WO2023203181A1 - Methods and products for the diagnosis and prognosis of a coronavirus infection - Google Patents

Abstract

The application discloses methods for determining or predicting the severity of a disease caused by a coronavirus infection based on the detection of fat mass and obesity associated (FTO) in a subject. Also methods for monitoring clinical progression of a disease caused by coronavirus infection and for assessing the efficacy of a therapeutic treatment of a disease caused by coronavirus infection are included and based on the use of FTO as clinical biomarker.

Description

METHODS AND PRODUCTS FOR THE DIAGNOSIS AND PROGNOSIS OF A CORONAVIRUS INFECTION

FIELD OF THE INVENTION

The present invention relates to methods for determining or predicting the severity of a disease caused by a coronavirus infection, for monitoring clinical progression of said disease and for assessing the efficacy of a therapeutic treatment of said disease.

BACKGROUND OF THE INVENTION

Nowadays, the world is facing a Coronavirus Disease 2019 (COVID-19) pandemic, resulting from a newly emerged virus named Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), which has spread at an unprecedented alarming rate. This pandemic causes a broad range of diseases, from unnoticeable or mild symptoms like common cold (cough, fever, fatigue, body aches), to lifethreatening illness such as pneumonia, acute respiratory distress, chest pain, strokes and multiple organ failure, killing millions of people worldwide. Despite preventing measures and progress in vaccination campaigns, its continuing resurgence represents a threat to both public health and economy. Hence, reducing COVID-19 burden is a worldwide priority.

Until recently, during each pandemic peak, hospitals have been overwhelmed with COVID-19 patients requiring intensive care. Particularly, diagnosis of COVID-19 severity is presently relying on clinical parameters and chest radiographic examination obtained on hospital admission. Nevertheless, these stratification approaches, including changes in inflammatory markers such as leukocytes, proteins and metabolites, have some limitations and are not sufficient to predict which patients will become the sickest. First, they are complex and may account for diverse inflammatory conditions. Second, they differ negligibly between severe and mild groups and only enable clinical risk assessment taken as a whole. Last, such analyses are time consuming, require specific expertise and rise healthcare costs. Accordingly, there persists a need to establish rapid, economical and reliable quantitative methods able to accurately sort patients based on severity status and thereby provide an opportunity for better healthcare outcomes. Identification of important biomarkers and developing a uniform evaluation method is hence critical to optimize clinical COVID-19 patient triage, which will ultimately lead to significant financial gains.

In light of ongoing efforts to develop tools to counteract COVID-19, novel breakthroughs highlighted the arising importance of epitranscriptomics. For example, N6-methyladenosine (m⁶A) and proteins of the methyltransferase complex involved in the modifications of m6A, such as METTL3, METTL14, FTO and ALKBH5, have been investigated for their role in modulating SARS-CoV-2 life cycle and COVID- 19 aftermath (Liu et al., 2021, Cell Res., 31(4): 404-414; Zhang et al., 2021, Cell Discov 7, 7; Li et al., 2021, Cell Reports, 109091; Liu et al, 2014, Nat Chem Biol, 10, 93-95; Jia et al., 2011, Nat Chem Biol, 7, 885-887; Zheng et al., 2013, Mol Cell 49: 18-29).

SUMMARY

As corroborated in the experimental section, the inventors of the present application found that in COVID-19 patients down-regulation of fat mass and obesity associated (FTO) correlates with higher SARS-CoV-2 expression and a more severe disease status. The present invention thus identified FTO as a biomarker for the disease severity or prognosis of a disease caused by a coronavirus infection.

In an aspect, the present invention provides a method for determining or predicting the severity of a disease caused by a coronavirus infection in a subject. Said method comprises the method comprising detecting FTO, and more specific measuring the quantity and/or activity of FTO in a sample from the subject. In some embodiments, the method allows to categorize the subject as asymptomatic or mild symptomatic, or as severe symptomatic requiring hospitalization or intensive care treatment. In some embodiments, the subject is categorized as not having or not being at risk of developing a severe or critical form of the disease, or as having or being at risk of developing a severe or critical form of the disease.

In a related aspect, a method for monitoring clinical progression of a disease caused by coronavirus infection in a subject is provided. Said method comprises detecting FTO, and more specific measuring the quantity and/or activity of FTO, in samples from the subject obtained at two or more different time points and comparing the FTO quantity and/or activity detected at the different time points.

In another related aspect, a method for assessing the efficacy of a therapeutic treatment of a disease caused by coronavirus infection in a subject is provided. Said method comprises detecting FTO, and more specific measuring the quantity and/or activity of FTO, in a sample from the subject obtained at a time point before the start of the treatment and at one or more time points after the start of the treatment, and comparing the FTO quantity and/or activity detected at the different time points.

In some embodiments, the disease caused by a coronavirus infection is a disease caused by a - coronavirus, preferably a Sarbecovirus.

In some embodiments, the coronavirus is selected from the Middle East respiratory syndrome-related coronavirus (MERS-CoV), the Severe Acute Respiratory virus (SARS-CoV), the Severe Acute Respiratory 2 virus (SARS-CoV-2) or a SARS-CoV-2 mutant virus, preferably wherein the coronavirus is the SARS- CoV-2 virus or a SARS-CoV-2 mutant virus. The above and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject-matter of appended claims is hereby specifically incorporated in this specification.

BRIEF DESCRIPTION OF DRAWINGS

The following description of the figures of specific embodiments of the invention is merely exemplary in nature and is not intended to limit the present teachings, their application or uses.

Fig. 1. FTO down-regulation correlates with higher SARS-CoV-2 expression. (A) Scheme of single-cell RNA sequencing (scRNA-seq) in COVID-19 patients. 44 patients were involved in this cohort with 13 control (ctrl) and 31 COVID-19 patients (5 and 26 with Mild or Severe symptoms, respectively). Lung cells from airway epithelium and alveoli were collected by Broncho-Alveolar Lavages (BAL) and subjected to scRNA-seq analysis. (B, C) Dot plots show the differential gene expression of FTO and SARS-CoV-2 between Ctrl and COVID-19 patients' cells in either the entire BAL dataset (B) or in Myeloid, Lymphoid and Epithelial cells identified in BAL (C). (D, E) Dot plots display the differential gene expression of FTO and SARS-CoV-2 between Mild and Severe COVID-19 patient's cells in either the entire BAL dataset (D) or in Myeloid, Lymphoid and Epithelial cells domains identified in BAL (E). For gene expression results in dot plots, the dot size represents the proportion of cells within the respective cell type expressing the gene of interest and the dot color (white, grey or black) represents the average gene expression level within the particular cell type, ranging from low (white) to high (black). (F) FTO and SARS-CoV-2 expression anti-correlates in epithelial cells from Mild and Severe COVID-19 patients. Density plots illustrate cells distribution for each gene expression. One-tailed Spearman's test was used for the correlation analyses.

Fig. 2. FTO classifies COVID-19 severity in lung's epithelial cells. (A) Classifier construction was performed using a k-Nearest Neighbor (kNN) machine learning approach on single cell RNA-seq dataset, especially gene expression from lung's epithelial cells, obtained from a patient cohort with 13 Control (Ctrl) and 31 COVID-19 patients (5 Mild and 26 Severe) subjected to Broncho-Alveolar Lavage (BAL) sampling. (B, C) Receiver Operating Characteristic (ROC) curves show the ability of FTO as a COVID-19 severity diagnostic classifier against I FI6, I LIB, IL1R2 (B) or against CCR2, CCR5, IL6 (C).

(D) ROC curves show the ability of a 2-gene signature (FTO and I FI6) in classifying COVID-19 severity.

(E) ROC curves show the ability of m⁶A enzymes as COVID-19 severity diagnostic classifiers. The mean of the Area Under the ROC Curve (AUC) as well as the 95% confidence interval are indicated for each gene. Fig. 3. FTO classifies COVID-19 severity in nasal swab. (A) Classifier construction was performed using a kNN machine learning approach on RNA-seq dataset obtained from a patient cohort with 93 Control (Ctrl) and 80 COVID-19 patients (72 Mild and 8 Severe) subjected to nasal swab sampling. (B, C) ROC curves show the ability of FTO as a COVID-19 severity diagnostic classifier against I FI6, I LIB, IL1R2 (B) or against CCR2, CCR5, IL6 (C). (D) ROC curves show the ability of a 2-gene signature (FTO and IFI6) in classifying COVID-19 severity. (E) ROC curves show the ability of a 3-gene signature (FTO, IL1B and CCR2) in classifying COVID-19 severity. (F) ROC curves show the ability of m⁶A enzymes as COVID-19 severity diagnostic classifiers. The mean of the Area Under the ROC Curve (AUC) as well as the 95% confidence interval are indicated for each gene.

Fig. 4. FTO classifies COVID-19 severity in blood's leukocytes. (A) Classifier construction was performed using a kNN machine learning approach on RNA-seq dataset obtained from a patient cohort with 26 Control (Ctrl) and 100 COVID-19 patients (50 Mild and 50 Severe) subjected to blood drawing. (B, C) ROC curves show the ability of FTO as a COVID-19 severity diagnostic classifier against IFI6, 1 LIB, IL1R2 (B) or against CCR2, CCR5, IL6 (C). (D) ROC curves show the ability of a 2-gene signature (FTO and I F 16) in classifying COVID-19 severity. (E) ROC curves show the ability of a 3-gene signature (FTO, IL1R2 and CCR5) in classifying COVID-19 severity. (F) ROC curves show the ability of m⁶A enzymes as COVID-19 severity diagnostic classifiers. The mean of the Area Under the ROC Curve (AUC) as well as the 95% confidence interval are indicated for each gene.

DESCRIPTION OF EMBODIMENTS

Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one or ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation is meant to apply throughout this specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined.

As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass "consisting of" and "consisting essentially of", which enjoy well-established meanings in patent terminology.

The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

The terms "about" or "approximately" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/-10% or less, preferably +/-5% or less, more preferably +/-1% or less, and still more preferably +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" refers is itself also specifically, and preferably, disclosed.

Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order, unless specified. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

Whereas the terms "one or more" or "at least one", such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, "one or more" or "at least one" may refer to 1, 2, 3, 4, 5, 6, 7 or more.

The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Reference throughout this specification to "one embodiment", "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

As corroborated by the experimental section, which illustrates certain representative embodiments of the invention, the inventors found that the quantity and/or activity of FTO can be used as a clinical marker for determining or predicting the severity of a disease caused by a coronavirus infection.

Accordingly, an aspect relates to a method for determining or predicting the severity of a disease caused by coronavirus infection in a subject, wherein the method comprises detecting fat mass and obesity associated (FTO) in a sample from the subject, more specific measuring the quantity and/or activity of FTO in a sample from the subject.

The phrases "determining severity" and "predicting severity" of a disease may be used interchangeably herein.

The terms "predicting", "prediction" or "predictive" as used herein refer to an advance declaration, indication or foretelling of the severity of a disease caused by coronavirus infection. For example, a prediction of the severity of the disease caused by coronavirus infection in a subject may indicate that the subject is categorized as asymptomatic or mild symptomatic or as severe symptomatic requiring hospitalization or intensive care treatment. Or, a prediction of the severity of the disease caused by coronavirus infection in a subject may indicate that the subject is categorized as not having or not being at risk of developing a severe or critical form of the disease, or as having or being at risk of developing a severe or critical from of the disease. Thus, in some embodiments, a method for determining or predicting the severity of a disease caused by a coronavirus infection in a subject is provided, wherein the method comprises detecting FTO, in particular measuring FTO quantity and/or activity, in a sample from the subject and wherein the method allows the subject to be categorized as asymptomatic or mild symptomatic, or as severe symptomatic requiring hospitalization and/or intensive care unit (ICU) treatment. In some embodiments, a method for determining or predicting the severity of a disease caused by a coronavirus infection in a subject is provided, wherein the method comprises detecting FTO, in particular measuring FTO quantity and/or activity, in a sample from the subject and wherein the method allows the subject to be categorized as not having or not being at risk of developing a severe or critical form of the disease, or as having or being at risk of developing a severe or critical form of the disease. Particularly, a severe or critical form of the disease is a disease wherein hospitalization and/or ICU treatment is necessary. By means of an example and without limitation, the method may allow to determine or predict, in an individual patient who has tested positive for a coronavirus infection, and optionally who has presented at a general practitioner or may even have been admitted to a hospital, whether that subject has or is likely to develop a severe or critical form of the disease. This knowledge may help the medical practitioner manage the patient adequately, such as recommending hospitalisation of the patient, prescribing coronavirus infection treatments typically reserved for patients who are at a greater risk of deterioration, ensuring particularly attentive monitoring of the patient, or even directing the patient to an ICU unit, etc.

In certain embodiments, the methods or uses as taught herein are useful for the categorization or stratification of subjects having a disease caused by a coronavirus infection. Hence, a population of subjects having a disease caused by coronavirus infection may be stratified or categorized, i.e. divided or separated into subgroups or strata, based on the quantity and/or activity of FTO in samples from the subjects. In certain embodiments, a subject may be allocated or categorized to a given subgroup or stratum when the subject displays a quantity and/or activity of FTO corresponding to or encompassed by said subgroup or stratum. In certain embodiments, the subgroups are selected from asymptomatic subjects, mild symptomatic subjects, or severe symptomatic subjects requiring hospitalization or intensive care treatment. In certain embodiments, the subgroups are selected from subjects not having or not being at risk of developing a severe or critical form of the disease caused by coronavirus infection, or subjects having or being at risk of developing a severe or critical form of the disease caused by coronavirus infection. In certain embodiments, a severe or critical form of the disease is to be understood as a disease that requires hospitalization or intensive care treatment.

As evidenced by the experimental data, the inventors found an inverse correlation between FTO expression and the severity of disease caused by a coronavirus infection. Therefore, in certain embodiments, decreased quantity and/or activity (downregulation for reasons of brevity) of FTO in the sample indicates greater severity of the disease caused by a coronavirus infection. In certain embodiments, normal or increased quantity and/or activity of FTO in the sample indicates a less severe disease caused by the coronavirus infection. Such normal, increased or decreased quantity and/or activity of FTO may be assessed compared to a suitable reference value (i.e., a reference value of the quantity or activity of FTO) that represents one or more reference subjects with a known diagnosis and/or prognosis of the disease, or with a known categorization of the disease, or with a known risk of having or developing a severe or critical form of a disease caused by a coronavirus infection. In some embodiments, the reference value may correspond to the quantity and/or activity of FTO in a healthy subject or in a healthy tissue, for example, in a tissue or sample from a healthy subject that is of the same tissue type as the tissue that is afflicted by a pathology or of the same sample type as the sample of the diseased subject, or in a tissue from the diseased subject that is of the same tissue type as the afflicted tissue but is not afflicted by the pathology.

In certain embodiments, reduced quantity and/or activity of FTO in the sample, such as in particular a quantity and/or activity of FTO that is lower than the reference value representative of a subject not having a disease caused by coronavirus infection or a subject having only a mild disease caused by coronavirus infection, indicates that the subject has a severe disease caused by coronavirus infection that requires hospitalization or intensive care treatment.

In certain embodiments, reduced quantity and/or activity of FTO in the sample, such as in particular a quantity and/or activity of FTO that is lower than the reference value representative of a subject not having a disease caused by coronavirus infection or a subject having only a mild disease caused by coronavirus infection, indicates that the subject is at risk of developing a severe or critical form of the disease caused by coronavirus infection.

Also provided is a method for monitoring clinical progression of a disease caused by coronavirus infection in a subject wherein FTO is detected, in particular the quantity and/or activity of FTO is measured, in sample obtained from said subject at two or more different time points and wherein the FTO that is detected, or the FTO quantity and/or activity that is measured, is compared at the different time points.

In certain embodiments, an increase in FTO activity and/or quantity at the later time point compared to the earlier time point (upregulation for reasons of brevity) indicates a favourable clinical progression of the disease in the subject, whereas a decrease in FTO activity and/or quantity at the later time point compared to the earlier time point (downregulation for reasons of brevity) indicates progression of the subject towards greater severity of the disease, such as wherein the subject has progressed to a severe disease that requires hospitalization or intensive care treatment or wherein the subject has a severe disease that requires extension of hospitalization or extension of the intensive care treatment. Such upregulation or downregulation of FTO between the different time points may be assessed by comparing the intra-patient measurements obtained at the different time points directly, or alternatively by comparing each of the measurements to a suitable reference value as explained above.

In some embodiments, the methods as taught herein for the determination or prediction of severity of a disease caused by coronavirus infection in a subject allow to select a therapeutic treatment for the subject. For example, in case more severe disease caused by coronavirus infection is determined or predicted in the subject, a therapeutic treatment specifically indicated for or typically reserved for severe disease can be selected. On the other hand, when a mild disease caused by coronavirus infection is determined or predicted in the subject, no therapeutic treatment or a therapeutic treatment more suitable for mild or moderate disease can be selected (such as for example, rest, and/or symptomatic treatments to alleviate common coronavirus infection symptoms such as fever and cough). The present methods and uses may thus allow to stratify patients having a disease caused by coronavirus infection for a specific therapeutic treatment.

A related aspect further provides a method for assessing the efficacy of a therapeutic treatment of a disease caused by a coronavirus infection in a subject. Said method comprises detecting FTO, in particular measuring FTO quantity and/or activity, in a sample from the subject obtained at a time point before the start of the treatment and at one or more time points after the start of the treatment, and comparing the FTO detected at the different time points, or comparing the FTO quantity and/or activity measured at the different time points. In some embodiments, the methods or uses as taught herein may thus allow to predict an outcome of a therapeutic treatment for a disease caused by coronavirus infection. An increase in FTO activity and/or quantity at the later time point compared to the earlier time point (upregulation for reasons of brevity) indicates a clinical response to the therapeutic treatment, whereas a decrease in FTO activity and/or quantity at the later time point compared to the earlier time point (downregulation for reasons of brevity) indicates no response to the treatment or even worsening of the disease, such as wherein the subject, despite the therapeutic treatment, has progressed to a severe disease that requires hospitalization or intensive care treatment or wherein the subject has a severe disease that requires extension of hospitalization or extension of the intensive care treatment. Such upregulation or downregulation of FTO between the different time points may be assessed by comparing the intra-patient measurements obtained at the different time points directly, such as at the start and after one, two, three or more weeks after the start of the therapeutic treatment, or alternatively by comparing each of the measurements to a suitable reference value as explained above. Based on the prediction or the assessment of the efficacy of the therapeutic treatment, the therapeutic treatment for the disease can be initiated, continued or adapted. In certain embodiments, the methods and uses as taught herein are thus provided to evaluate whether the subject is sensitive or responsive or susceptible to a particular therapeutic treatment for the disease cause by coronavirus infection. In certain embodiments, the methods and uses as taught herein are provided to evaluate whether the subject is insensitive, unresponsive or resistant to a particular therapeutic treatment for the disease caused by coronavirus infection.

The terms "sensitivity", "responsiveness" or "susceptibility" may be used interchangeably herein and refer to the quality that predisposes a subject having a disease caused by coronavirus infection to be sensitive or reactive to a particular therapeutic treatment. A subject is "sensitive", "responsive" or "susceptible" (which terms may be used interchangeably) to treatment with a particular therapeutic agent if the subject will have benefit from the treatment.

The terms "insensitivity", "unresponsiveness", "insusceptibility" or "resistance" may be used interchangeably herein and refer to the quality that predisposes a subject having a disease caused by coronavirus infection to a minimal (e.g. insignificant) or no response to treatment with a particular therapeutic agent. A subject is "insensitive", "unresponsive", "unsusceptible" or "resistant" (which terms may be used interchangeably) to treatment with a particular therapeutic agent if the subject will have no clinical benefit from the treatment.

As illustrated in the examples, the inventors thus demonstrated that FTO can be used as a clinical marker for determining or predicting the severity of a disease caused by coronavirus infection, or for monitoring the clinical progression of a subject with a disease caused by coronavirus infection, or for assessing the efficacy of a therapeutic treatment of a disease caused by coronavirus infection in a subject.

The present methods as taught herein thus also allow predicting survival or prognosis of a disease caused by coronavirus in a subject using FTO as a clinical marker. Also provided is the use of FTO as a biomarker useful for predicting survival or for prognosis of a disease caused by coronavirus infection in a subject. In certain embodiments of the methods or uses as taught herein, normal or increased quantity and/or activity of FTO in the sample as compared to a reference value of a healthy or to a value of a sample that was taken on an earlier time point indicates an increased chance of survival of the subject or a favourable prognosis in the subject. In certain embodiments, decreased quantity and/or activity (downregulation for reasons of brevity) of FTO in the sample as compared to a reference value of a healthy subject or to a value of a sample that was taken on an earlier time point indicates a reduced chance of survival of the subject or an unfavourable prognosis in the subject. Such normal quantity and/or activity of FTO or upregulation or downregulation of FTO may be assessed compared to a suitable reference value (i.e., a reference value of the quantity and/or activity of FTO) that represents one or more reference subjects with favourable survival or prognosis, whereby normal quantity and/or activity of FTO may then refer to a quantity and/or activity that is substantially the same as the reference value. In certain embodiments, the reference value may correspond to the quantity and/or activity of FTO in a healthy tissue, for example, in a tissue or sample from a healthy subject that is the same tissue type as the tissue that is afflicted by a pathology in a diseased subject or of the same sample type as in the diseased subject, or in tissue from the diseased subject that is of the same tissue type as the afflicted tissue but is not afflicted by the pathology.

In certain embodiments, detecting FTO in the sample includes measuring the quantity and/or activity of FTO in the sample. In some embodiments, detecting FTO in the sample includes measuring the quantity of FTO in the sample. In some embodiments, detecting FTO in the sample includes measuring the activity of FTO in the sample. In some other embodiments, detecting FTO in the sample includes measuring both the quantity of FTO and the activity of FTO in the sample. In some preferred embodiments, detecting FTO in the sample comprises measuring FTO gene expression in the sample, such as for example measuring FTO messenger RNA (mRNA) in the sample.

The terms "amount", "quantity" and "level" are synonymous and as used herein refer to but are not limited to the absolute or relative amount of FTO, and any other value or parameter associated with the latter or which can derive therefrom. Such values or parameters comprise signal intensity values obtained by direct or indirect measurement, for example, in quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The readout may be a mean, average, median or the variance or other statistically or mathematically-derived value associated with the measurement. The absolute values obtained for the FTO values under identical conditions will display a variability that is inherent in live biological systems and also reflects individual FTO quantity variability as well as the variability inherent between individuals.

An absolute quantity of a marker, peptide, polypeptide, protein, or nucleic acid in a sample may be advantageously expressed as weight or as molar amount, or more commonly as a concentration, e.g., weight per volume or mol per volume.

A relative quantity of a marker, peptide, polypeptide, protein, or nucleic acid in a sample may be advantageously expressed as an increase or decrease or as a fold-increase or fold-decrease relative to said another value, such as relative to a reference value as taught herein. Performing a relative comparison between first and second parameters (e.g., first and second quantities) may but need not require determining first the absolute values of said first and second parameters. For example, a measurement method may produce quantifiable readouts (such as, e.g., signal intensities) for said first and second parameters, wherein said readouts are a function of the value of said parameters, and wherein said readouts may be directly compared to produce a relative value for the first parameter vs. the second parameter, without the actual need to first convert the readouts to absolute values of the respective parameters.

Reference to the activity of a protein, polypeptide, or peptide may generally encompass any one or more aspects of the biological activity of the protein, polypeptide, or peptide, such as without limitation any one or more aspects of its biochemical activity, enzymatic activity, signaling activity, interaction activity, ligand activity, and/or structural activity, e.g., within a cell, tissue, organ or an organism.

Depending on factors that can be evaluated and decided on by a skilled person, such as inter alia the type of a marker (e.g., peptide, polypeptide, protein, or nucleic acid), the type of the tested object (e.g., a cell, cell population, tissue, organ, or organism, e.g., the type of biological sample of a subject, e.g., whole blood, tissue biopsy), the expected abundance of the marker in the tested object, the type, robustness, sensitivity and/or specificity of the detection method used to detect the marker, etc., the quantity and/or activity of a marker may be measured directly in the tested object, or the tested object may be subjected to one or more processing steps aimed at achieving an adequate measurement of the marker.

The reference to "Fat mass and obesity associated", "alpha-ketoglutarate-dependent dioxygenase FTO" or "FTO" denotes the FTO markers, peptide, polypeptide, protein, or nucleic acid, as commonly known under said designation in the art. By means of additional guidance, FTO is also known as GDFD, ALKBH9, and BMIQ14. The terms denote FTO nucleic acids, as well as FTO peptides, polypeptides and proteins, as apparent from the context. The term "FTO polypeptide" as used herein is synonymous with "FTO protein".

By means of an example, human FTO mRNA is annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) accession numbers NM_001080432.3 ("isoform 3"), NM_001363891.1 ("isoform 2), NM_001363894.1 ("isoform 1), NM_001363896.1 ("isoform 4), NM_001363897.1 ("isoform 5"), NM_001363898.1 ("isoform 6), NM_001363899.1 ("isoform 7"), NM_001363900.1 ("isoform 8"), NM_001363901.1 ("isoform 9"), NM_001363901.1 ("isoform 10"), NM_001363905.1 ("isoform 11"), "NM_001363905.1 ("isoform 11"), NM_001363988.1 ("isoform 12"), XM_011523316.3 ("predicted transcript variant isoform X6"), XM_017023656.1 ("predicted transcript variant isoform X7"), XM_011523314.3 ("predicted transcript variant isoform X4"), XM_017023654.2 ("predicted transcript variant isoform X2"), XM_017023655.2 ("predicted transcript variant isoform X3"), XM_011523315.3 ("predicted transcript variant isoform X5"), XM_017023657.2 ("predicted transcript variant isoform X8"), or XM_024450437.1 ("predicted transcript variant isoform X9").

Nucleotides 30 (start codon) to 1547 (stop codon) of NM_001080432.3 constitute the FTO coding sequence (CDS). By means of an example, nucleotides 30 to 1547 of NM_001080432.3 are reproduced below: atgaagcgcaccccgactgccgaggaacgagagcgcgaagctaagaaactgaggcttcttgaagagcttgaagacacttggctcccttatctga cccccaaagatgatgaattctatcagcagtggcagctgaaatatcctaaactaattctccgagaagccagcagtgtatctgaggagctccataa agaggttcaagaagcctttctcacactgcacaagcatggctgcttatttcgggacctggttaggatccaaggcaaagatctgctcactccggtatc tcgcatcctcattggtaatccaggctgcacctacaagtacctgaacaccaggctctttacggtcccctggccagtgaaagggtctaatataaaac acaccgaggctgaaatagccgctgcttgtgagaccttcctcaagctcaatgactacctgcagatagaaaccatccaggctttggaagaacttgct gccaaagagaaggctaatgaggatgctgtgccattgtgtatgtctgcagatttccccagggttgggatgggttcatcctacaacggacaagatga agtggacattaagagcagagcagcatacaacgtaactttgctgaatttcatggatcctcagaaaatgccatacctgaaagaggaaccttattttg gcatggggaaaatggcagtgagctggcatcatgatgaaaatctggtggacaggtcagcggtggcagtgtacagttatagctgtgaaggccctga agaggaaagtgaggatgactctcatctcgaaggcagggatcctgatatttggcatgttggttttaagatctcatgggacatagagacacctggttt ggcgataccccttcaccaaggagactgctatttcatgcttgatgatctcaatgccacccaccaacactgtgttttggccggttcacaacctcggttt agttccacccaccgagtggcagagtgctcaacaggaaccttggattatattttacaacgctgtcagttggctctgcagaatgtctgtgacgatgtg gacaatgatgatgtctctttgaaatcctttgagcctgcagttttgaaacaaggagaagaaattcataatgaggtcgagtttgagtggctgaggca gttttggtttcaaggcaatcgatacagaaagtgcactgactggtggtgtcaacccatggctcaactggaagcactgtggaagaagatggagggt gtgacaaatgctgtgcttcatgaagttaaaagagaggggctccccgtggaacaaaggaatgaaatcttgactgccatccttgcctcgctcactgc acgccagaacctgaggagagaatggcatgccaggtgccagtcacgaattgcccgaacattacctgctgatcagaagccagaatgtcggccata ctgggaaaaggatgatgcttcgatgcctctgccgtttgacctcacagacatcgtttcagaactcagaggtcagcttctggaagcaaaaccctag

(SEQ ID NO: 1).

By means of an example, human FTO protein sequence is annotated under NCBI Genbank accession numbers NP_001073901.1, NP_001350820.1, NP_001350823.1, NP_001350825.1, NP_001350826.1, NP_001350827.1, NP_001380828.1, NP_001350829.1, NP_001350830.1, NP_001350832.1, NP_001350834.1, NP_001350917.1, XP_011521618.1, XP_016879145.1, XP_011521616.1, XP_016879144.1, XP_011521617.1, XP_016879146.1, XP_016879147.1.

As an example, amino acid sequence of NP_001073901.1 is further reproduced below:

MKRTPTAEEREREAKKLRLLEELEDTWLPYLTPKDDEFYQQWQLKYPKLILREASSVSEELHKEVQEAFLTLHKHGCL FRDLVRIQGKDLLTPVSRILIGNPGCTYKYLNTRLFTVPWPVKGSNIKHTEAEIAAACETFLKLNDYLQIETIQALEELA AKEKANEDAVPLCMSADFPRVGMGSSYNGQDEVDIKSRAAYNVTLLNFMDPQKMPYLKEEPYFGMGKMAVSW HHDENLVDRSAVAVYSYSCEGPEEESEDDSHLEGRDPDIWHVGFKISWDIETPGLAIPLHQGDCYFMLDDLNATH QHCVLAGSQPRFSSTHRVAECSTGTLDYILQRCQLALQNVCDDVDNDDVSLKSFEPAVLKQGEEIHNEVEFEWLRQ FWFQGNRYRKCTDWWCQPMAQLEALWKKM EGVTNAVLHEVKREGLPVEQRNEILTAILASLTARQNLRREWH ARCQSRIARTLPADQKPECRPYWEKDDASMPLPFDLTDIVSELRGQLLEAKP (SEQ ID NO: 2).

By means of an example, human FTO gene is annotated under NCBI Genbank Gene ID 79068.

A skilled person can appreciate that any sequences represented in sequence databases or in the present specification may be of precursors of markers, peptides, polypeptides, proteins, or nucleic acids and may include parts which are processed away from mature molecules.

In some embodiments, measurement or determination of the quantity and/or activity of FTO may be combined with one or more other clinical markers. In some embodiments, said additional clinical marker may be a marker that is already known as a clinical marker that correlates to the severity, clinical progression or therapeutic efficacy of a disease caused by a coronavirus infection. In some embodiments, said additional clinical marker may be a marker that was not yet known as a clinical marker to correlate with the severity, clinical progression or therapeutic efficacy of a disease caused by a coronavirus infection. In some embodiments, the clinical marker may be selected from the group comprising or consisting of IFI6, IL1B, IL1R2, CCR2, CCR5, IL6, METTL3, METTL14, ALKBH5, and combinations thereof. In some embodiments, the detection of FTO may be combined with detection of IFI6 (interferon alpha inducible protein 6). In some other embodiments, the detection of FTO may be combined with the detection of CCR2 (C-C chemokine receptor type 2) and I LIB (interleukin-lB). In some other embodiments, the detection of FTO may be combined with the detection of METTL3 (methyltransferase 3, N6-adenosine), METTL14 (methyltransferase 14, N6-adenosine)and ALKBH5 (AlkB homolog 5).

The reference to "IFI6" denotes the IFI6 markers, peptide, polypeptide, protein, or nucleic acid, as commonly known under said designation in the art. By means of additional guidance, IFI6 is also known as "interferon alpha inducible protein 6". The terms denote IFI6 nucleic acids, as well as IFI6 peptides, polypeptides and proteins, as apparent from the context. The term "IFI6 polypeptide" as used herein is synonymous with "IFI6 protein". By means of an example, human IFI6 mRNA is annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) accession number NM_002038.4, NM_022872.3, or NM_022873.3. By means of an example, human IFI6 protein sequence is annotated under NCBI Genbank accession numbers NP_002029.3, NP_075010.1, or NP_075011.1.

The reference to "IL1B" denotes the IL1B markers, peptide, polypeptide, protein, or nucleic acid, as commonly known under said designation in the art. By means of additional guidance, IL1B is also known as "interleukin 1 beta". The terms denote IL1B nucleic acids, as well as IL1B peptides, polypeptides and proteins, as apparent from the context. The term "I LIB polypeptide" as used herein is synonymous with "I LIB protein". By means of an example, human I LIB mRNA is annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) accession number NM_008851.1. By means of an example, human IL1B protein sequence is annotated under NCBI Genbank accession number NP_000567.1

The reference to "IL1R2" denotes the IL1R2 markers, peptide, polypeptide, protein, or nucleic acid, as commonly known under said designation in the art. By means of additional guidance, IL1R2 is also known as "interleukin 1 receptor type 2". The terms denote IL1R2 nucleic acids, as well as IL1R2 peptides, polypeptides and proteins, as apparent from the context. The term "IL1R2 polypeptide" as used herein is synonymous with "IL1R2 protein". By means of an example, human IL1R2 mRNA is annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) accession number NM_001261419.2 or NM_004633.4. By means of an example, human IL1R2 protein sequence is annotated under NCBI Genbank accession number NP_001248348.1 or NP_004624.1.

The reference to "CCR2" denotes the CCR2 markers, peptide, polypeptide, protein, or nucleic acid, as commonly known under said designation in the art. By means of additional guidance, CCR2 is also known as "C-C motif chemokine receptor 2". The terms denote CCR2 nucleic acids, as well as CCR2 peptides, polypeptides and proteins, as apparent from the context. The term "CCR2 polypeptide" as used herein is synonymous with "CCR2 protein". By means of an example, human CCR2 mRNA is annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) accession number NM_001123041.3, or NM_001123396.4. By means of an example, human CCR2 protein sequence is annotated under NCBI Genbank accession number NP_001116513.2 or NP_001116868.1.

The reference to "CCR5" denotes the CCR5 markers, peptide, polypeptide, protein, or nucleic acid, as commonly known under said designation in the art. By means of additional guidance, CCR5 is also known as "C-C motif chemokine receptor 5". The terms denote CCR5 nucleic acids, as well as CCR5 peptides, polypeptides and proteins, as apparent from the context. The term "CCR5 polypeptide" as used herein is synonymous with "CCR5 protein". By means of an example, human CCR5 mRNA is annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) accession number NM_000579.4, NM_001100168.2, or NM_001394783.1. By means of an example, human CCR5 protein sequence is annotated under NCBI Genbank accession number NP_000570.1, NP_001093638.1, or NP_001381712.1.

The reference to "IL6" denotes the IL6 markers, peptide, polypeptide, protein, or nucleic acid, as commonly known under said designation in the art. By means of additional guidance, IL6 is also known as "interleukin 6". The terms denote IL6 nucleic acids, as well as IL6 peptides, polypeptides and proteins, as apparent from the context. The term "IL6 polypeptide" as used herein is synonymous with "IL6 protein". By means of an example, human IL6 mRNA is annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) accession number NM_000600.5, NM_001318095.2, or

NM_001371096.1. By means of an example, human IL6 protein sequence is annotated under NCBI Genbank accession numbers NP_000591.1, NP_001305024.1, or NP_001358025.1.

The reference to "METTL3" denotes the METTL3 markers, peptide, polypeptide, protein, or nucleic acid, as commonly known under said designation in the art. By means of additional guidance, METTL3 is also known as "methyltransferase 3 N6-adenosine-methyltransferase complex catalytic subunit". The terms denote METTL3 nucleic acids, as well as METTL3 peptides, polypeptides and proteins, as apparent from the context. The term "METTL3 polypeptide" as used herein is synonymous with "METTL3 protein". By means of an example, human METTL3 mRNA is annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) accession number NM_019852.5. By means of an example, human METTL3 protein sequence is annotated under NCBI Genbank accession number NP_062826.2.

The reference to "METTL14" denotes the METTL14 markers, peptide, polypeptide, protein, or nucleic acid, as commonly known under said designation in the art. By means of additional guidance, METTL14 is also known as "methyltransferase 14 N6-adenosine-methyltransferase complex catalytic subunit". The terms denote METTL14 nucleic acids, as well as METTL14 peptides, polypeptides and proteins, as apparent from the context. The term "METTL14 polypeptide" as used herein is synonymous with "METTL14 protein". By means of an example, human METTL14 mRNA is annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) accession number NM_020961.4. By means of an example, human METTL14 protein sequence is annotated under NCBI Genbank accession number NP_066012.1.

The reference to "ALKBH5" denotes the ALKBH5 markers, peptide, polypeptide, protein, or nucleic acid, as commonly known under said designation in the art. By means of additional guidance, ALKBH5 is also known as "alkB homolog 5, RNA demethylase". The terms denote ALKBH5 nucleic acids, as well as ALKBH5 peptides, polypeptides and proteins, as apparent from the context. The term "ALKBH5 polypeptide" as used herein is synonymous with "ALKBH5 protein". By means of an example, human ALKBH5 mRNA is annotated under NCBI Genbank (http://www.ncbi.nlm.nih.gov/) accession number NM_017758.4. By means of an example, human ALKBH5 protein sequence is annotated under NCBI Genbank accession number NP_060228.3. The term "marker" is widespread in the art and commonly broadly denotes a biological molecule, more particularly an endogenous biological molecule, and/or a detectable portion thereof, whose qualitative and/or quantitative evaluation in a test object (e.g., in or on a cell, cell population, tissue, organ, or organism, e.g., in a biological sample of a subject) is predictive or informative with respect to one or more aspects of the tested object's phenotype and/or genotype. The terms "marker" and "biomarker" may be used interchangeably throughout this specification.

In certain embodiments, markers as intended herein may be peptide-, polypeptide- and/or proteinbased. In certain embodiments, markers as intended herein may be nucleic acid-based. For example, a marker may be comprised of peptide(s), polypeptide(s) and/or protein(s) encoded by a given gene, or of detectable portions thereof. Further, whereas the term "nucleic acid" generally encompasses DNA, RNA and DNA/RNA hybrid molecules, in the context of markers the term may typically refer to heterogeneous nuclear RNA (hnRNA), pre-mRNA, messenger RNA (mRNA), or copy DNA (cDNA), or detectable portions thereof. Such nucleic acid species are particularly useful as markers, since they contain qualitative and/or quantitative information about the expression of the gene. Particularly preferably, a nucleic acid-based marker may encompass mRNA of a given gene, or cDNA made of the mRNA, or detectable portions thereof. In certain preferred embodiments, the quantity of FTO mRNA is measured in a sample or samples, preferably wherein said quantity of FTO mRNA is measured by RNA sequencing or quantitative RT-PCR.

The term "gene" is well-known in the art and in general refers to a locatable region of genomic sequence, corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed regions and/or other functional sequence regions. Genes typically comprise a coding sequences encoding a gene product, such as an RNA molecule or a polypeptide.

The term "protein" as used throughout this specification generally encompasses macromolecules comprising one or more polypeptide chains, i.e., polymeric chains of amino acid residues linked by peptide bonds. The term may encompass naturally, recombinantly, semi-synthetically or synthetically produced proteins. The term also encompasses proteins that carry one or more co- or post- expression-type modifications of the polypeptide chain(s), such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N- terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes protein variants or mutants which carry amino acid sequence variations vis-a-vis a corresponding native proteins, such as, e.g., amino acid deletions, additions and/or substitutions. The term contemplates both full-length proteins and protein parts or fragments, e.g., naturally- occurring protein parts that ensue from processing of such full-length proteins. The term "polypeptide" as used throughout this specification generally encompasses polymeric chains of amino acid residues linked by peptide bonds. Hence, especially when a protein is only composed of a single polypeptide chain, the terms "protein" and "polypeptide" may be used interchangeably herein to denote such a protein. The term is not limited to any minimum length of the polypeptide chain. The term may encompass naturally, recombinantly, semi-synthetically or synthetically produced polypeptides. The term also encompasses polypeptides that carry one or more co- or post-expression- type modifications of the polypeptide chain, such as, without limitation, glycosylation, acetylation, phosphorylation, sulfonation, methylation, ubiquitination, signal peptide removal, N-terminal Met removal, conversion of pro-enzymes or pre-hormones into active forms, etc. The term further also includes polypeptide variants or mutants which carry amino acid sequence variations vis-a-vis a corresponding native polypeptide, such as, e.g., amino acid deletions, additions and/or substitutions. The term contemplates both full-length polypeptides and polypeptide parts or fragments, e.g., naturally-occurring polypeptide parts that ensue from processing of such full-length polypeptides.

The term "peptide" as used throughout this specification preferably refers to a polypeptide as used herein consisting essentially of 50 amino acids or less, e.g., 45 amino acids or less, preferably 40 amino acids or less, e.g., 35 amino acids or less, more preferably 30 amino acids or less, e.g., 25 or less, 20 or less, 15 or less, 10 or less or 5 or less amino acids.

The reference to any marker, peptide, polypeptide, protein, or nucleic acid, corresponds to the marker, peptide, polypeptide, protein, or nucleic acid, commonly known under the respective designations in the art. The terms encompass such markers peptides, polypeptides, proteins, or nucleic acids, of any organism where found, and particularly of animals, preferably warm-blooded animals, more preferably vertebrates, yet more preferably mammals, including humans and nonhuman mammals, still more preferably of humans.

The terms particularly encompass such markers, peptides, polypeptides, proteins, or nucleic acids, with a native sequence, i.e., ones of which the primary sequence is the same as that of the markers, peptides, polypeptides, proteins, or nucleic acids found in or derived from nature. A skilled person understands that native sequences may differ between different species due to genetic divergence between such species. Moreover, native sequences may differ between or within different individuals of the same species due to normal genetic diversity (variation) within a given species. Also, native sequences may differ between or even within different individuals of the same species due to somatic mutations, or post-transcriptional or post-translational modifications. Any such variants or isoforms of markers, peptides, polypeptides, proteins, or nucleic acids are intended herein. Accordingly, all sequences of markers, peptides, polypeptides, proteins, or nucleic acids found in or derived from nature are considered "native". The terms encompass the markers, peptides, polypeptides, proteins, or nucleic acids when forming a part of a living organism, organ, tissue or cell, when forming a part of a biological sample, as well as when at least partly isolated from such sources. The terms also encompass markers, peptides, polypeptides, proteins, or nucleic acids when produced by recombinant or synthetic means.

In certain embodiments, markers, peptides, polypeptides, proteins, or nucleic acids, may be human, i.e., their primary sequence may be the same as a corresponding primary sequence of or present in a naturally occurring human markers, peptides, polypeptides, proteins, or nucleic acids. Hence, the qualifier "human" in this connection relates to the primary sequence of the respective markers, peptides, polypeptides, proteins, or nucleic acids, rather than to their origin or source. For example, such markers, peptides, polypeptides, proteins, or nucleic acids may be present in or isolated from samples of human subjects or may be obtained by other means (e.g., by recombinant expression, cell- free transcription or translation, or non-biological nucleic acid or peptide synthesis).

Unless otherwise apparent from the context, reference herein to any marker, peptide, polypeptide, protein, or nucleic acid, or fragment thereof may generally also encompass modified forms of said marker, peptide, polypeptide, protein, or nucleic acid, or fragment thereof, such as bearing postexpression modifications including, for example, phosphorylation, glycosylation, lipidation, methylation, cysteinylation, sulphonation, glutathionylation, acetylation, oxidation of methionine to methionine sulphoxide or methionine sulphone, and the like.

Fragments of any marker, peptide, polypeptide, protein, or nucleic acid, are also envisaged herein.

Hence, for example, the reference herein to measuring (or measuring the quantity of) any one marker, peptide, polypeptide, protein, or nucleic acid, may encompass measuring the marker, peptide, polypeptide, protein, or nucleic acid, and/or measuring one or more fragments thereof. For example, any marker, peptide, polypeptide, protein, or nucleic acid, and/or one or more fragments thereof may be measured collectively, such that the measured quantity corresponds to the sum amounts of the collectively measured species. In another example, any marker, peptide, polypeptide, protein, or nucleic acid, and/or one or more fragments thereof may be measured each individually.

The term "fragment" as used throughout this specification with reference to a peptide, polypeptide, or protein generally denotes a portion of the peptide, polypeptide, or protein, such as typically an N- and/or C-terminally truncated form of the peptide, polypeptide, or protein. Preferably, a fragment may comprise at least about 30%, e.g., at least about 50% or at least about 70%, preferably at least about 80%, e.g., at least about 85%, more preferably at least about 90%, and yet more preferably at least about 95% or even about 99% of the amino acid sequence length of said peptide, polypeptide, or protein. For example, insofar not exceeding the length of the full-length peptide, polypeptide, or protein, a fragment may include a sequence of > 5 consecutive amino acids, or > 10 consecutive amino acids, or > 20 consecutive amino acids, or > 30 consecutive amino acids, e.g., >40 consecutive amino acids, such as for example > 50 consecutive amino acids, e.g., > 60, > 70, > 80, > 90, > 100, > 200, > 300, > 400, > 500 or > 600 consecutive amino acids of the corresponding full-length peptide, polypeptide, or protein.

For example, a fragment of FTO polypeptide may include a sequence of > 5 consecutive amino acids, or > 10 consecutive amino acids, or > 20 consecutive amino acids, or > 30 consecutive amino acids, e.g., >40 consecutive amino acids, such as for example > 50 consecutive amino acids, e.g., > 60, > 70,

> 80, > 90, > 100, > 110, > 120, > 130, > 140, > 150, > 160, > 170, > 180, > 190, > 200, > 250, > 300, > 350, > 400, > 450, > 500, > 510, > 520, > 530, or > 540 consecutive amino acids of the corresponding full-length FTO polypeptide.

The term "fragment" with reference to a nucleic acid (polynucleotide) generally denotes a 5'- and/or 3' -truncated form of a nucleic acid. Preferably, a fragment may comprise at least about 30%, e.g., at least about 50% or at least about 70%, preferably at least about 80%, e.g., at least about 85%, more preferably at least about 90%, and yet more preferably at least about 95% or even about 99% of the nucleic acid sequence length of said nucleic acid. For example, insofar not exceeding the length of the full-length nucleic acid, a fragment of an FTO nucleic acid may include a sequence of > 5 consecutive nucleotides, or > 10 consecutive nucleotides, or > 20 consecutive nucleotides, or > 30 consecutive nucleotides, e.g., >40 consecutive nucleotides, such as for example > 50 consecutive nucleotides, e.g.,

> 60, > 70, > 80, > 90, > 100, > 200, > 300, > 400, > 500, > 600, > 700, > 800, > 900, > 1000, > 1100, > 1200 > 1300, > 1400, or > 1500 consecutive nucleotides of the corresponding full-length FTO nucleic acid, in particular FTO mRNA.

The terms encompass fragments arising by any mechanism, in vivo and/or in vitro, such as, without limitation, by alternative transcription or translation, exo- and/or endo-proteolysis, exo- and/or endonucleolysis, or degradation of the peptide, polypeptide, protein, or nucleic acid, such as, for example, by physical, chemical and/or enzymatic proteolysis or nucleolysis.

Where the present specification refers to or encompasses fragments of proteins, polypeptides or peptides, this in particular denotes such fragments which are biologically active. The term "biologically active" is interchangeable with terms such as "functionally active" or "functional", denoting that the fragment at least partly retains the biological activity or intended functionality of the respective or corresponding protein, polypeptide, or peptide. Preferably, a functionally active fragment may retain at least about 20%, e.g., at least about 25%, or at least 30%, or at least about 40%, or at least about 50%, e.g., at least 60%, more preferably at least about 70%, e.g., at least 80%, yet more preferably at least about 85%, still more preferably at least about 90%, and most preferably at least about 95% or even about 100% of the intended biological activity or functionality compared with the corresponding protein, polypeptide, or peptide.

By means of an example and not limitation, a biologically active fragment of a FTO polypeptide as disclosed herein shall at least partly retain the biological activity of the FTO polypeptide. For example, it may retain one or more aspects of the biological activity of the FTO polypeptide.

By means of an example and not limitation, reference to the activity of the FTO polypeptide or functionally active fragment thereof may particularly denote its m6A RNA demethylase activity, i.e., its ability to demethylate a ribonucleic acid (RNA) molecule comprising one or more N⁶- methyladenosines (m6A). For example, the demethylase activity of FTO can be measured by existing methodologies for measuring the quantity of m⁶A RNA modification as described herein, such as mass spectrometry, RIP-seq or RIP-qPCR analysis.

In aspects of the invention, the methods as taught herein comprise detecting FTO in a sample from the subject. In certain embodiments, the quantity and/or activity of FTO in the sample is measured. In certain preferred embodiments, the quantity of FTO in the sample or samples is measured. For example, the quantity of FTO mRNA in the sample is measured. Measurement of FTO mRNA expression levels can for example be done using RT-qPCR or RNA sequencing (RNA-seq).

A marker, peptide, polypeptide, protein or nucleic acid is "detected" or "measured" in a sample when the presence or absence, quantity and/or activity of said marker, peptide, polypeptide, protein, or nucleic acid is determined or measured in the sample, preferably substantially to the exclusion of other markers, peptides, polypeptides, proteins, or nucleic acids.

In certain embodiments, the method as taught herein may comprise measuring the quantity and/or activity of FTO, i.e., the quantity of FTO, the activity of FTO, or both. Preferably, the method as taught herein comprise measuring the quantity of FTO.

In certain embodiments of the methods as taught herein, FTO nucleic acid or fragment thereof, FTO polypeptide or fragment thereof, or both may be detected or measured. Preferably, FTO nucleic acid or fragment thereof is detected or measured.

In certain preferred embodiments, the methods as taught herein comprise measuring the quantity of FTO nucleic acid or fragment thereof.

Any existing, available or conventional separation, detection and/or quantification method may be used to measure the presence or absence (e.g., readout being present vs. absent; or detectable amount vs. undetectable amount) and/or quantity (e.g., readout being an absolute or relative quantity) of markers (such as FTO) in a tested object (e.g., in or on a cell, cell population, tissue, organ, or organism, e.g., in a biological sample from a subject).

In some examples, detection methods may include affinity-based assay methods, wherein the ability of an assay to separate, detect and/or quantify a marker, peptide, polypeptide, protein, or nucleic acid is conferred by specific binding between a separable, detectable and/or quantifiable binding agent and i) the marker peptide, polypeptide, protein, or nucleic acid, or ii) a label or tag comprised by (e.g., covalently bound to or conjugated with) the marker peptide, polypeptide, protein, or nucleic acid.

The level of biomarkers at the nucleic acid level, more particularly RNA level, e.g., at the level of hnRNA, pre-mRNA, mRNA, or cDNA, may be detected using standard quantitative RNA or cDNA measurement tools known in the art. Non-limiting examples include hybridization-based analysis, microarray expression analysis, digital gene expression (DGE), RNA-in-situ hybridization (RISH), Northern-blot analysis and the like; PCR, RT-PCR, RT-qPCR, end-point PCR, digital PCR or the like; supported oligonucleotide detection, pyrosequencing, polony cyclic sequencing by synthesis, simultaneous bi-directional sequencing, single-molecule sequencing, single molecule real time sequencing, true single molecule sequencing, hybridization-assisted nanopore sequencing, sequencing by synthesis, or the like. In preferred embodiments, the quantity of the marker (e.g., FTO) is measured using RNA sequencing or quantitative RT-PCR.

Numerous different PCR or qPCR protocols are known in the art. Generally, in PCR, a target polynucleotide sequence is amplified by reaction with a pair of oligonucleotide primers. The primers hybridize to complementary regions of a target nucleic acid and a DNA polymerase extends the primers to amplify the target sequence, generating an amplification product. The amplification cycle is repeated to increase the concentration of the amplification product.

The reaction can be performed in any thermocycler commonly used for PCR. However, preferred are cyclers with real-time fluorescence measurement capabilities, for example, Smartcycler® (Cepheid, Sunnyvale, CA), ABI PRISM 7700® (Applied Biosystems, Foster City, CA), Rotor-Gene™ (Corbett Research, Sydney, Australia), Lightcycler® (Roche Diagnostics Corp, Indianapolis, IN), iCycler® (Biorad Laboratories, Hercules, CA), MX4000® (Stratagene, La Jolla, CA), and CFX96 Real-Time PCR system (Biorad).

As used herein, "quantitative PCR" (or "real-time qPCR") refers to the direct monitoring of the progress of a PCR amplification as it is occurring without the need for repeated sampling of the reaction products. In QPCR, the reaction products may be monitored via a signaling mechanism (e.g., fluorescence) as they are generated and are tracked after the signal rises above a background level but before the reaction reaches a plateau. The number of cycles required to achieve a detectable or "threshold" level of fluorescence ("cycle threshold", "CT") varies directly with the concentration of amplifiable targets at the beginning of the PCR process, enabling a measure of signal intensity to provide a measure of the amount of target nucleic acid in a sample in real time.

By means of example and not limitation, real-time amplification, especially real-time PCR, as intended herein encompasses fully conventional systems, such as, e.g., the TaqMan™ system developed by Applied Biosystems, which relies on the release and detection of a fluorogenic probe during each round of DNA amplification (Holland et al. 1991. Detection of specific polymerase chain reaction product by utilizing the 5'— 3' exonuclease activity of Thermus aquaticus DNA polymerase. PNAS 88: 7276-80). The method uses the 5' exonuclease activity of Taq polymerase during primer extension to cleave a dual-labelled, fluorogenic probe hybridized to the target DNA between the PCR primers. Prior to cleavage, a reporter fluorophore, such as 6-carboxyfluorescein (6-FAM) at the 5' end of the probe is quenched by 6-carboxy-tetramethylrhodaniine (TAMRA) through fluorescent resonance energy transfer (FRET). Following digestion, FAM is released. The resulting fluorescence measured in realtime at around 518 nm during the log phase of product accumulation is proportional to the number of copies of the target sequence.

Further real-time amplification, especially real-time PCR, detection systems can also utilize FRET, such as, e.g., systems based on molecular beacons. Molecular beacons are single-stranded polynucleotide probes that possess a stem-and-loop hairpin structure. The loop portion is a probe sequence complementary to a sequence within an amplicon to be evaluated, and the stem is formed by short complementary sequences located at the opposite ends of the molecular beacon. The molecular beacon is labelled with a fluorophore (e.g., 6-FAM) at one end and a quencher (e.g., TAMRA) at the other end. When free in solution, the stem keeps the fluorophore and the quencher in close proximity, causing the fluorescence of the fluorophore to be quenched by FRET. However, when bound to its complementary target, the probe-target hybrid forces the stem to unwind, separating the fluorophore from the quencher, and restoring the fluorescence. Accordingly, when the quantity of an amplicon increases during amplification, this can be monitored as an increase in the fluorescence of the corresponding beacon (see, e.g., Manganelli et al. 2001. Real-time PCR using molecular beacons. Methods Mol Med 54: 295-310; Marras SAE. 2006. Selection of fluorophore and quencher pairs for fluorescent nucleic acid hybridization probes. Methods Mol Biol 335: 3-16; Marras SAE et al. 2006. Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes. Clin Chim Acta 363: 48-60 for further discussion of molecular beacons detection).

An alternative real-time PCR amplification and detection system is the Light Upon Extension (LUX™) system commercialized by Invitrogen (Carlsbad, CA) and described in detail in Nazarenko et al. 2002 (Nucleic Acids Research 30: e37) and Nazarenko et al. 2002 (Nucleic Acids Research 30: 2089-2095). This system employs primer pairs in which usually one of the primers of said primer pair is labelled by a fluorophore (such as, e.g., FAM or JOE or Alexa Fluor 546). The particular structure of the "free" primer quenches the signal of the fluorophore bound thereto, whereas the fluorophore's signal intensity increases when the primer assumes an extended conformation once incorporated into the amplification product. The sequence of primers may be tailored to perform with the LUX™ technology, following instructions of the above publications of Nazarenko et al. 2002 or using software tools provided by Invitrogen on www.invitrogen.com/lux. The LUX™ technology is particularly well suited for multiplexing (/.e., performing in a single reaction) of two or more amplifications using different primer sets, since each of the primer sets may be marked using a different fluorophore.

For description of additional ways to detect and evaluate amplification products in real-time (e.g., using adjacent probes; 5'-nuclease probes such as Taqman™; Light-up probes; Duplex scorpion primers; Amplifluor primers; and further alternative fluorescent hybridization probe formats; see, e.g., Marras SAE et al. 2006. Real-time assays with molecular beacons and other fluorescent nucleic acid hybridization probes; Clin Chim Acta 363: 48-60, esp. section 6 and references therein).

In some methods of detection of the activity and/or quantity of an FTO protein, the binding agent may be i) an immunological binding agent (antibody) or ii) a non-immunological binding agent. Examples of antibodies capable of binding to human FTO include without limitation those available from the following vendors ("#" stands for catalogue number): R&D Systems (#AF7208, polyclonal sheep; #MAB7208, mouse monoclonal); Cell Signaling Technology (#14386, rabbit polyclonal); OriGene (#TA809392, mouse monoclonal); Invitrogen (# PA5-18942, goat polyclonal; Cat # PA5-20736, rabbit polyclonal); GeneTex (#GTX131517, rabbit polyclonal; #GTX82693, mouse monoclonal); ProteoGenix (#PTX12945, rabbit polyclonal); and Santa Cruz (#sc-515411, #sc-515410, mouse monoclonals). Further example of i) include anti-myc antibodies, which specifically bind to a myc tag (EQKLISEEDL), or anti-FLAG antibodies, which specifically bind to a FLAG tag (DYKDDDDK). Non-limiting examples of ii) include streptavidin, which specifically binds to biotin; metal ion (e.g., Ni²⁺), which specifically binds to his-tag; and maltose binding protein (MBP), which specifically binds maltose. Affinity-based assay methods, such immunological assay methods, include without limitation immunohistochemistry, immunocytochemistry, flow cytometry, mass cytometry, fluorescence activated cell sorting (FACS), fluorescence microscopy, fluorescence based cell sorting using microfluidic systems, (immuno)affinity adsorption based techniques such as affinity chromatography, magnetic particle separation, magnetic activated cell sorting or bead based cell sorting using microfluidic systems, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) and ELISPOT based techniques, radioimmunoassay (RIA), Western blot, etc. In further examples, such methods may include mass spectrometry analysis methods. Generally, any mass spectrometric (MS) techniques that are capable of obtaining precise information on the mass of peptides, and preferably also on fragmentation and/or (partial) amino acid sequence of selected peptides (e.g., in tandem mass spectrometry, MS/MS; or in post source decay, TOF MS), may be useful herein for separation, detection and/or quantification of markers (such as, preferably, peptides, polypeptides, or proteins). Suitable peptide MS and MS/MS techniques and systems are well-known per se (see, e.g., Methods in Molecular Biology, vol. 146: "Mass Spectrometry of Proteins and Peptides", by Chapman, ed., Humana Press 2000, ISBN 089603609x; Biemann 1990. Methods Enzymol 193: 455-79; or Methods in Enzymology, vol. 402: "Biological Mass Spectrometry", by Burlingame, ed., Academic Press 2005, ISBN 9780121828073) and may be used herein. MS arrangements, instruments and systems suitable for biomarker peptide analysis may include, without limitation, matrix-assisted laser desorption/ionisation 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; atmospheric pressure photoionization mass spectrometry (APPI-MS); APPI-MS/MS; and APPI- (MS)ⁿ. Peptide ion fragmentation in tandem MS (MS/MS) arrangements may be achieved using manners established in the art, such as, e.g., collision induced dissociation (CID). Detection and quantification of markers by mass spectrometry may involve multiple reaction monitoring (MRM), such as described among others by Kuhn et al. 2004 (Proteomics 4: 1175-86). MS peptide analysis methods may be advantageously combined with upstream peptide or protein separation or fractionation methods, such as for example with the chromatographic and other methods.

In other examples, such methods may include chromatography methods. The term "chromatography" encompasses methods for separating substances, such as chemical or biological substances, e.g., markers, such as preferably peptides, polypeptides, or proteins, referred to as such and vastly available in the art. In a preferred approach, chromatography refers to a process in which a mixture of substances (analytes) carried by a moving stream of liquid or gas ("mobile phase") is separated into components as a result of differential distribution of the analytes, as they flow around or over a stationary liquid or solid phase ("stationary phase"), between said mobile phase and said stationary phase. The stationary phase may be usually a finely divided solid, a sheet of filter material, or a thin film of a liquid on the surface of a solid, or the like. Chromatography is also widely applicable for the separation of chemical compounds of biological origin, such as, e.g., amino acids, proteins, fragments of proteins or peptides, etc.

Chromatography may be preferably columnar (i.e., wherein the stationary phase is deposited or packed in a column), preferably liquid chromatography, and yet more preferably HPLC. While particulars of chromatography are well known in the art, for further guidance see, e.g., Meyer M., 1998, ISBN: 047198373X, and "Practical HPLC Methodology and Applications", Bidlingmeyer, B. A., John Wiley & Sons Inc., 1993. Exemplary types of chromatography include, without limitation, high- performance liquid chromatography (HPLC), normal phase HPLC (NP-HPLC), reversed phase HPLC (RP- HPLC), ion exchange chromatography (IEC), such as cation or anion exchange chromatography, hydrophilic interaction chromatography (HILIC), hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) including gel filtration chromatography or gel permeation chromatography, chromatofocusing, affinity chromatography such as immunoaffinity, immobilized metal affinity chromatography, and the like.

Further techniques for separating, detecting and/or quantifying markers, such as preferably peptides, polypeptides, or proteins, may be used, optionally in conjunction with any of the above described analysis methods. Such methods include, without limitation, chemical extraction partitioning, isoelectric focusing (IEF) including capillary isoelectric focusing (CIEF), capillary isotachophoresis (CITP), capillary electrochromatography (CEC), and the like, one-dimensional polyacrylamide gel electrophoresis (PAGE), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), capillary gel electrophoresis (CGE), capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), free flow electrophoresis (FFE), etc.

In some embodiments, reagents such as binding agents (for example oligonucleotide primers) as taught herein may comprise a detectable label. The term "label" refers to any atom, molecule, moiety or biomolecule that may be used to provide a detectable and preferably quantifiable read-out or property, and that may be attached to or made part of an entity of interest, such as a binding agent. Labels may be suitably detectable by for example mass spectrometric, spectroscopic, optical, colorimetric, magnetic, photochemical, biochemical, immunochemical or chemical means. Labels include without limitation dyes; radiolabels such as ³²P, ³³P, ³⁵S, ¹²⁵l, ¹³¹l; electron-dense reagents; enzymes (e.g., horse-radish peroxidase or alkaline phosphatase as commonly used in immunoassays); binding moieties such as biotin-streptavidin; haptens such as digoxigenin; luminogenic, phosphorescent or fluorogenic moieties; mass tags; and fluorescent dyes alone or in combination with moieties that may suppress or shift emission spectra by fluorescence resonance energy transfer (FRET). 1

In some embodiments, binding agents may be provided with a tag that permits detection with another agent (e.g., with a probe binding partner). Such tags may be, for example, biotin, streptavidin, his-tag, myc tag, maltose, maltose binding protein or any other kind of tag known in the art that has a binding partner. Example of associations which may be utilized in the probe:binding partner arrangement may be any, and includes, for example bioti streptavidin, his-tag:metal ion (e.g., Ni²⁺), maltose:maltose binding protein, etc.

The biomarker - binding agent conjugate may be associated with or attached to a detection agent to facilitate detection. Examples of detection agents include, but are not limited to, luminescent labels; colorimetric labels, such as dyes; fluorescent labels; or chemical labels, such as electroactive agents (e.g., ferrocyanide); enzymes; radioactive labels; or radiofrequency labels. The detection agent may be a particle. Examples of such particles include, but are not limited to, colloidal gold particles; colloidal sulphur particles; colloidal selenium particles; colloidal barium sulfate particles; colloidal iron sulfate particles; metal iodate particles; silver halide particles; silica particles; colloidal metal (hydrous) oxide particles; colloidal metal sulfide particles; colloidal lead selenide particles; colloidal cadmium selenide particles; colloidal metal phosphate particles; colloidal metal ferrite particles; any of the above-mentioned colloidal particles coated with organic or inorganic layers; protein or peptide molecules; liposomes; or organic polymer latex particles, such as polystyrene latex beads. Preferable particles may be colloidal gold particles.

The quantity or activity of FTO in a sample from a subject may refer to an absolute quantity and/or activity of FTO in the sample from the subject.

The quantity or activity of FTO in a sample from a subject may refer to a relative quantity and/or activity of FTO in the sample from the subject, i.e., the quantity or activity of FTO in the sample from the subject compared with the quantity or activity of FTO in a reference sample, for instance a sample, e.g., of non-diseased tissue, from the same subject or from an unrelated subject. For instance, the relative quantitative quantity of FTO nucleic acid or a fragment thereof, e.g., as measured by RT-PCR, may range from 0 to 1 or more.

In certain embodiments, the reference value for FTO quantity and/or activity may correspond to the quantity and/or activity of FTO in a healthy tissue, for example, in a tissue or sample from a healthy subject that is of the same tissue type as the tissue that is afflicted by a pathology in a diseased subject or that is the same sample type as the sample of the diseased subject, or in tissue from the diseased subject that is of the same tissue type as the afflicted tissue but is not afflicted by the pathology. In preferred embodiments, the reference value for FTO quantity and/or activity corresponds to the quantity and/or activity of FTO in a healthy subject.

In certain embodiments, the methods as taught herein rely on comparing the quantity and/or activity of FTO measured in samples from subjects wherein the samples are obtained at two or more different time points in order to monitor the clinical progression of a disease caused by coronavirus infection. In certain embodiments, the quantity and/or activity of FTO in a sample of the subject can then be compared to the quantity and/or activity of FTO in a different sample from the subject. For example, the quantity and/or activity of FTO in one or more subsequent samples of the subject can be compared to the quantity and/or activity of FTO detected in a first sample of the subject in order to monitor clinical progression of the subject. In certain embodiments, the quantity and/or activity of FTO in samples obtained at two or more different timepoints can be compared to the quantity and/or activity of FTO of a reference value, such as for example the quantity and/or activity of FTO in a sample of a healthy subject.

In certain embodiments, the method and uses as taught herein may rely on comparing the quantity and/or activity of FTO measured in samples of a subject having a disease caused by coronavirus infection collected before the start of a therapeutic treatment and at one or more time points after the start of the treatment, wherein comparing the FTO detected at the different time points allows to assess the efficacy of a particular therapeutic treatment.

In a further example, a reference value can represent a sample obtained from a healthy subject. In some examples, a reference value can represent a sample obtained from a subject having an asymptomatic or mild symptomatic disease caused by a coronavirus infection.

In another example, a reference value of FTO quantity and/or activity may represent a known chance of survival or a known prognosis of the disease caused by coronavirus infection in the subject, such as the prediction of an increased chance of survival of the subject, or a good or favorable prognosis or clinical progression in the subject, or the prediction of a reduced chance of survival of the subject, or a poor or unfavorable prognosis or clinical progression in the subject.

Such comparison may generally include any means to determine the presence or absence of at least one difference or deviation and optionally of the size of such difference or deviation between values being compared. A comparison may include a visual inspection, an arithmetical or statistical comparison of measurements. Such statistical comparisons include, but are not limited to, applying a rule.

Reference values may be established according to known procedures. For example, a reference value may be established in a reference subject or individual or a population of individuals who are healthy (i.e., not afflicted by coronavirus infection), or who are characterized by a particular severity (e.g. a low severity) of a disease caused by coronavirus infection, or by a particular clinical progression (e.g. a favorable progression) of a disease caused by coronavirus infection. A population may comprise without limitation 2 or more, 10 or more, or even several hundred or more individuals.

For example, a reference value may be established in tissue or sample from a healthy individual or tissue samples from a population of healthy individuals, wherein the tissue is of the same tissue type as the tissue that is afflicted by a pathology in a diseased subject. Such population may comprise without limitation 2 or more, 10 or more, 100 or more, or even several hundred or more individuals.

For example, a reference value may be established in one or more (e.g., 2, 3, 4 or 5) samples from a tissue from the diseased subject that is of the same tissue type as the afflicted tissue but is not afflicted by the pathology.

A "deviation" of a first value from a second value may generally encompass any direction (e.g., increase: first value > second value; or decrease: first value < second value) and any extent of alteration.

For example, a deviation may encompass a decrease in a first value by, without limitation, at least about 10% (about 0.9-fold or less), or by at least about 20% (about 0.8-fold or less), or by at least about 30% (about 0.7-fold or less), or by at least about 40% (about 0.6-fold or less), or by at least about 50% (about 0.5-fold or less), or by at least about 60% (about 0.4-fold or less), or by at least about 70% (about 0.3-fold or less), or by at least about 80% (about 0.2-fold or less), or by at least about 90% (about 0.1-fold or less), relative to a second value with which a comparison is being made.

For example, a deviation may encompass an increase of a first value by, without limitation, at least about 10% (about 1.1-fold or more), or by at least about 20% (about 1.2-fold or more), or by at least about 30% (about 1.3-fold or more), or by at least about 40% (about 1.4-fold or more), or by at least about 50% (about 1.5-fold or more), or by at least about 60% (about 1.6-fold or more), or by at least about 70% (about 1.7-fold or more), or by at least about 80% (about 1.8-fold or more), or by at least about 90% (about 1.9-fold or more), or by at least about 100% (about 2-fold or more), or by at least about 150% (about 2.5-fold or more), or by at least about 200% (about 3-fold or more), or by at least about 500% (about 6-fold or more), or by at least about 700% (about 8-fold or more), or like, relative to a second value with which a comparison is being made.

Preferably, a deviation may refer to a statistically significant observed alteration. For example, a deviation may refer to an observed alteration, which falls outside of error margins of reference values in a given population (as expressed, for example, by standard deviation or standard error, or by a predetermined multiple thereof, e.g., ±lxSD or ±2xSD or ±3xSD, or ±lxSE or ±2xSE or ±3xSE). Deviation may also refer to a value falling outside of a reference range defined by values in a given population (for example, outside of a range which comprises >40%, > 50%, >60%, >70%, >75% or >80% or >85% or >90% or >95% or even >100% of values in said population).

In a further embodiment, a deviation may be concluded if an observed alteration is beyond a given threshold or cut-off. Such threshold or cut-off may be selected as generally known in the art to provide for a chosen sensitivity and/or specificity of the prediction methods, e.g., sensitivity and/or specificity of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90%, or at least 95%.

For example, receiver-operating characteristic (ROC) curve analysis can be used to select an optimal cut-off value, e.g. of the quantity and/or activity of FTO, for clinical use of the methods as taught herein, based on acceptable sensitivity and specificity, or related performance measures which are well-known per se, such as positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), negative likelihood ratio (LR-), Youden index, or similar. By means of an example, a cut-off value may be selected such as to provide for AUC value higher than 50%, or higher than 55%, or higher than 60%, or higher than 65%, or higher than 70%, or higher than 75%, or higher than 80%, or higher than 85%, or higher than 90%, or higher than 95%.

By extensive research, the present inventors have found the subjects having a severe symptomatic disease requiring hospitalization or intensive care treatment caused by coronavirus infection or subjects that have an unfavorable clinical progression of the disease show a reduced quantity of FTO or show downregulation of FTO, such as of the quantity of FTO nucleic acid or fragment thereof (e.g., as determined by measuring FTO gene expression). On the other hand, subjects that have an asymptomatic disease or a mild symptomatic disease not requiring hospitalization, or subjects that show a favorable clinical progression of the disease show an increased or similar quantity of FTO or show an upregulation of FTO, such as of the quantity of FTO nucleic acid or fragment thereof (e.g., as determined by measuring FTO gene expression).

The reference to "upregulation of FTO" comprises an increase of FTO, such as an increased quantity and/or activity of FTO, e.g., an increased quantity of FTO gene expression.

The reference to "downregulation of FTO" comprises a decrease of FTO, such as a decreased quantity and/or activity of FTO, e.g., a decreased quantity of FTO gene expression.

Such upregulation or downregulation may be determined relative to a suitable reference value, for example a reference value corresponding to the level or activity of FTO in a healthy tissue or sample from a healthy subject, for example, in a sample or tissue from a healthy subject that is of the same tissue type as the tissue that is afflicted by a pathology in a diseased subject or that is the same sample type as the sample of the diseased subject, or in tissue from the diseased subject that is of the same tissue as the afflicted tissue but is not afflicted by the pathology.

In certain embodiments of the methods as taught herein, the method for determining or predicting the severity of a disease caused by coronavirus infection in a subject may comprises the steps of:

(a) measuring the quantity and/or activity of FTO in the sample from the subject;

(b) comparing the quantity and/or activity of FTO as measured in (a) with a reference value of FTO quantity or activity, said reference value representing a known severity status of the disease caused by coronavirus infection in a reference subject;

(c) finding a deviation or no deviation of the quantity and/or activity of FTO as measured in (a) from said reference value; and

(d) attributing said finding of deviation or no deviation to a particular determination of the severity of the disease caused by coronavirus infection, such as asymptomatic disease, mild symptomatic disease, or severe symptomatic disease requiring hospitalization or intensive care treatment, in the subject.

In certain embodiments, the reference value represents a reference subject that is not affected by a coronavirus infection or that is affected by a coronavirus infection but wherein the subject has no or only mild symptoms, such as for example a healthy subject,, and wherein: a reduced quantity and/or activity of FTO as measured in (a) compared with the reference value indicates that the subject has a more severe disease caused by coronavirus infection, particularly a severe symptomatic disease requiring hospitalization or intensive care treatment; the same or increased quantity and/or activity of FTO as measured in (a) compared with the reference value indicates that the subject has a less severe disease caused by coronavirus infection, such as an asymptomatic disease or a mild symptomatic disease not requiring hospitalization.

In certain embodiments of the methods as taught herein, the method for monitoring clinical progression of a disease caused by coronavirus infection in a subject may comprise the steps of:

(a) measuring the quantity and/or activity of FTO in samples from the subject wherein the samples are obtained at a first time point and at one or more later time points; (b) comparing the quantity and/or activity of FTO as measured in (a) in the one or more samples obtained at the later time points with the quantity and/or activity of FTO as measured in the sample of the subject obtained at the first time point;

(c) finding a deviation or no deviation of the quantity and/or activity of FTO as measured in (a) from said sample obtained at the first time point; and

(d) attributing said finding of deviation or no deviation to a particular clinical progression of the disease caused by coronavirus infection, such as a favorable clinical progression towards a disease not requiring hospitalization or an unfavorable clinical progression towards a severe symptomatic disease requiring hospitalization or intensive care treatment.

In certain embodiments, in the methods to monitor the clinical progression of a disease caused by coronavirus infection as taught herein above: a reduced quantity and/or activity of FTO as measured in (a) in the one or more samples obtained at the later time points as compared with the quantity and/or activity of FTO as measured in the sample obtained at the first time point in the subject indicates that the subject shows an unfavorable clinical progression, such as a progression towards a severe symptomatic disease requiring hospitalization or intensive care treatment; or the same or increased quantity and/or activity of FTO as measured in (a) in the one or more samples obtained at the later time points as compared with the quantity and/or activity of FTO as measured in the sample obtained at the first time point in the subject indicates that the subject show a favorable clinical progression, such as a progression towards an asymptomatic or mild symptomatic disease not requiring hospitalization.

In certain embodiments of the methods as taught herein, the method for assessing the efficacy of a therapeutic treatment of a disease caused by coronavirus infection in a subject may comprise the steps of:

(a) measuring the quantity and/or activity of FTO in samples from the subject wherein the samples are obtained at a timepoint before the start of the treatment and at one or more time points after the start of the treatment;

(b) comparing the quantity and/or activity of FTO as measured in (a) in the one or more samples obtained at different time points after the start of the treatment with the quantity or activity of FTO as measured in the sample obtained before the start of the treatment;

(c) finding a deviation or no deviation of the quantity and/or activity of FTO as measured in (a) from said first sample obtained before the start of the treatment; and (d) attributing said finding of deviation or no deviation to a particular efficacy of the therapeutic treatment, such as a clinical response to the therapeutic treatment or no clinical response to the therapeutic treatment.

In certain embodiments, in the methods to assess the efficacy of a therapeutic treatment of a disease caused by coronavirus infection as taught herein above: a reduced quantity and/or activity of FTO as measured in (a) in the one or more samples obtained after the therapeutic treatment as compared with the quantity or activity of FTO as measured in the sample obtained before the therapeutic treatment in the subject indicates that the therapeutic treatment has a reduced or poor efficacy, such as wherein the subject does not respond to the therapeutic treatment or does not respond enough to the therapeutic treatment, for example wherein the disease in the subject progresses towards or remains a severe symptomatic disease requiring hospitalization or intensive care treatment despite the therapeutic treatment; or the same or increased quantity and/or activity of FTO as measured in the one or more samples obtained after the therapeutic treatment as compared with the quantity and/or activity of FTO as measured in the sample obtained before the therapeutic treatment in the subject indicates that the therapeutic treatment has an improved or good efficacy, such as wherein the subject responds to the therapeutic treatment, for example wherein the disease in the subject progresses towards or remains an asymptomatic disease or a mild symptomatic disease not requiring hospitalization or intensive care treatment due to the therapeutic treatment.

As taught herein, the present methods and uses are provided for determining the severity of a disease caused by a coronavirus infection, or for monitoring clinical progression of said disease, or for assessing the therapeutic treatment of said diseased. In some embodiments, the coronavirus is a - coronavirus, such as a coronavirus selected from the Middle East respiratory syndrome-related coronavirus (MERS-CoV), the Severe Acute Respiratory virus (SARS-CoV), the Severe Acute Respiratory 2 virus (SARS-CoV-2) or a SARS-CoV-2 mutant virus. In some preferred embodiments, the coronavirus is a Sarbecovirus, preferably a Sarbecovirus selected from the Severe Acute Respiratory virus (SARS- CoV), the Severe Acute Respiratory 2 virus (SARS-CoV-2) or a SARS-CoV-2 mutant virus. In some preferred embodiments, the coronavirus is the SARS-CoV-2 virus or a SARS-CoV-2 mutant virus.

The terms "sample" or "biological sample" as used throughout this specification include any biological specimen obtained (isolated, removed) from a subject. Samples may include without limitation organ tissue (e.g. lung tissue), whole blood, plasma, serum, whole blood cells, red blood cells, white blood cells (e.g. peripheral blood mononuclear cells), saliva, naso-pharyngeal fluid, oropharyngeal fluid, bronchoalveolar fluid, sputum, urine, stool (faeces), tears, sweat, sebum, nipple aspirate, ductal lavage, synovial fluid, cerebrospinal fluid, lymph, fine needle aspirates, amniotic fluid, any other bodily fluid, exudate or secretory fluid, cell lysates, cellular secretion products, inflammation fluid, semen and vaginal secretions. Preferably, a sample may be readily obtainable by non-invasive or minimally invasive methods, such as blood collection ("liquid biopsy"), saliva collection, naso-pharyngeal swab collection, oropharyngeal swab collection, bronchoalveolar lavage collection, sputum collection, urine collection, faeces collection, tissue (e.g. lung) biopsy or fine-needle aspiration, allowing the provision, removal or isolation of the sample from a subject. The term "tissue" as used herein encompasses all types of cells of the body including cells of organs but also including blood and other body fluids recited above. The tissue may be healthy or affected by pathological alterations, e.g. inflammation or infection. The tissue may be from a living subject or may be cadaveric tissue.

In certain embodiments, the sample is selected from a blood-derived sample, a saliva sample, a nasopharyngeal sample, an oropharyngeal swab sample, a bronchoalveolar lavage sample or a sputum sample. In some embodiments, the blood-derived sample is a sample comprising peripheral blood mononuclear cells (PMBCs). PBMCs can be extracted from a blood sample using techniques known in the art (e.g. using the Ambion® LeukoLOCK™ Fractionation & Stabilization kit). In preferred embodiments, the sample is a naso-pharyngeal swab sample.

The biological sample can be obtained from a subject in any way typically used in clinical settings for obtaining a sample comprising the required cells or nucleic acid including RNA (such as mRNA), genomic DNA, mitochondrial DNA, and protein-associated nucleic acids. For example, the sample can be obtained from fresh, frozen or paraffin-embedded samples or biopsies of an organ or tissue comprising the suitable cells or nucleic acids to be tested. In some embodiments, the sample comprises live cells. In some embodiments, the sample comprises a single cell. If desired, the sample can be mixed with a fluid or purified or amplified or otherwise treated. For example, samples may be treated in one or more purification steps in order to increase the purity of the desired cells or nucleic acids in the sample, or they may be examined without any purification steps. Any nucleic acid specimen in purified or non-purified form obtained from such sample can be utilized in the methods as taught herein.

In certain embodiments of the methods or uses as taught herein, RNA is isolated from the sample, such as a blood-derived sample, a saliva sample, a naso-pharyngeal sample, an oropharyngeal swab sample, a bronchoalveolar lavage sample or a sputum sample, for m6A RNA modification analysis. RNA may be isolated by any means known in the art, such as techniques involving one-step RNA isolation, glass binding, acid phenol chloroform extraction, CsCI cushion isolation, or oligo dT Selection, including the use of commercially available kits, such as RNAqueous™ Total RNA Isolation Kit (e.g., cat.

# AM1912, Invitrogen), or RNeasy Total RNA Isolation Kits (e.g., cat. # 74104, Qiagen).

The terms "subject", "individual" or "patient" are used interchangeably throughout this specification, and typically and preferably denote humans, but may also encompass reference to non-human animals, preferably warm-blooded animals, even more preferably mammals, such as, e.g. non-human primates, rodents, canines, felines, equines, ovines, porcines, and the like. The term "non-human animals" includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, and nonmammals such as chicken, amphibians, reptiles, etc. In certain embodiments, the subject is a mammal. In some embodiments, the subject is a non-human mammal. In some preferred embodiments of the methods and uses as taught herein, the subject is a human subject. In other embodiments, the subject is an experimental animal or animal substitute as a disease model. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as foetuses, whether male or female, are intended to be covered.

Suitable subject may include without limitation subjects presenting to a physician for a screening for a coronavirus infection, subjects presenting to a physician with symptoms and signs indicative of a coronavirus infection or a disease caused by a coronavirus infection, subjects already diagnosed with a coronavirus infection or a disease caused by a coronavirus infection, subject who received already therapeutic treatment for a disease caused by coronavirus infection, subjects currently undergoing therapeutic treatment for a disease caused by coronavirus infection, and subjects that previously received therapeutic treatment for a disease caused by coronavirus infection.

As used throughout this specification, the terms "therapy" or "treatment" refer to the alleviation or measurable lessening of one or more symptoms or measurable markers of a pathological condition such as a disease or disorder. The terms encompass primary treatments as well as neo-adjuvant treatments, adjuvant treatments and adjunctive therapies. The terms "therapy" or "treatment" broadly refer to the alleviation or measurable lessening of one or more symptoms or measurable markers of a disease caused by a coronavirus infection. Measurable lessening includes any statistically significant decline in a measurable marker or symptom. Generally, the terms encompass both curative treatments and treatments directed to reduce symptoms and/or slow progression of the disease. The terms encompass both the therapeutic treatment of an already developed pathological condition, as well as prophylactic or preventative measures, wherein the aim is to prevent or lessen the chances of incidence of a pathological condition. In certain embodiments, the terms may relate to therapeutic treatments. In certain other embodiments, the terms may relate to preventative treatments. Treatment of a chronic pathological condition during the period of remission may also be deemed to constitute a therapeutic treatment. The term may encompass ex vivo or in vivo treatments.

Also disclosed is a method comprising determining the quantity and/or activity of FTO in a sample of a subject at risk of developing a severe disease caused by coronavirus infection. Further disclosed is a method comprising determining the quantity and/or activity of FTO in a sample of a subject suspected of having a coronavirus infection. In certain embodiments, the sample is selected from a blood-derived sample, a saliva sample, a naso-pharyngeal sample, an oropharyngeal swab sample, a bronchoalveolar lavage sample or a sputum sample. In preferred embodiments, the sample is a naso-pharyngeal swab sample.

In another aspect, a method of treatment of a severe disease caused by coronavirus infection is provided. Said method comprises detecting the quantity and/or activity of FTO in a sample of a subject and administering a therapeutically effective amount of a therapeutic agent to the subject. In some embodiments, the quantity and/or activity of FTO in the sample of the subject is compared to a reference value of FTO quantity and/or activity, said reference value representing a known severity status of the disease caused by coronavirus infection in a reference subject, and wherein a deviation or no deviation of the quantity and/or activity of FTO as measured in the sample of the subject from the reference value is indicative for the subject to receive a therapeutically effective amount of a therapeutic agent to treat the disease caused by coronavirus infection. In some embodiments, the reference value represents a reference subject that is not affected by a coronavirus infection and wherein reduced quantity and/or activity of FTO as measured in the sample of the subject compared with the reference value indicates that the subject has a more severe disease caused by coronavirus infection, particularly a severe symptomatic disease requiring hospitalization or intensive care treatment and wherein the subject needs treatment with the therapeutic agent.

The methods of treatment as taught herein may specifically relate to prophylactic and/or therapeutic treatment of a disease resulting from coronavirus infection. In particular embodiments, said method relates to a therapeutic treatment of a disease resulting from coronavirus infection. The therapeutic agent administered to the subject can be a therapeutic agent known to be effective against a severe disease caused by a coronavirus infection. For example, such therapeutic agent can be any agent that is known to be effective in treatment of a disease caused by coronavirus infection. In some embodiments, the therapeutic agent is an antiviral agent, such as for example Paxlovid™ (Pfizer) or Molnupiravir (Merck). In some embodiments, the therapeutic agent is an anti-SARS-CoV-2 monoclonal antibody. For example, the anti-SARS-CoV2 monoclonal antibody can be selected from the group comprising bebtelovimab, sotrovimab, bamlanivimab, etesevimab, casirivimab, or imdevimab.

The present application also provides aspects and embodiments as set forth in the following Statements. In these statements, the wording "The [subject] according to Statement [number] wherein ... " or The [subject] according to any one of Statements [numbers] wherein ... " also discloses and may be replaced by the simple wording "In certain embodiments..".

Statement 1. A method for determining or predicting the severity of a disease caused by coronavirus infection in a subject, the method comprising detecting fat mass and obesity associated (FTO), in particular measuring the quantity and/or activity of FTO, in a sample from the subject.

Statement 2. The method according to Statement 1, whereby the subject is categorised as asymptomatic or mild symptomatic, or as severe symptomatic requiring hospitalization or intensive care treatment.

Statement 3. The method according to Statement 1, wherein the subject is categorised as not having or not being at risk of developing a severe or critical form of the disease, or as having or being at risk of developing a severe or critical form of the disease.

Statement 4. The method according to any one of Statements 1 to 3, wherein the determination or prediction of the severity of the disease in the subject allows to select a therapeutic treatment for the subject.

Statement 5. A method for monitoring clinical progression of a disease caused by coronavirus infection in a subject, the method comprising detecting FTO, in particular measuring the activity and/or quantity of FTO, in samples from the subject obtained at two or more different time points and comparing the FTO detected, or the FTO quantity and/or activity measured, at the different time points.

Statement 6. A method for assessing the efficacy of a therapeutic treatment of a disease caused by coronavirus infection in a subject, the method comprising detecting FTO, in particular measuring the activity and/or quantity of FTO, in a sample from the subject obtained at a time point before the start of the treatment and at one or more time points after the start of the treatment, and comparing the FTO detected, or the FTO quantity and/or activity measured, at the different time points.

Statement 7. The method according to any one of Statements 1 to 6, wherein the quantity and/or activity of FTO in the sample or samples is measured, preferably wherein at least the quantity of FTO in the sample or samples is measured.

Statement 8. The method according to any one of Statements 1 to 7, wherein the quantity of FTO mRNA in the sample or samples is measured, preferably by RNA sequencing or quantitative RT-PCR.

Statement 9. The method according to any one of Statements 7 or 8, wherein comparatively reduced quantity and/or activity of FTO indicates greater severity of the disease.

Statement 10. The method of any one of the Statements 1 to 9, wherein the coronavirus is a - coronavirus, preferably a Sarbecovirus.

Statement 11. The method of any one of the Statements 1 to 10, wherein the coronavirus is selected from the Middle East respiratory syndrome-related coronavirus (MERS-CoV), the Severe Acute Respiratory virus (SARS-CoV), the Severe Acute Respiratory 2 virus (SARS-CoV-2) or a SARS-CoV-2 mutant virus, preferably wherein the coronavirus is the SARS-CoV-2 virus or a SARS-CoV-2 mutant virus.

Statement 12. The method of any one of the Statements 1 to 11, wherein the subject is a mammal, preferably a human.

Statement 13. The method according to any one of Statements 1 to 12, wherein the sample is selected from a blood-derived sample, a saliva sample, a naso-pharyngeal swab sample, an oropharyngeal swab sample, a bronchoalveolar lavage sample or a sputum sample.

Statement 14. The method according to any one of Statements 1 to 13, wherein the sample is a nasopharyngeal swab sample. It is apparent that there have been provided in accordance with the invention products, methods, and uses, that provide for substantial advantages as set forth above. While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and broad scope of the appended claims.

The above aspects and embodiments are further supported by the following non-limiting examples.

EXAMPLES EXAMPLE 1

MATERIALS AND METHODS

Data collection

Single cell RNA-sequencing data were retrieved from Wauters et al. (2021, Cell Res., 31, 272-290). This cohort is composed of 13 non-COVID-19 and 22 COVID-19 pneumonia patients, collected from the University Hospitals Leuven, between March 31st 2020 and May 4th 2020, and processed scRNA-seq data on COVID-19 BAL fluid from 9 patients by Liao et al. (2020, Nat. Med., 26, 842-844). Disease severity was defined as 'mild' or 'severe', based on the level of respiratory support at the time of sampling. Specifically, 'mild' patients required no respiratory support or supplemental oxygen through a nasal cannula, whereas 'severe' patients were mechanically ventilated or received extracorporeal membrane oxygenation. Regarding analysis, we retrieved normalized gene count of our genes of interest from an object analyzed with the Seurat package (v3.1.4). Cells from either control or COVID- 19 samples were additionally defined according to their SARS-CoV-2 expression status (at least one read on SARS-CoV-2 genome required for positive status). For the classifier analysis we filtered the dataset in order to keep the epithelial cells and more precisely the AT2, Ciliated, Secretory and Hillock type cells.

RNA-seq data from nasal swab samples were retrieved from Ng et al. (GSE163151) (Sci Adv. 2021). This cohort contains 93 control patients, defined by the non-pathogenic status, and 147 SARS-CoV-2 positive patients. The severity status was known from only 80 SARS-CoV-2 positive patients. Specifically, 8 patients were categorized as 'severe' patients requiring Intensive Care Unit (ICU) support, whereas 72 patients were categorized as 'mild' patients without the need for hospitalization or only non-ICU hospitalization. Regarding analysis, Transcripts Per Million (TPM) gene expressions were calculated using the HTseq tool. RNA-seq data from blood draw were retrieved from Overmyer et al. (GSE157103) (Cell Syst., 2021, 12(1): 23-40, e7). This cohort consists of 26 non-COVID-19 and 100 COVID-19 patients. Disease severity was defined as 'mild' or 'severe', based on ICU needs. Specifically, 'severe' patients required Intensive Care Unit (ICU) support, whereas 'mild' patients were non-ICU hospitalized. TPM gene expressions were directly used to build the classifier.

Single cell RNA-seq analysis

Single cell RNA-seq data were retrieved from Wauters et al. (2021, Cell Res., 31, 272-290). Normalized gene counts of FTO were obtained from an object analyzed with the Seurat package (v3.1.4.). Cells from either control or COVID-19 samples were additionally defined according to their SARS-CoV-2 expression status.

Classifier construction

Classifier for COVID-19 severity status were built using a K-Nearest Neighbors (k-NN) model approach. In brief, to classify mild from severe COVID-19 patients, a k-NN algorithm (as implemented in the R package FNN_1.1.3), was trained with genes of interest expression from the above-mentioned datasets. Hundred bootstrap replicates of tri-fold cross-validation were performed to reduce model error and obtain a better estimation of the Area Under the Receiver Operating Characteristic (ROC) Curve (AUC). Results were represented by ROC curves using the R packages pROC_1.18.0. FTO performance as a severity classifier was benchmarked against expression of well-known cytokines and interferon-stimulated genes (ISG). Among those, we used the interferon-stimulated gene IFI6, which is strongly induced by SARS-CoV-2 in the upper airway cells, the pro-inflammatory cytokines IL1B, IL1R2 and IL6, which production is increased in macrophages during severe COVID-19, as well as the chemokine receptors CCR2 and CCR5, mediating monocyte infiltration in inflammatory diseases (Mick et al., 2020, Nat Commun 11, 5854; Merad and Martin, 2020, Nat Rev Immunol, 20: 355-362).

RESULTS

FTO down regulation correlates with SARS-CoV-2 infection and COVID-19 severity in patients

To test whether FTO may impact on SARS-CoV-2 viral cycle and affect the course of COVID-19 in patients, we retrieved single cell RNA-seq data from bronchioalveolar lavage (BAL) samples that were collected from 44 hospitalized patients with pneumonia. Of those patients, 31 tested positive for SARS-CoV-2 after RT-qPCR on nasopharyngeal swab or lower respiratory tract sample, referred to as "COVID-19", and 13 tested negative, referred to as "Control" (ctrl) (Fig. 1A). We first investigated the relationship of FTO and SARS-CoV-2 in the BAL data set. Taking all cells into account, we observed, as expected, SARS-CoV-2 expression in COVID-19, but not in Control samples (Fig. IB). FTO expression, in contrast, appeared strongly decreased in COVID-19 as compared to Control samples, while, notably, the fraction of cells expressing FTO remained unchanged. We made similar observations in the distinct BAL cell types (Fig. 1C). Indeed, SARS-CoV-2 expression was detected exclusively in myeloid, lymphoid and epithelial cells from COVID-19 samples, while FTO expression was decreased in the same cells from COVID-19 as compared to Control samples. Of note, the fraction of FTO expressing epithelial cells was slightly increased in COVID-19 as compared to Control samples, but the overall FTO expression level remained lower in COVID-19 than in Control samples.

Next, we studied FTO and SARS-CoV-2 expression with respect to COVID-19 severity. In BAL samples from patients with mild and severe COVID-19, we found FTO expression to be slightly decreased in severe vs. mild cases, with 4% and 16% of cells expressing FTO, respectively, while SARS-CoV-2 expression was increased in severe samples with no changes in the fraction of infected cells between mild and severe cases (Fig. ID). Similar results were obtained in the distinct BAL cell types (Fig. IE) and strikingly, in epithelial BAL cells, where we found FTO to be strongest expressed, we observed the biggest decrease in FTO expression between severe and mild cases. These results suggest an inverse relationship between FTO expression and SARS-CoV-2 infection that seems to be linked to COVID-19 severity in patients.

Finally, to further support this conclusion, we measured the correlation between FTO and SARS-CoV- 2 expression in mild and severe cases (Fig. IF). This revealed a significant anti-correlation that depends on severity status. Indeed, mild and severe COVID-19 cases exhibited distinct expression profiles, with mild samples having higher FTO and lower SARS-CoV-2 expression and severe cases having lower FTO and higher SARS-CoV-2 expression.

In summary, our results in patient samples reveal an inverse correlation between FTO expression and SARS-CoV-2 infection or COVID-19 severity and point to a potential role of FTO-mediated m⁶A demethylation in the course of COVID-19.

Efficacy of FTO as a COVID-19 severity marker from broncho-alveolar lavage

Using a machine learning approach, we first evaluated the ability of FTO expression to predict COVID- 19 severity in epithelial cells from single cell RNA-seq on broncho-alveolar lavage (BAL) in COVID-19 patients (Fig. 2A). We observed an area under the "Receiver Operating Characteristic" (ROC) curve (AUC) of 0.768 with an 95% confidence interval (Cl) of 0.704 - 0.833 (Fig. 2B and 2C), indicating that FTO effectively classifies mild disease (not requiring hospitalization) from severe disease (requiring hospitalization and/or ICU) with a mean accuracy of 76.8%. Moreover, in order to benchmark FTO prediction accuracy, we compared with several reported COVID-19 biomarkers (cfr. Materials and Methods - Classifier construction). We found that IFI6, IL1B, IL1R2, CCR2, CCR5 and IL6 achieved an AUC of 0.732, 0.509, 0.476, 0.519, 0.523 and 0.493, respectively (Fig. 2B and 2C). Hence, while IL1B, IL1R2, CCR2, CCR5 and IL6 do not significantly classify COVID-19 severity in epithelial cells, FTO is slightly more accurate than I FI6 in discriminating COVID-19 severity. We then combined FTO and I FI6 genes expression and found that this 2-gene signature operated even better with an area under the curve (AUC) of 0.793 and a 95% Cl between 0.718 to 0.869 (Fig. 2D), underlining the complementary information carried by both genes. We further assessed other members of the m⁶A machinery (Fig. 2E). We uncovered that only the m⁶A demethylases significantly differentiate mild from severe patients, FTO achieving the strongest AUC values. Therefore, FTO expression could be used as a reliable diagnosis and prognosis classifier of COVID-19 severity from BAL samples upon patient hospitalization, with 70.4-83.3% accuracy.

Efficacy of FTO as a COVID-19 severity marker from nasal swab

To further extend the relevance and practicability of the usage of FTO expression to predict COVID-19 severity, we investigated whether FTO prediction accuracy in single cell RNA-seq from BAL could be recapitulated in an RNA-seq from nasopharyngeal swab from COVID-19 patients (Fig. 3A). Interestingly, we found that, while I FI6, IL1R2, CCR5 and IL6 genes could not significantly distinguish between patients with mild and severe status; FTO, IL1B and CCR2 efficiently predicted COVID-19 severity in patients with accuracy of 64.8%, 84.8% and 90%, respectively (Fig. 3B and 3C). Moreover, the 2-gene signature identified on single cell data (i.e. FTO and IFI6) showed lower and in this experiment insignificant AUC values compared to FTO taken alone (Fig. 3D). In addition, the combination of FTO, I LIB and CCR2 achieved a significant AUC of 0.723, which was lower than I LIB (0.848) and CCR2 (0.900) but higher than FTO alone (0.648) (Fig. 3E), pointing out the potential interest of a 3-gene signature in this experiment. Looking at m⁶A enzymes performance, we found that only FTO demonstrates an effective capability of severity stratification (Fig. 3F). Altogether, these results highlight the promising potential to integrate FTO into diagnostic assays to identify COVID-19 severity in patients at the time of admission.

Efficacy of FTO as a COVID-19 severity marker from blood draw As a standard of care, clinicians routinely use blood routine test on hospital admission to initially assess the severity of COVID-19 and optimize patient triage and resources allocation. Therefore, we assessed FTO performance in predicting COVID-19 severity in patients' blood draw samples (Fig. 4A). We show that FTO gene expression achieved a mean accuracy of 75.8% with an Cl of 60% - 91.5%, higher than IFI6, I LIB, CCR2 and IL6 which were not found significant. IL1R2 and CCR5 showed higher capability than FTO, with an AUC of 0.910 and 0.851, respectively (Fig. 4B and 4C). Furthermore, looking at the 2-gene signature identified on single cell data (i.e. FTO and IFI6), we found that FTO prediction performance surpassed both IFI6 and the combination (Fig. 4D). Additionally, FTO alone operated even better than the 3-gene signature composed of FTO, IL1R2 and CCR5 (Fig. 4E). Looking at m⁶A enzymes performance, we found that FTO and METTL3 demonstrate an effective capability of severity stratification (Fig. 4F), even though FTO shows the strongest prediction ability. All in all, these results underline once more the promising use of FTO in COVID-19 severity assessment, such as in COVID-19 severity predictive diagnostics upon hospital admission.

Conjointly, these examples establish FTO as a prognosis biomarker to discriminate COVID-19 severity status in patients, from different biological sample types. Importantly, FTO appears to be the only reliable biomarker in all biological contexts in comparison to the other genes benchmarked. Therefore, measuring FTO sustains a novel universal simple accurate diagnostic tool to be used in clinical practice, helping patient prioritization and improving resource management and disease outcome.

EXAMPLE 2: Evaluation of FTO expression in BAL, swab or blood samples of patients hospitalized for covid infection.

Materials and Methods

Sample Collection, RNA isolation and Storage

At hospital admission, either a bronchoalveolar lavage (BAL) sample, a naso-pharyngeal swab swab sample or a blood sample is collected from a patient infected with COVID-19.

For BAL, retrieved lavage volume is separated into aliquots by the performing endoscopist. The aliquots are immediately put on ice and transported to a Biosafety Level 3 Laboratory. BAL fluid is centrifuged, the cellular fraction is resuspended in ice-cold PBS, filtered using a 40 pm nylon mesh and centrifuged. The cell pellet is resuspended in red blood cell lysis buffer, centrifuged, resuspended in PBS containing UltraPure™ BSA (Thermofisher) and filtered over Flowmi® 40 pm cell strainers (Sigma Aldrich) using wide-bore 1 mL low-retention filter tips. RNA is isolated from the cells using Trizol™ reagent following manufacturer's protocol (Thermofisher) and can be stored at -80 °C for further analysis.

For nasal swab, samples are pre-treated with a 1:1 ratio of DNA/RNA Shield before extraction. RNA is extracted using the Mag-Bind® Viral DNA/RNA 96 kit (Omega Bio-Tek) on a KingFisher™ Flex instrument according to the manufacturer's instructions (Thermofisher). Extracted RNA can be stored at -80°C degrees for later analysis.

For blood draw, samples are collected in plasma preparation tubes. One tube is processed through LeukoLOCK® filters (Thermofisher) to isolate peripheral blood mononuclear cells (PBMCs) and to extract RNA from the leukocytes following manufacturer recommendation. Eluted RNA can be stored at -80°C degrees for later analysis.

RNA-sequencing and Analysis

Extracted RNA from either BAL, swab or blood samples is treated with a nuclease cocktail of TURBO™ DNase (Thermofisher) and Baseline-Zero™ DNase (Lucigen, Thermofisher) and purified using AM Pure XP beads (Beckman). Purified RNA is used for library preparation using the SMART-Seq® Stranded kit (Takarabio) and purified using AMPure XP beads. Libraries are quantified using the Qubit dsDNA HS Assay on the Qubit Flex (Thermofisher), and sequenced on the NovaSeq 6000 (Illumina) using 150- base pair paired-end sequencing. Included in each sequencing run are negative controls (nuclease- free water) to monitor for laboratory and reagent contamination and a Human Reference RNA Standard to monitor for sequencing efficiency. Quality control is performed on the fastq files to ensure that sequencing reads met pre-established cutoffs for number of reads and quality using FastQC. Quality filtering and adapter trimming is performed using Trimmomatic tools. Remaining reads are aligned to the ENSEMBL GRCh38 human reference genome assembly using STAR, and gene frequencies were counted using HTseqCounts.

RT-qPCR and Analysis

Isolated RNA is converted to cDNA using the SuperScrip™ II Reverse Transcriptase (Thermofisher) with random primer. All PCR reactions are carried out using the LightCycler® 480 SYBR Green I Master according to the manufacturer's protocol (Roche Diagnostics) and quantified on an LightCycler® 480 II Real-Time PCR System (Roche Diagnostics). In all cases, average threshold cycles are determined from at least triplicate reactions, and relative gene expression levels were calculated following the 2- AACt method. RNA expression levels are normalized to the mean expression of housekeeping genes (GAPDH and Tubulin). The primer pairs used for qPCR are: FTO (forward: CGAGAGCGCGAAGCTAAGAA; SEQ ID NO: 3; reverse: CAGCTGCCACTGCTGATAGA; SEQ ID NO: 4), GAPDH (forward: TGCACCACCAACTGCTTAGC; SEQ ID NO: 5; reverse: GGCATGGACTGTGGTCATGAG; SEQ ID NO: 6), Tubulin (forward: AGCAGCCTCATCTGTTGGAC; SEQ ID NO: 7; reverse: GCACAAGGGAAGCTGGAGAT; SEQ ID NO: 8). Decision Making

From RNA-seq, decision is taken from a predefined expression threshold for FTO set on a cohort of COVID-19 non-hospitalized patients. If FTO count of a disease sample is lower than this threshold, then this patient is at higher risk to develop a severe COVID-19 and to be hospitalized.

From RT-qPCR, decision is taken from a predefined expression threshold for FTO set on a cohort of COVID-19 non-hospitalized patients. If FTO cycle threshold (CT) value of a disease sample's is higher than this threshold, then this patient is at higher risk to develop a severe COVID-19 and to be hospitalized.

Claims

1. A method for determining or predicting the severity of a disease caused by coronavirus infection in a subject, the method comprising measuring the quantity and/or activity of fat mass and obesity associated (FTO) in a sample from the subject.

2. The method according to claim 1, comprising the steps of:

(a) measuring the quantity and/or activity of FTO in the sample from the subject;

(b) comparing the quantity and/or activity of FTO as measured in (a) with a reference value of FTO quantity and/or activity, said reference value representing a known severity status of the disease caused by coronavirus infection in a reference subject;

(c) finding a deviation or no deviation of the quantity and/or activity of FTO as measured in (a) from said reference value; and

(d) attributing said finding of deviation or no deviation to a particular determination of the severity of the disease caused by coronavirus infection.

3. The method according to claim 1 or 2, whereby the subject is categorised as asymptomatic or mild symptomatic, or as severe symptomatic requiring hospitalisation or intensive care treatment.

4. The method according to claim 1 or 2, wherein the subject is categorised as not having or not being at risk of developing a severe or critical form of the disease, or as having or being at risk of developing a severe or critical form of the disease.

5. The method according to any one of claims 1 to 4, wherein the determination or prediction of the severity of the disease in the subject allows to select a therapeutic treatment for the subject.

6. A method for monitoring clinical progression of a disease caused by coronavirus infection in a subject, the method comprising measuring the quantity and/or activity of FTO in samples from the subject obtained at two or more different time points and comparing the FTO quantity and/or activity measured at the different time points.

7. The method according to claim 6, comprising the steps of:

(a) measuring the quantity and/or activity of FTO in the sample from the subject wherein the samples are obtained at a first time point and at one or more later time points; (b) comparing the quantity and/or activity of FTO as measured in (a) in the one or more samples obtained at the later time points with the quantity and/or activity of FTO as measured in the sample of the subject obtained at the first time point;

(c) finding a deviation or no deviation of the quantity and/or activity of FTO as measured in (a) from said sample obtained at the first time point; and

(d) attributing said finding of deviation or no deviation to a particular clinical progression of the disease caused by coronavirus infection.

8. A method for assessing the efficacy of a therapeutic treatment of a disease caused by coronavirus infection in a subject, the method comprising measuring the quantity and/or activity of FTO in a sample from the subject obtained at a time point before the start of the treatment and at one or more time points after the start of the treatment, and comparing the FTO quantity and/or activity measured at the different time points.

9. The method of claim 8, comprising the steps of:

(a) measuring the quantity and/or activity of FTO in the sample from the subject wherein the samples are obtained at a first time point before the start of the treatment and at one or more time points after the start of the treatment;

(b) comparing the quantity and/or activity of FTO as measured in (a) in the one or more samples obtained at different time points after the start of treatment with the quantity and/or activity of FTO as measured in the sample obtained before the start of the treatment;

(c) finding a deviation or no deviation of the quantity and/or activity of FTO as measured in (a) from said first sample obtained before the start of the treatment; and

(d) attributing said finding of deviation or no deviation to a particular efficacy of the therapeutic treatment.

10. The method according to any one of claims 1 to 9, wherein at least the quantity of FTO in the sample or samples is measured.

11 The method according to any one of claims 1 to 10, wherein the quantity of FTO mRNA in the sample or samples is measured, preferably by RNA sequencing or quantitative RT-PCR.

12. The method according to any one of claims 10 or 11, wherein comparatively reduced quantity and/or activity of FTO indicates greater severity of the disease.

13. The method of any one of the claims I to 12, wherein the coronavirus is a p-coronavirus, preferably a Sarbecovirus.

14. The method of any one of the claims 1 to 13, wherein the coronavirus is selected from the Middle

East respiratory syndrome-related coronavirus (MERS-CoV), the Severe Acute Respiratory virus (SARS- CoV), the Severe Acute Respiratory 2 virus (SARS-CoV-2) or a SARS-CoV-2 mutant virus, preferably wherein the coronavirus is the SARS-CoV-2 virus or a SARS-CoV-2 mutant virus.

15. The method of any one of the claims I to 14, wherein the subject is a mammal, preferably a human.

16. The method according to any one of claims 1 to 15, wherein the sample is selected from a blood- derived sample, a saliva sample, a naso-pharyngeal swab sample, an oropharyngeal swab sample, a bronchoalveolar lavage sample or a sputum sample.

17. The method according to any one of claims 1 to 16, wherein the sample is a naso-pharyngeal swab sample.