is composed of oligonucleotides and / or DNA and / or RNA fragments, which are hybridized with one or more gene products of a subset of a biomarker or a subset of a biomarker provided herein or any combination thereof. In some embodiments, such a kit may include primers for PCR of a biomarker or a subset of a biomarker RNA product or a cDNA copy of an RNA product or both. In some embodiments, such a kit may include primers for PCR and probes for qPCR. In some embodiments, such a kit may include multiple primers and multiple probes, some of which have different fluorophores to allow simultaneous measurement of multiple gene products of a subset of a biomarker or a subset of a biomarker provided herein. In some embodiments, such a kit may further include materials and reagents for producing cDNA from RNA. In some embodiments, such a kit may include antibodies specific to the protein products of a subset of a biomarker or a subset of a biomarker provided herein. Such a test kit may additionally include materials and reagents for isolating RNA and / or protein from a biological sample. In addition, such test kits may include materials and reagents for synthesizing cDNA from RNA isolated from a biological sample. In certain embodiments, such test kits may include a computer program product embedded in a computer-readable medium for performing various functions according to the method. In certain embodiments, the test kit may include a computer program product embedded in a computer-readable medium and instructions.

[0223] In some embodiments, such kits measure the expression of one or more nucleic acid products of a subset of biomarkers or biomarkers provided herein. According to this embodiment, the kit may include materials and reagents necessary for measuring the expression of a specific nucleic acid product of a subset of biomarkers or biomarkers provided herein. For example, a microarray or RT-PCR kit can be produced for specific conditions and only includes those reagents and materials necessary for measuring the level of a specific RNA transcript of a subset of biomarkers or biomarkers provided herein, to predict whether the patient is clinically sensitive to the compound. Alternatively, in some embodiments, the kit may include materials and reagents necessary for measuring the expression of a specific nucleic acid product of a gene other than a biomarker provided herein. For example, in certain embodiments, the kit includes the materials and reagents necessary to measure the expression level of 1 gene, 2 genes, 3 genes, 4 genes, 5 genes, 6 genes, 7 genes, 8 genes, 9 genes, 10 genes, 15 genes, 20 genes, 25 genes, 30 genes, 35 genes, 40 genes, 45 genes, 50 genes or more of the genes of the biomarkers provided herein, as well as the reagents and materials necessary to measure the expression level of at least 1 gene, at least 2 genes, at least 3 genes, at least 4 genes, at least 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 15 genes, at least 20 genes, at least 25 genes, at least 30 genes, at least 35 genes, at least 40 genes, at least 45 genes, at least 50 genes or more in addition to the biomarkers provided herein. In other embodiments, the kit contains at least 1 gene, at least 2 genes, at least 3 genes, at least 4 genes, at least 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 15 genes, at least 20 genes, at least 25 genes, at least 30 genes, at least 35 genes, at least 40 genes, at least 45 genes, at least 50 genes or more of the biomarkers provided herein, as well as 1 gene, 2 genes, 3 genes, 4 genes, 5 genes that are not biomarkers provided herein. , 10 genes, 15 genes, 20 genes, 25 genes, 30 genes, 35 genes, 40 genes, 45 genes, 50 genes, 55 genes, 60 genes, 65 genes, 70 genes, 75 genes, 80 genes, 85 genes, 90 genes, 95 genes, 100 genes, 125 genes, 150 genes, 175 genes, 200 genes, 225 genes, 250 genes, 300 genes, 350 genes, 400 genes, 450 genes or more genes.In certain embodiments, the kit contains at least 1 gene, at least 2 genes, at least 3 genes, at least 4 genes, at least 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 15 genes, at least 20 genes, at least 25 genes, at least 30 genes, at least 35 genes, at least 40 genes, at least 45 genes, at least 50 genes or more of the genes that measure the biomarkers provided herein, as well as 1-10 genes, 1-100 genes, 1-15 genes or more that are not biomarkers provided herein. The reagents and materials necessary for measuring the expression level of 10 genes, 1-200 genes, 1-300 genes, 1-400 genes, 1-500 genes, 1-1000 genes, 25-100 genes, 25-200 genes, 25-300 genes, 25-400 genes, 25-500 genes, 25-1000 genes, 100-150 genes, 100-200 genes, 100-300 genes, 100-400 genes, 100-500 genes, 100-1000 genes or 500-1000 genes.

[0224] For nucleic acid microarray kit, the kit generally includes probes attached to the solid support surface. In one such embodiment, the probe can be an oligonucleotide or a longer probe, including probes ranging in length from 150 nucleotides to 800 nucleotides. The probe can be labeled with a detectable label. In a specific embodiment, the probe is specific to one or more gene products of the biomarkers provided herein. The microarray kit may include instructions for measuring and methods for explaining and analyzing the data generated by measuring. In a specific embodiment, the kit includes instructions for predicting whether the patient is clinically sensitive to the compound. The kit may also include hybridization reagents and / or reagents necessary for detecting the signal generated when the probe hybridizes with the target nucleic acid sequence. Typically, the materials and reagents used for the microarray kit are in one or more containers. Each component of the kit is typically in its own suitable container.

[0225] In certain embodiments, the nucleic acid microarray kit includes the materials and reagents necessary to measure the expression level of 1 gene, 2 genes, 3 genes, 4 genes, 5 genes, 6 genes, 7 genes, 8 genes, 9 genes, 10 genes, 15 genes, 20 genes, 25 genes, 30 genes, 35 genes, 40 genes, 45 genes, 50 genes or more or a combination thereof of the genes of the biomarkers provided herein, as well as the reagents and materials necessary to measure the expression level of at least 1 gene, at least 2 genes, at least 3 genes, at least 4 genes, at least 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 15 genes, at least 20 genes, at least 25 genes, at least 30 genes, at least 35 genes, at least 40 genes, at least 45 genes, at least 50 genes or more genes in addition to the genes of the biomarkers provided herein. In other embodiments, the nucleic acid microarray kit contains at least 1 gene, at least 2 genes, at least 3 genes, at least 4 genes, at least 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 15 genes, at least 20 genes, at least 25 genes, at least 30 genes, at least 35 genes, at least 40 genes, at least 45 genes, at least 50 genes, or more, or any combination thereof, of the genes that measure the biomarkers provided herein, as well as 1 gene, 2 genes, 3 genes, 4 genes, or more that are not genes of the biomarkers provided herein. The invention also provides the reagents and materials necessary to increase the expression level of 5 genes, 5 genes, 10 genes, 15 genes, 20 genes, 25 genes, 30 genes, 35 genes, 40 genes, 45 genes, 50 genes, 55 genes, 60 genes, 65 genes, 70 genes, 75 genes, 80 genes, 85 genes, 90 genes, 95 genes, 100 genes, 125 genes, 150 genes, 175 genes, 200 genes, 225 genes, 250 genes, 300 genes, 350 genes, 400 genes, 450 genes or more genes.In another embodiment, the nucleic acid microarray kit contains at least 1 gene, at least 2 genes, at least 3 genes, at least 4 genes, at least 5 genes, at least 6 genes, at least 7 genes, at least 8 genes, at least 9 genes, at least 10 genes, at least 15 genes, at least 20 genes, at least 25 genes, at least 30 genes, at least 35 genes, at least 40 genes, at least 45 genes, at least 50 genes, or more, or any combination thereof, of the genes that measure the biomarkers provided herein, and 1-10 genes, 1-100 genes that are not genes of the biomarkers provided herein. The present invention also relates to reagents and materials necessary for regulating the expression level of 1-150 genes, 1-200 genes, 1-300 genes, 1-400 genes, 1-500 genes, 1-1000 genes, 25-100 genes, 25-200 genes, 25-300 genes, 25-400 genes, 25-500 genes, 25-1000 genes, 100-150 genes, 100-200 genes, 100-300 genes, 100-400 genes, 100-500 genes, 100-1000 genes or 500-1000 genes.

[0226] For quantitative PCR, the kit generally includes preselected primers specific to a particular nucleic acid sequence. The quantitative PCR kit may also include enzymes (e.g., polymerases, such as Taq polymerases), deoxynucleotides, and the buffer required for the amplification reaction that are applicable to nucleic acid amplification. The quantitative PCR kit may also include probes specific to nucleic acid sequences that are associated with or indicate a disease. The probe may be labeled with or without a fluorophore. The probe may be labeled with or without a quencher molecule. In certain embodiments, the quantitative PCR kit also includes components applicable to reverse transcription RNA, including enzymes (e.g., reverse transcriptases, such as AMV, MMLV, etc.) and primers for reverse transcription, as well as deoxynucleotides and the buffer required for the reverse transcription reaction. Each component of the quantitative PCR kit is generally placed in a suitable container. Therefore, these kits generally include different containers applicable to each individual reagent, enzyme, primer, and probe. In addition, the quantitative PCR kit may include instructions for reacting and methods for explaining and analyzing the data generated by reacting. In a specific embodiment, the kit contains instructions for predicting whether a patient is clinically sensitive to a compound.

[0227] For antibody-based kits, the kit may include, for example: (1) a first antibody that binds to a peptide, polypeptide, or protein of interest (which may or may not be attached to a solid support); and, optionally, (2) a second, different antibody that binds to the first antibody or the peptide, polypeptide, or protein and is conjugated to a detectable label (e.g., a fluorescent label, a radioisotope, or an enzyme). In specific embodiments, the peptide, polypepti...

Claims

1. A method of identifying a subject suffering from chronic fatigue syndrome (CFS) or verifying 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 cerebellum-related protein (CAP) selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2, and CD3D; and (b) if the expression level of the biomarker is higher than a reference expression level of the biomarker, identifying or validating the subject as having CFS.

2. The method of claim 1, wherein the subject has reported chronic debilitating fatigue, restless sleep, mental and / or physical pain, neurological and cognitive impairment, and / or autoimmunity or immunodeficiency.

3. A method for determining the severity of 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 cerebellum-related 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 CFS in the subject based on the comparison in step (b).

4. The method of claim 3, wherein the severity of the CFS is determined to be severe if the expression level of the biomarker is above a reference expression level of the biomarker, optionally wherein the reference expression level of the biomarker is the expression level of the biomarker in a subject with mild CFS or in a cohort of subjects with mild CFS.

5. A method of identifying a subject who is likely or unlikely to respond to a CFS treatment or predicting the responsiveness of a subject to a CFS treatment, the method comprising: (a) determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is cerebellum-related protein (CAP) selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2, and CD3D; and (b) if the expression level of the biomarker is higher than a reference expression level of the biomarker, then the subject is identified or predicted as being likely to respond to the treatment.

6. The method of claim 5, wherein the method further comprises administering the treatment to the subject identified or predicted to be likely to respond to the treatment.

7. A method of selectively treating a subject suffering from or suspected of suffering from CFS with a therapy, the method comprising: (a) determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is cerebellum-related protein (CAP) selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2, and CD3D; (b) identifying or predicting the subject as likely to respond to treatment for CFS if the expression level of the biomarker is above a reference expression level of the biomarker; and (c) administering the treatment to the subject identified or predicted to be likely to respond to the treatment.

8. The method of any one of claims 1 to 7, wherein the reference expression level of the biomarker is a predetermined expression level of the biomarker.

9. The method of any one of claims 1 to 8, wherein the reference expression level of the biomarker is the expression level of the biomarker in a subject not suffering from CFS or in a cohort of subjects not suffering from CFS.

10. The method of any one of claims 1 to 8, wherein the reference expression level of the biomarker is the expression level of the biomarker in a healthy subject or a cohort of healthy subjects.

11. The method of any one of claims 1 to 8, wherein the reference expression level of the biomarker is the expression level of the biomarker in a subject with mild CFS or in a cohort of subjects with mild CFS.

12. The method of any one of claims 1 to 11, wherein the biomarker is HSPA8 or ABCE1, optionally wherein the reference expression level of the biomarker is the expression level of the biomarker in healthy subjects or subjects not suffering from CFS, or in a cohort of healthy subjects or subjects not suffering from CFS.

13. The method of any one of claims 1 to 12, wherein the method comprises determining the expression levels of two, three, four, five or all biomarkers selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2 and CD3D.

14. The method of claim 13, wherein the method comprises determining the expression level of: (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.

15. The method of claim 13, wherein the method comprises determining the expression levels of IKZF2, IKZF3, ABCE1, BACH2, CD3D, and HSPA8.

16. The method of any one of claims 13 to 15, wherein the method comprises comparing the expression level of each of the biomarkers to their respective reference expression levels.

17. The method of any one of claims 13 to 15, wherein the method comprises obtaining a composite score based on the expression levels of the biomarkers, and comparing the composite score with a reference score derived from reference expression levels of the biomarkers.

18. A method of determining or monitoring the effectiveness of a treatment in a subject suffering from CFS, 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 cerebellum-related protein (CAP) selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, 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.

19. The method of claim 18, wherein the method comprises: If the second expression level is lower than the first expression level, the treatment is determined to be effective.

20. The method of claim 18 or 19, wherein the method further comprises determining or adjusting the dosage of the treatment to the subject.

21. A method of screening a treatment for effectiveness in treating CFS, the method comprising: (a) determining a first expression level of a biomarker in a sample prior to administering the treatment to the sample, wherein the biomarker is cerebellum-related protein (CAP) selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, 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.

22. The method of any one of claims 18 to 21, wherein the biomarker is HSPA8 or ABCE1.

23. The method of any one of claims 18 to 22, wherein the method comprises determining the first expression level and the second expression level of two, three, four, five or all biomarkers selected from the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2 and CD3D.

24. The method of claim 23, wherein the method comprises determining the first expression level and the second expression level of: (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.

25. The method of claim 23, wherein the method comprises determining the first expression level and the second expression level of all biomarkers in the group consisting of HSPA8, ABCE1, IKZF2, IKZF3, BACH2, and CD3D.

26. The method of any one of claims 23 to 25, wherein the method comprises comparing the first expression level of each of the biomarkers to their respective second expression levels.

27. The method of any one of claims 23 to 25, wherein the method comprises obtaining a first comprehensive score based on the first expression level of the biomarker and a second comprehensive score based on the second expression level of the biomarker, and comparing the first comprehensive score to the second comprehensive score.

28. The method of any one of claims 5 to 27, wherein the treatment comprises an immunomodulatory drug (IMiD).

29. The method of any one of claims 5 to 27, wherein the treatment comprises a cerebellar protein (CRBN) modulator or a compound capable of binding to CRBN and / or inducing a conformational change thereof.

30. The method of any one of claims 5 to 27, wherein the treatment comprises an agent that depletes B cells.

31. The method of any one of claims 1 to 30, wherein the CFS is associated with an autoimmune disease or a viral infection.

32. A method of identifying a subject with long COVID or verifying 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 cerebellum-related protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; and (b) if the expression level of the biomarker is higher than a reference expression level of the biomarker, the subject is identified or verified as having long COVID.

33. A method of identifying a subject who is likely or unlikely to respond to a long COVID treatment or predicting the responsiveness of a subject to a long COVID treatment, the method comprising: (a) determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is cerebellum-related protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; and (b) if the expression level of the biomarker is higher than a reference expression level of the biomarker, then the subject is identified or predicted as being likely to respond to the treatment.

34. The method of claim 33, wherein the method further comprises administering the treatment to the subject identified or predicted as likely to respond to the treatment.

35. A method of selectively treating a subject having or suspected of having long COVID with a therapy, the method comprising: (a) determining the expression level of a biomarker in a sample from the subject, wherein the biomarker is cerebellum-related protein (CAP) selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D; (b) identifying or predicting the subject as likely to respond to long COVID treatment if the expression level of the biomarker is above a reference expression level of the biomarker; and (c) administering the treatment to the subject identified or predicted to be likely to respond to the treatment.

36. The method of any one of claims 32 to 35, wherein the reference expression level of the biomarker is a predetermined expression level of the biomarker.

37. The method of any one of claims 32 to 35, wherein the reference expression level of the biomarker is 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.

38. The method of any one of claims 32 to 35, wherein the reference expression level of the biomarker is the expression level of the biomarker in a healthy subject or a cohort of healthy subjects.

39. The method of any one of claims 32 to 35, wherein the reference expression level of the biomarker is the expression level of the biomarker in a subject with acute COVID or a cohort of subjects with acute COVID.

40. The method of any one of claims 32 to 39, wherein the biomarker is HSPA8 or IKZF3, optionally wherein the reference expression level of the biomarker is the expression level of the biomarker in healthy subjects or subjects without long COVID, or in a cohort of healthy subjects or subjects without long COVID.

41. The method of any one of claims 32 to 39, wherein the method comprises determining the expression levels of two, three, four, five or all biomarkers selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2 and CD3D.

42. The method of claim 41, wherein the method comprises determining the expression level of: (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.

43. The method of claim 41, wherein the method comprises determining the expression levels of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D.

44. The method of any one of claims 41 to 43, wherein the method comprises comparing the expression level of each of the biomarkers to their respective reference expression levels.

45. The method of any one of claims 41 to 43, wherein the method comprises obtaining a composite score based on the expression levels of the biomarkers, and comparing the composite score to a reference score derived from reference expression levels of the biomarkers.

46. ​​A method of determining or monitoring the effectiveness of a treatment in a subject with 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 cerebellum-related 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.

47. The method of claim 46, wherein the method comprises: If the second expression level is lower than the first expression level, the treatment is determined to be effective.

48. The method of claim 46 or 47, wherein the method further comprises determining or adjusting the dosage of the treatment to the subject.

49. A method of screening a treatment for effectiveness in treating long COVID, the method comprising: (a) determining a first expression level of a biomarker in a sample prior to administering the treatment to the sample, wherein the biomarker is cerebellum-related 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.

50. The method of any one of claims 46-49, wherein the biomarker is HSPA8 or IKZF3.

51. The method of any one of claims 46 to 50, wherein the method comprises determining the first expression level and the second expression level of two, three, four, five or all biomarkers selected from the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2 and CD3D.

52. The method of claim 51, wherein the method comprises determining the first expression level and the second expression level of: (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.

53. The method of claim 51, wherein the method comprises determining the first expression level and the second expression level of all biomarkers in the group consisting of HSPA8, IKZF3, ABCE1, IKZF2, BACH2, and CD3D.

54. The method of any one of claims 51 to 53, wherein the method comprises comparing the first expression level of each of the biomarkers to their respective second expression levels.

55. The method of any one of claims 51 to 53, wherein the method comprises obtaining a first comprehensive score based on the first expression level of the biomarker and a second comprehensive score based on the second expression level of the biomarker, and comparing the first comprehensive score to the second comprehensive score.

56. The method of any one of claims 33 to 55, wherein the treatment comprises an immunomodulatory drug (IMiD).

57. The method of any one of claims 33 to 55, wherein the treatment comprises a CRBN modulator or a compound capable of binding to CRBN and / or inducing a conformational change thereof.

58. The method of any one of claims 33 to 55, wherein the treatment comprises an agent that depletes B cells.

59. The method of any one of claims 32 to 58, wherein the subject has had coronavirus disease 2019 (COVID-19).

60. The method of any one of claims 1 to 59, wherein the expression level of the biomarker is determined by measuring the mRNA level of the biomarker.

61. The method of claim 60, wherein the mRNA level is determined by using quantitative reverse transcriptase PCR (RT-qPCR), microarray, Northern blot, or RNA sequencing.

62. The method of any one of claims 1 to 59, wherein the expression level of the biomarker is determined by measuring the protein level of the biomarker.

63. The method of claim 62, wherein 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).

64. A kit for performing the method of any one of claims 1 to 63, the kit 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.

65. The kit of claim 64, wherein the kit further comprises means for obtaining the sample.

66. The kit of claim 64 or 65, wherein the kit further comprises instructions for interpreting the determined expression levels.

67. The kit of any one of claims 64 to 66, wherein the kit further comprises a reference expression level of the biomarker.

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