WO2022271201A2

WO2022271201A2 - Ace2 and tmprss2 gene expression as predictive markers of covid-19 severity

Info

Publication number: WO2022271201A2
Application number: PCT/US2022/011532
Authority: WO
Inventors: Sotiris Nikolopoulos; Michael PAPACHARLAMBOUS; Georgette ALITHINOS
Original assignee: International Advantage, Inc.
Priority date: 2021-01-07
Filing date: 2022-01-07
Publication date: 2022-12-29
Also published as: WO2022271201A3

Abstract

A method of determining a prognosis of a subject with a COVID-19 infection is provided, comprising measuring the expression levels of ACE2 and TMPRSS2 nucleic acids or fragments thereof and one or more control nucleic acids or fragments thereof in a sample from the subject, comparing the expression levels of the nucleic acids, and determining the prognosis of COVID-19 infection in the subject based on altered expression of ACE2 and TMPRSS2 compared to the expression level of the one or more control nucleic acids, wherein an increase in expression is indicative that the COVID-19 infection is likely to be severe. When the COVID-19 infection is likely to be severe, the method also involves treating the subject with a therapeutically effective amount of a corticosteroid, a monoclonal antibody-based therapy, and/or an antiviral agent. Diagnostic kits and point-of-care devices for predicting the prognosis of a COVID-19 infection are also provided.

Description

ACE2 AND TMPRSS2 GENE EXPRESSION AS PREDICTIVE MARKERS

OF COVID-19 SEVERITY

FIELD OF THE INVENTION

The present invention relates to methods and kits for measuring ACE2 and TMPRSS2 gene expression to identify individuals at high-risk for developing a severe COVID-19 infection.

BACKGROUND OF TIΪE INVENTION COVID-19 (Coronavirus Disease 2019) is an infectious disease caused by Severe Acute Respiratory Symptom Coronavirus 2 (SARS-Cov2; Chikhale et al., 2020). Common symptoms of COVID-19 include fever, cough, and difficulty breathing. In the majority of cases, patients infected with COVID-19 experience mild to moderate symptoms that do not require hospitalization. However, in severe cases, COVID-19 can cause pneumonia, severe acute respiratory syndrome, multiple organ failure, and death. COVID-19 was declared a pandemic by the World Health Organization (WHO) because of the global spread of SARS- CoV-2 and the millions of deaths caused by COVID-19.

The present invention generally addresses the need for diagnostic assays to determine the prognosis of patients with COVID-19. In particular, the present invention is directed to methods and kits for measuring A CE2 and TMPRSS2 gene expression to identify individuals at high-risk for developing a severe COVID-19 infection.

SUMMARY OF THE INVENTION

To address the foregoing problems, in whole or in part, and/or other problems that may have been observed by persons skilled in the art, the present disclosure provides compositions and methods as described by way of example as set forth below'.

In one embodiment, the present invention relates to a method of determining a prognosis of a subject with a COVID-19 infection comprising the steps of: a) measuring the expression level of two or more target nucleic acids and one or more control nucleic acids in a test sample from the subject, wherein the two or more target nucleic acids comprise ACE2 and TMPRSS2 nucleic acids or fragments thereof, and wherein the one or more control nucleic acids comprise GAPDH, ACTB, and/or RPL13A nucleic acids or fragments thereof; b) comparing the expression level of the two or more target nucleic acids to the expression level of the one or more control nucleic acids; c) determining the prognosis of COVID-19 infection in the subject based on altered expression of the two or more target nucleic acids as compared to the expression level of the one or more control nucleic acids, wherein an increase in expression is indicative that the COVID-19 infection is likely to be severe; and d) when the COVID-19 infection is likely to be severe, treating the subject with a therapeutically effective amount of a corticosteroid, a monoclonal antibody- based therapy, and/or an antiviral agent.

In some aspects, step (a) further comprises the steps of: i) contacting the sample with a primer that specifically hybridizes to the ACE2, TMPRSS2 , GAPDH, ACTB, and/or RPL13A nucleic acid or fragment thereof; li) amplifying the ACE2, TMPRSS2, GAPDH, ACTB , and/or RPL13A nucleic acid or fragment thereof to produce an ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid amplification product, iii) sequencing the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid amplification product; and tv) detecting the expression level of the ACE2, TMPRSS2 , GAPDH, ACTB, and/or RPL13A nucleic acid.

In other aspects, the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid is mRNA. In further aspects, the sample is selected from the group consisting of blood, serum, plasma, urine, and saliva. In still further aspects, the invention relates to a diagnostic kit for determining the prognosis of a subject with a COVID-19 infection, comprising means for earning out the method along with instructions for use.

In another embodiment, the present invention relates to a point-of-care device for determining a prognosis of a subject with a COVID-19 infection, comprising: a) a housing: b) a power supply disposed within the housing; c) a memory disposed within the housing; d) a user interface attached to or integrated into the housing; e) one or more communication interfaces disposed within, attached to, or integrated into the housing; and f) a test cartridge interface disposed within, attached to, or integrated into the housing, wherein the test cartridge is configured to: i) measure the expression level of two or more target nucleic acids and one or more control nucleic acids in a test sample from the subject, wherein the two or more target nucleic acids comprise A CE2 and TMPRSS2 or fragments thereof, and wherein the one or more control nucleic acids comprise GAPDH, ACTB , and/or RPL13A nucleic acids or fragments thereof; ii) compare the expression level of the two or more target nucleic acids to the expression level of the one or more control nucleic acids; and iii) produce a report via the user interface that indicates: aa) the expression level of the two or more target nucleic acids compared to the expression level of the control nucleic acids, wherein an increase in expression is indicative that the COVID-19 infection is likely to be severe; and bb) a treatment plan, wherein when the COVID-19 infection is likely to be severe, the treatment plan comprises treating the subject with a therapeutically effective amount of a corticosteroid, a monoclonal antibody -based therapy, and/or an antiviral agent.

In some aspects, step (f)(i) further comprises the steps of: aa) contacting the sample with a primer that specifically hybridizes to the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid; bb) amplifying the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid to produce mACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid amplification product: cc) sequencing the ACE2 , TMPRSS2 , GAPDH , ACTB, and/or RPL13A nucleic acid amplification product; and dd) detecting the expression level of the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid.

In other aspects, the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid is mRNA. In further aspects, the sample is selected from the group consisting of blood, serum, plasma, urine, and saliva.

Additional features of the invention will be or will become apparent to one with skill in the art upon examination of the following figures and detailed description. It is intended that all such additional features and advantages be included within this description, be within the scope of the invention, and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the subject matter of the present invention in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale, and wherein:

FIG. 1 is a schematic diagram showing how the attachment protein "spike" of SARS- CoV-2 uses the host cell receptor ACE2 for its attachment and the transmembrane protease TMPRSS2 for its activation.

FIG. 2 shows real-time PCR data from blood samples collected from two different blood donors. FIG. 2A show's the DNA synthesis and quantitation curves that correspond to ACE2 and TMPRSS2 expression. FIG. 2B show's the melt curves that correspond to the expected and correct product for each gene examined. A ~250 fold difference in the expression of both ACE2 and TMPRSS2 was observed between the two blood samples.

FIG. 3 is a graphical representation of ACE2 (FIG. 3 A) and TMPRSS2 (FIG. 3B) gene expression distribution in different blood samples. Each dot represents the gene expression level for an individual. The y axis is in logarithmic scale. A more than 4000-fold difference in ACE2 and TMPRSS2 expression among different individuals was observed.

FIG. 4 is a graphical representation of ACE2 (FIG. 4A) and TMPRSS2 (FIG. 4B) gene expression distribution in blood samples from different individuals.

DETAILED DESCRIPTION

The subject matter of the present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the subject matter of the present invention are shown. Like numbers refer to like elements throughout. The subject matter of the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure wall satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the subject matter of the present invention set forth herein will come to mind to one skilled in the art to which the subject matter of the present invention pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. All illustrations of the drawings are for the purpose of describing selected versions of the present invention and are not intended to limit the scope of the present invention. Therefore, it is to be understood that the subject matter of the present invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

ACE 2 And TMPRSS2 Gene Expression as Predictive Markers of COVID-19 Severity

The present invention relates to methods and kits for measuring A CE2 and TMPRSS2 gene expression to identify individuals at high-risk for developing a severe COVID-19 infection.

Since the end of 2019, public health has been in serious threat because of SARS- CoV-2 (Chikhale et al., 2020). COVID-19 was declared a pandemic by the World Health Organization (WHO) because of the global spread of SARS-CoV-2 and the millions of deaths caused by this disease.

The SARS-CoV-2 virus continues to mutate and present new variants in the human population. A lineage is a genetically closely related group of virus variants derived from a common ancestor. A variant has one or more mutations that differentiate it from other variants of the SARS-CoV-2 viruses. Multiple variants of SARS-CoV-2 have been documented in the United States and globally throughout this pandemic. The U.S. government SARS-CoV-2 Interagency Group (SIG) has classified the following SARS-CoV- 2 variants: Alpha (B.1.1.7 and Q lineages); Beta (B.1.351 and descendant lineages); Gamma (P.1 and descendent lineages); Delta (B.1.617.2 and AY lineages); Epsilon (B.1.427 and B.1.429); Eta (B.1.525); Iota (B.1.526); Kappa (B.l.617.1); 1.617.3; Mu (B.1.621,

B.1.621.1); Omicron (B.1.1.529 and BA lineages); and Zeta (P.2) (see www.cdc.gov/coronavirus/2019-ncov/variants/variant-classifications.html, last accessed Jan. 4, 2022). Accordingly, throughout the specification and claims, reference to SARS-CoV-2 includes all variants of SARS-CoV-2 and reference to a COVID-19 infection includes any infection caused by a SARS-CoV-2 variant.

Genome analysis has revealed that SARS-CoV-2 is distinguished from other coronaviruses in that SARS-CoV -2 exhibits high affinity for the Angiotensin Converting Enzyme 2 (ACE2) receptor and a cleavage site at the S1/S2 spike junction that determines infectivity and host range (Andersen et al, 2020). Research has also confirmed that SARS- CoV-2 uses the ACE2 receptor and the protease Transmembrane Serine Protease 2 (TMPRSS2) for host cell entry (Stopsack et al, 2020; FIG. 1).

The ACE2 receptor on host cells acts as an entry point for SARS-CoV-2 (Choudhary et al., 2021). Thus, it can be considered that the ACE2 gene is a genetic risk factor for viral infection. However, its role is not limited there, as it is a key component of the Renin- Angiotensin System (RAS) which plays crucial role in maintaining blood pressure homeostasis (Kuba et al., 2006). Moreover, ACE2 activity is critical for a variety of diseases, such as hypertension, diabetes, etc. Loss of ACE2 expression helps the development and the progression of a disease, whereas increased expression may benefit the treatment (Tikellis & Thomas, 2012). Another important function of ACE2 is the reduction of oxidati v e stress (Gopallawa & Uhal, 2014).

The TMPRSS2 gene encodes a protein of the serine protease family. This protein is involved in many physiological and pathological processes. Nowadays, one of the most interesting and well-studied functions of TMPRSS2 is that facilitates the entry of many viruses, including SARS-CoV-2, into host cells by activating viral proteins (Baughn et al,, 2020). Specifically, TMPRSS2 is employed by SARS-CoV-2 for spike (S) protein priming in order to enter into host cells (Wang et al., 2020). TMPRSS2 expression is essential for viral spread and pathogenesis (Zarubin et al., 2021; Hoffmann et al, 2020). After being activated, vims binds to the ACE2 receptor.

Many studies have revealed the high range of ACE2 and TMPRSS2 expression among people (Wang et al., 2020), trying to correlate them with age, sex, smoking and other pathological states or even ethnicity that makes people more susceptible to infection (Piva et al., 2.021). Iwata-Yoshikawa and co-workers studied TMPRS S2-deficient mice and observed that lack of TMPRSS2 affected the start of the infection and the viral spread (Iwata- Yoshikawa et al., 2019). Another study in cell lines revealed that low level of TMPRSS2 expression accompanied by reduced infection and replication of human coronaviruses (Shirato et al., 2018). Sharif- Askari and colleagues (2020) studied the ACE2 and TMPRSS2 expression levels of children and adults in order to explain why children are less susceptible to SARS-CoV-2 infection (Sharif- Askari et al., 2020). The results revealed that the expression levels of both receptors were lower in nasal and bronchial epithelial tissue of children compared with those of adults. It was hypothesized that the high range in ACE2 and TMPRSS2 expression may be associated with the wide variance in the illness’ severity caused by SARS-CoV-2.

As described more fully in the Examples section below, it was discovered that the gene expression of ACE2 and TMPRSS2 in blood samples by quantitative real-time PCR (qRT-PCR) showed that ACE2 and TMPRSS2 gene expression differs significantly among different individuals. The present inventors applied this discovery to develop a fast and reliable molecular assay based on this gene expression analysis which helps to determine individuals who are at high risk for developing severe COVlD-19 infection. Specifically, the present invention provides a method of identifying individuals who are at high-risk for severe COVID-19 infection by determining the level of expression from the following gene sequences in a test sample (e.g., a blood sample):

1. ACE2 (Angiotensin-Converting Enzyme 2)

2. TMPRSS2 (Transmembrane Serine Protease 2)

3. GAPDH (Glyceraldehyde-Phosphate Dehydrogenase)

The level of expression of each gene may be determined by measuring the level of mRNA. Methods of detecting mRNA concentrations are described in the Definitions section below and are also well-known in the art. For example, the level of expression may be determined by real time PCR. In one embodiment, the assay utilizes double-stranded binding dyes such as SYBR™ Green I.

The quantifying step may also be performed by generating complementary DNA from the isolated RNA. From the complementary DNA that is generated, the genes for ACE2 and TMPRSS2 (or a fragment thereof) are amplified. The amplified product is then quantified.

The quantity of amplified product is correlated with the risk of COVID-19 severity' in the event that the subject does have or would have COVID-19.

A further aspect of the invention provides an assay kit comprising means for specifically determining the level of expression of three or more genes in the blood sample selected, wherein the three or more genes comprise ACE2, TMPRSS2, and GAPDH. The assay kit comprises means to specifically determine the level of expression from A CE2 and TMPRSS2 along with the housekeeping gene GADPH as a control. The housekeeping gene may also be ACTB (β-actin) or RPL13A (Ribosomal Protein L13a). The kit may comprise means for detecting the level of expression of the forementioned genes with a pair of primers specific for each gene for use in real time PCR.

Accordingly, the present invention relates to a method of determining a prognosis of a subject with a COVlD-19 infection comprising the steps of: a) measuring the expression level of two or more target nucleic acids and one or more control nucleic acids in a test sample from the subject, wherein the two or more target nucleic acids comprise A CE2 and TMPRSS2 nucleic acids or fragments thereof, and wherein the one or more control nucleic acids comprise GAPDH, ACTB, and/or RPL13A nucleic acids or fragments thereof; b) comparing the expression level of the two or more target nucleic acids to the expression level of the one or more control nucleic acids; c) determining the prognosis of COVID-19 infection in the subject based on altered expression of the two or more target nucleic acids as compared to the expression level of the one or more control nucleic acids, wherein an increase in expression is indicative that the COVID-19 infection is likely to be severe; and d) when the COVID-19 infection is likely to be severe, treating the subject with a therapeutically effective amount of a corticosteroid, a monoclonal antibody- based therapy, and/or an antiviral agent.

In some aspects, step (a) further comprises the steps of: i) contacting the sample with a primer that specifically hybridizes to the ACE2 , TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid; ii) amplifying the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid to produce an ACE2, TMPRSS2 , GAPDH, ACTB, and/or RPL13A nucleic acid amplification product; hi) sequencing the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic add amplification product; and iv) detecting the expression level of the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid.

In other aspects, the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid is mRMA In further aspects, the sample is selected from the group consisting of blood, serum, plasma, urine, and saliva, in still further aspects, the invention relates to a diagnostic kit for determining the prognosis of a subject with a COVID-19 infection, comprising means for carrying out the method along with instructions for use.

In another embodiment, the present invention relates to a point-of-care device for determining a prognosis of a subject with a COVID-19 infection, comprising: a) a housing; b) a power supply disposed within the housing; c) a memory disposed within the housing; d) a user interface attached to or integrated into the housing; e) one or more communication interfaces disposed within, attached to, or integrated into the housing; and f) a test cartridge interface disposed within, attached to, or integrated into the housing, wherein the test cartridge is configured to: i) measure the expression level of two or more target nucleic acids and one or more control nucleic acids in a test sample from the subject, wherein the two or more target nucleic acids comprise ACE2 and TMPRSS2 nucleic acids or fragments thereof or fragments thereof, and wherein the one or more control nucleic acids comprise GAPDH, ACTB , and/or RPL13A nucleic acids or fragments thereof; ii) compare the expression level of the two or more target nucleic acids to the expression level of the one or more control nucleic acids; and iii) produce a report via the user interface that indicates: aa) the expression level of the two or more target nucleic acids compared to the expression level of the control nucleic acids, wherein an increase in expression is indicative that the COVID-19 infection is likely to be severe; and bb) a treatment plan, wherein when the COVID-19 infection is likely to be severe, the treatment plan comprises treating the subject with a therapeutically effective amount of a corticosteroid, a monoclonal antibody-based therapy, and/or an antiviral agent.

In some aspects, step (f)(i) further comprises the steps of: aa) contacting the sample with a primer that specifically hybridizes to the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid; bb) amplifying the ACE2, TMPRSS2 , GAPDH, ACTB, and/or RPL13A nucleic acid to produce an ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid amplification product; cc) sequencing the ACE2 , TMPRSS2 , GAPDH, ACTB, and/or RPL13A nucleic acid amplification product; and dd) detecting the expression level of the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid.

In other aspects, the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid is rnRNA. In further aspects, the sample is selected from the group consisting of blood, serum, plasma, urine, and saliva.

Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide one skilled in the art with a general guide to many of the terms used in the present application: Allen et al., Remington: The Science and Practice of Pharmacy 22^nded., Pharmaceutical Press (Sep. 15, 2012); Homyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3 ^rded., revised ed., J. Wiley & Sons (New York, N.Y. 2006); Smith, March 's Advanced Organic Chemistry Reactions , Mechanisms and Structure 7^th ed., J. Wiley & Sons (New York, N.Y. 2013);

Singleton, Dictionary ofDNA and Genome Technology 3^rd ed., Wiley-Blackwell (Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed.. Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. 2012). For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2^nded.. Cold Spring Harbor Press (Cold Spring Harbor N.Y., 2013); Kohler and Milstein, Deri vation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J Immunol. (1976), 6(7):511-9; and Riechmann et al, Reshaping human antibodies for therapy, Nature (1988), 332(6162 ): 323-7.

Nucleic acids and proteins can be detected within the methods of the present invention using various techniques known in the art. For example, genes and mRNA can be detected by genotyping assays, polymerase chain reaction (PCR), Reverse transcription PCR, real-time PCR, microarray, DNA sequencing, and/or RNA sequencing techniques. Proteins can be detected by various techniques such as immunohistochemistry, Western blots, and/or protein arrays. In various embodiments, the detection agents are oligonucleotide probes, nucleic acids, DNAs, RNAs, aptamers, peptides, proteins, antibodies, or small molecules, or a combination thereof. In various embodiments, microarrays are used to detect the expression of certain genes, in some embodiments, the microarray is an oligonucleotide microarray,

DNA microarray, cDNA microarrays, RNA microarray, peptide microarray, protein microarray, or antibody microarray, or a combination thereof.

As used herein, the term “nucleic acid” or “polynucleotide” generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. As used herein, the term “oligonucleotide” refers to a relatively short polynucleotide. This includes, without limitation, single-stranded deoxynbonucleotides, single- or double-stranded ribonucleotides, RNA:DNA hybrids and double-stranded DNAs.

As used herein, the term “fragment” or “polynucleotide fragment” means a polynucleotide of reduced length relative to a reference polynucleotide and comprising, over the common portion, a nucleotide sequence identical to that of the reference polynucleotide. Such a polynucleotide fragment may he, where appropriate, included in a larger polynucleotide of which it is a constituent. Such polynucleotide fragments comprise, or alternatively consist of, polynucleotides ranging in length from at least 6, 8, 9, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51, 54, 57, 60, 63, 66, 70, 75, 78, 80, 90,

100, 105, 120, 135, 150, 200, 300, 500, 720, 900, 1000 or 1500 consecutive nucleotides of a reference polynucleotide. Polynucleotide fragments include, for example, DNA fragments and RN A fragments.

As used herein, the term “primer” denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence. A primer serves as an initiation point for nucleotide polymerization catalyzed by DNA polymerase, RNA polymerase, or reverse transcriptase.

As used herein, the term “probe” denotes a defined nucleic acid segment which can be used to identify a specific polynucleotide sequence present in samples, wherein the nucleic acid segment comprises a nucleotide sequence complementary to the specific polynucleotide sequence to he identified.

As used herein, the terms “complementary'” or “complement thereof refer to the sequences of polynucleotides that are capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety- of the complementary region. For the purpose of the presently disclosed subject matter, a first polynucleotide is deemed to be complementary' to a second polynucleotide when each base in the first polynucleotide is paired with its complementary base. Complementary bases are, generally, A and T (or A and U), or C and G. “Complement” is used herein as a synonym from “complementary polynucleotide,” “complementary nucleic acid” and “complementary nucleotide sequence”. These terms are applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.

As used herein, the terms “amplify,” “amplification,” “nucleic acid amplification,” or the like, refer to the production of multiple copies of a nucleic acid template (e.g., a template DNA molecule), or the production of multiple nucleic acid sequence copies that are complementary to the nucleic acid template (e.g., a template DNA molecule).

As used herein, the term “level of expression” of a biomarker refers to the amount of biomarker detected. Levels of biomarker can be detected at the transcriptional level, the translational level, and the post-translational level, for example. “mRNA expression levels” refers to the amount of mRNA detected in a sample and “protein expression levels” refers to the amount of protein detected in a sample.

As used herein, the term “array” or “microarray” refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes, on a substrate.

As described above, assaying mRNA comprises using RNA sequencing, northern blot, in situ hybridization, hybridization array, serial analysis of gene expression (SAGE), reverse transcription PCR, real-time PCR, real-time reverse transcription PCR, quantitative PCR, or microarray, or a combination thereof. For example, mRNA may be detected by contacting the biological sample with polynucleotide probes capable of specifically hybridizing to mRNA of two or more target genes and thereby forming probe-target hybridization complexes. Hybridization-based RNA assays include, but are not limited to, traditional “direct probe” methods such as, northern blot or in situ hybridization. The methods can be used in a wide variety of formats including, but not limited to, substrate (e.g. membrane or glass) bound methods or array-based approaches. In a typical in situ hybridization assay, cells are fixed to a solid support, typically a glass slide. If a nucleic acid is to be probed, the cells are typically denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of labeled probes specific to the nucleic acid sequence encoding the protein. The targets (e.g., cells) are then typically washed at a predetermined stringency or at an increasing stringency until an appropriate signal to noise ratio is obtained. The probes are typically labeled, e.g., with radioisotopes or fluorescent reporters to enable detection and analysis with a variety of imaging equipment. Preferred probes are sufficiently long so as to specifically hybridize with the target nucleic acid(s) under stringent conditions. The preferred size range is from about 200 bases to about 1000 bases.

Also as described above, assaying a protein comprises using western blot, enzyme- linked immunosorbent assay (ELISA), radioimmunoassay, or mass spectrometry, or a combination thereof. For example, assaying a protein may comprise contacting the biological sample with antibodies capable of specifically binding to a target protein and thereby forming antigen-antibody complexes. Antibodies, both polyclonal and monoclonal, can be produced by a skilled artisan either by themselves using well known methods or they can be manufactured by sendee providers who specialize making antibodies based on known protein sequences. For example, production of monoclonal antibodies can be performed using the traditional hybridoma method by first immunizing mice with an antigen which may be an isolated protein of choice or fragment thereof and making hybridoma cell lines that each produce a specific monoclonal antibody. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen using, e.g., ELISA or Antigen Microarray Assay, or immuno-dot blot techniques. The antibodies that are most specific for the detection of the protein of interest can be selected using routine methods and using the antigen used for immunization and other antigens as controls. The antibody that most specifically detects the desired antigen and protein and no other antigens or proteins are selected for the processes, assays and methods described herein. The best clones can then be grown indefinitely in a suitable cell culture medium. They can also be injected into mice (in the peritoneal cavity, surrounding the gut) where they produce an antibody-rich ascites fluid from winch the antibodies can be isolated and purified. The antibodies can be purified using techniques that are well known to one of ordinary skill in the art.

Furthermore, the antibodies can he labeled. In some embodiments, the detection antibody is labeled by covalently linking to an enzyme, label with a fluorescent compound or metal, label with a chemiluminescent compound. For example, the detection antibody can be labeled with catalase and the conversion uses a colorimetric substrate composition comprises potassium iodide, hydrogen peroxide and sodium thiosulphate; the enzyme can be alcohol dehydrogenase and the conversion uses a colorimetric substrate composition comprises an alcohol, a pH indicator and a pH buffer, wherein the pH indicator is neutral red and the pH buffer is glycine-sodium hydroxide; the enzyme can also be hypoxanthine oxidase and the conversion uses a colorimetric substrate composition comprises xanthine, a tetrazolium salt and 4, 5-dihydroxy- 1,3-benzene disuiphonic acid. In one embodiment, the detection antibody is labeled by covalently linking to an enzyme, label with a fluorescent compound or metal, or label with a chemiluminescent compound.

Direct and indirect labels can be used in immunoassays. A direct label can be defined as an entity, which in its natural state, is visible either to the naked eye or with the aid of an optical filter and/or applied stimulation, e.g., ultraviolet light, to promote fluorescence. Examples of colored labels which can be used include metallic sol particles, gold sol particles, dye sol particles, dyed latex particles or dyes encapsulated in liposomes. Other direct labels include radionuclides and fluorescent or luminescent moieties, indirect labels such as enzymes can also be used according to the invention. Various enzymes are known for use as labels such as, for example, alkaline phosphatase, horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactate dehydrogenase and urease.

The antibody can be attached to a surface. Examples of useful surfaces on which the antibody can be attached for the purposes of detecting the desired antigen include nitrocellulose, PVDF, polystyrene, and nylon.

The terms “patient,” “individual,” or “subject” are used interchangeably herein, and refer to a mammal, particularly, a human. A “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. The patient may have mild, intermediate or severe disease. The patient may be treatment naive, responding to any form of treatment, or refractory. The patient may be an individual in need of treatment or in need of diagnosis based on particular symptoms or family history, in some cases, the terms may refer to treatment in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; and primates. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bo vines, e.g., cattle, oxen, and the like; ovmes, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects.

The terms “sample,” “patient sample,” “biological sample,” and the like, encompass a variety of sample types obtained from a patient, individual, or subject and can be used in a diagnostic or monitoring assay. The patient sample may be obtained from a healthy subject or a diseased patient. Moreover, a sample obtained from a patient can be di vided and only a portion may be used for diagnosis. Further, the sample, or a portion thereof, can be stored under conditions to maintain sample for later analysis. The definition specifically encompasses blood and other liquid samples of biological origin (including, but not limited to, peripheral blood, serum, plasma, cerebrospinal fluid, urine, saliva, stool, and synovial fluid), solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. In a specific embodiment, a sample comprises a blood sample, in another embodiment, a serum sample is used. The definition also includes samples that have been manipulated in any way after their procurement, such as by centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washed, or enriched for certain cell populations. The terms further encompass a clinical sample, and also include cells in culture, cell supernatants, tissue samples, organs, and the like. Samples may also comprise fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks, such as blocks prepared from clinical or pathological biopsies, prepared for pathological analysis or study by immunohistochemistry.

A “suitable control,” “appropriate control” or a “control sample” is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a “suitable control” or “appropriate control” is a value, level, feature, characteristic, property, and the like, determined in a cell, organ, or patient, e.g., a control or normal cell, organ, or patient, exhibiting, for example, normal traits.

In accordance with the invention, therapeutic agents may be administered using the appropriate modes of administration, for instance, the modes of administration recommended by the manufacturer for each of the therapeutic agents, in accordance with the invention, various routes may be utilized to administer the therapeutic agent of the claimed methods, including but not limited to aerosol, nasal, via inhalation, oral, transmucosal, transdermal, parenteral, enteral, topical or local. “Parenteral” refers to a route of administration that is generally associated with injection, including intracranial, intraventricular, intrathecal, epidural, intradural, intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, therapeutic agents may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders. Via the enteral route, the therapeutic agents can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release. Via the topical route, the therapeutic agents can he in the form of aerosol, lotion, cream, gel, ointment, suspensions, solutions, or emulsions. In accordance with the present invention, "administering” can be self- administering.

Typical dosages of a therapeutically effective amount of a therapeutic agent disclosed herein can be in the ranges recommended by the manufacturer where known therapeutic molecules or compounds are used, and also as indicated to the skilled artisan by the in vitro responses in cells or in vivo responses in animal models. Such dosages typically can be reduced by up to about an order of magnitude in concentration or amount without losing relevant biological activity. The actual dosage can depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of relevant cultured cells or histocultured tissue sample, or the responses observed in the appropriate animal models. In various embodiments, the therapeutic agent may be administered once a day (SID/QD), twice a day (BID), three times a day (TID), four times a day (QID), or more, so as to administer an effective amount of the therapeutic agent to the subject, where the effective amount is any one or more of the doses described herein.

In various embodiments, the therapeutic agent is administered at about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 mg/kg, or a combination thereof.

In various embodiments, the effecti v e amount of the therapeutic agent is any one or more of about 0.001-0.01, 0.01-0.1, 0 1-0.5. 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200- 300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 μg/kg/day, or a combination thereof. In various embodiments, the effective amount of the therapeutic agent is any one or more of about 0.001-0.01, 0.01-0.1, 0.1-0.5, 0.5-5, 5-10, 10-20, 20-50, 50-100, 100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, or 900-1000 mg/kg/day, or a combination thereof.

In some embodiments, the therapeutic agent may be administered at the prevention stage of a condition (i.e., when the subject has not developed the condition but is likely to or in the process to develop the condition). In other embodiments, the therapeutic agent may be administered at the treatment stage of a condition (i.e., when the subject has already developed the condition). To provide aspects of the present disclosure, embodiments may employ any number of programmable processing devices that execute software or stored instructions. Physical processors and/or machines employed by embodiments of the present disclosure for any processing or evaluation may include one or more networked (Internet, cloud, WAN, LAN, satellite, wired or wireless (RF, cellular, WiFi, Bluetooth, etc.)) or non-networked general purpose computer systems, microprocessors, filed programmable gate arrays (FPGAs), digital signal processors (DSPs), micro-controllers, smart devices (e.g., smart phones), computer tablets, handheld computers, and the like, programmed according to the teachings of the exemplary embodimentsI. In addition, the devices and subsystems of the exemplary embodiments can be implemented by the preparation of application-specific integrated circuits (ASICs) or by interconnecting an appropriate network of conventional component circuits. Thus, the exemplary embodiments are not limited to any specific combination of hardware circuitry and/or software.

Stored on any one or on a combination of computer readable media, the exemplary' embodiments of the present disclosure may include software for controlling the devices and subsystems of the exemplary embodiments, for driving the devices and subsystems of the exemplary embodiments, for enabling the devices and subsystems of the exemplary- embodiments to interact with a human user, and the like. Such software can include, but is not limited to, device drivers, firmware, operating systems, development tools, applications software, database management software, and the like. Computer code devices of the exemplary' embodiments can include any suitable interpretable or executable code mechanism, including hut not limited to scripts, interpretable programs, dynamic link libraries (DLLs), Java classes and applets, complete executable programs, and the like. Moreover, processing capabilities may be distributed across multiple processors for belter performance, reliability, cost, or other benefits.

Common forms of computer-readable media may include, for example, a floppy disk, a flexible disk, a hard disk, magnetic tape, any other suitable magnetic medium, a CD-ROM, CDRW, DVD, any other suitable optical medium, punch cards, paper tape, optical mark sheets, any other suitable physical medium with patterns of holes or other optically recognizable indicia, a RAM, a PROM, an EPROM, a FLASH-EPROM, any other suitable memory chip or cartridge, a carrier wave or any other suitable medium from which a computer can read. Such storage media can also be employed to store other types of data, e.g., data organized in a database, for access, processing, and communication by the processing devices.

Furthermore, terms and phrases used in this document, and variations thereof, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing: the term ‘including” should be read as mean “including, without limitation” or the like; the term “example” is used to provide exemplary instances of the item in discussion, not an exhaustive or limiting list thereof; and adjectives such as “conventional,” “traditional,” “standard,” “known” and terms of similar meaning should not be construed as limiting the item described to a given time period or to an item available as of a given time, but instead should be read to encompass conventional, traditional, normal, or standard technologies that may be available or known now' or at any time in the future. Likewise, a group of items linked with the conjunction “and” should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as “and/or” unless expressly stated otherwise. Similarly, a group of items linked with the conjunction “or” should not be read as requiring mutual exclusivity among that group, but rather should also be read as “and/or” unless expressly stated otherwise. Furthermore, although item, elements or components of the disclosure may be described or claimed in the singular, the plural is contemplated to he within the scope thereof unless limitation to the singular is explicitly stated. The presence of broadening w-ords and phrases such as “one or more,” “at least,” “but not limited to” or other like phrases in some instances shall not be read to mean that the narrower case Is intended or required in instances where such broadening phrases may be absent.

For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing amounts, sizes, dimensions, proportions, shapes, formulations, parameters, percentages, quantities, characteristics, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” even though the term “about” may not expressly appear with the value, amount, or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are not and need not be exact, but may be approximate and/or larger or smaller as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art depending on the desired properties sought to be obtained by the subject matter of the present invention. For example, the term “about,” when retelling to a value can be meant to encompass variations of, in some embodiments ± 100%, in some embodiments ± 50%, in some embodiments ± 20%, in some embodiments ± 10%, in some embodiments ± 5%, in some embodiments ± 1%, in some embodiments ± 0.5%, and in some embodiments ± 0.1% from the specified, amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries above and below the numerical values set forth. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

EXAMPLES

Example 1

The following study assessed the gene expression of ACE2 and TMPRSS2 in blood samples by quantitative real-time PCR (qRT-PCR), and demonstrated that .4CE2 and TMPRSS2 gene expression differs significantly among different individuals.

Blood Samples

Peripheral blood collection (1ml) was performed by an experienced nurse in tubes containing anti-coagulant (EDTA).

Gene Expression Analysis of Blood Samples

STEP 1 : RNA isolation

0.5 ml of the collected blood sample was mixed with RNAzol reagent in Eppendorf tubes. The isolation of RNA was performed according to the instructions of the manufacturer.

Successful blood sampling for this methodology yielded at least 3000ng RNA with excellent quality (A260/280 of 1.7 or higher).

STEP 2: cDNA Synthesis

The second step towards the gene-expression analysis was the conversion of the isolated RNA to complimentary DNA (cDNA). The last is performed by the use of a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), following exactly the instructions of the manufacturer. The final volume of the reaction was 20μl. STEP 3: Real-time PCR

The last step of the gene-expression analysis was real time PCR which was performed using Platinum® SYBR® Green qPCR SuperMix-UDG. This mixture contained SYBR® Green I fluorescent dye, Platinum® Taq DNA polymerase, Mg++, uracil-DNA glycosylase (UDG), proprietary stabilizers, and deoxyribonucleotide triphosphates (dNTPs), with dUTP instead of dTTP. The convenient SuperMix formulation delivered excellent sensitivity in the quantification of target sequences, with a linear dose response over a wide range of target concentrations. SYBR® Green I is a fluorescent dye that binds directly to double-stranded DNA (dsDNA). In qPCR, as dsDNA accumulates, the dye generates a signal that is proportional to the DNA concentration and that can be detected using real-time qPCR instruments. SYBR® Green I in this SuperMix formulation can quantify as few as 10 copies of a target gene in as little as 1 pg of template RNA. Platinum® Taq DNA polymerase is precomplexed with specific monoclonal antibodies that inhibit Taq DNA polymerase activity during reaction assembly at room temperature. Full polymerase activity is restored after the denaturation step in PCR cycling, providing an automatic hot start PCR. This significantly reduced nonspecific amplification and mispriming and increased amplification efficiency, sensitivity, and yield.

Reaction conditions for the real-time PCR reaction Cycling Program:

50°C for 2 min hold 95°C for 5 min hold 45 cycles of: 95°C, 15 s 58°C, 20 s 72°C, 30 s

After the end of the Cycling Program, a Melting Curve Analysis Program was performed in order to evaluate and confirm the correct real-time PCR products from their melting temperatures:

Melting Curve Analysis Program Ramp from 60 to 94 Rising by 0.2 degrees each step Wait for 90 seconds Wait for 1 second for each step afterwards STEP 4: Evaluation of Real-time PCR Results

From -90 blood samples analyzed, all of them were found to express both ACE2 and TMPRSS2 genes at various levels with the exception of one sample where no TMPRSS2 was detected and two samples where no ACE2 was detected. Results

FIG. 2 shows real-time PCR data from blood samples collected from two different blood donors. The DNA synthesis and quantitation curves that correspond to ACE2 and TMPRSS2 expression are shown in FIG. 2A. The melt curves that correspond to the expected and correct product for each gene examined are shown in FIG. 2B. A ~250 fold difference in the expression of both A CE2 and TMPRSS2 was observed between the two blood samples.

FIG. 3 is a graphical representation of ACE2 (FIG. 3A) and TMPRSS2 (FIG. 3B) gene expression distribution in different blood samples. Each dot represents the gene expression level for an individual. The y axis is in logarithmic scale. A more than 4000-fold difference in ACE2 and TMPRSS2 expression was observed among different individuals.

After studying the expression levels of ACE2 gene, the expression levels were further classified into five categories; very high (ΔCt < 8.5), high (8.5 < ΔCt < 10.5), moderate/high (10.5 < ΔCt < 12.5), moderate/low (12.5 < ΔCt < 14) and low (ΔCt > 14) risk group (FIG. 4A). Likewise, the expression levels of the TMPRSS2 gene were also classified into five categories; very high (ΔCt < 8), high (8 < ΔCt < 10), moderate/high (10 < ΔCt < 12), moderate/low (12 < ΔCt < 14) and low (ΔCt > 14) risk group (FIG. 4B). Low ACE2 and TMPRSS2 gene expression levels have been shown to be accompanied by less severe immunopathology (Sharif-Askari et al, 2020; Iwata- Yoshikaw'a et al., 2019). The majority of indi viduals tested had moderate gene expression levels, while a few' had either very high or low expression levels.

Conclusion

It was shown that the SARS-CoV-2 receptors ACE2 and TMPRSS2 were expressed at significantly higher levels in the blood of certain individuals. The observed differential level of expression of ACE2 and TMPRSS2 therefore provides a way of identifying the individuals that are prone to develop more severe symptoms and the individuals that will develop mild symptoms or will be asymptomatic upon COVID-19 infection.

REFERENCES

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2 Saheb Sharif-Askari N, Saheb Sharif-Askari F, Alabed M, et al. Airways Expression of SARS- CoV-2 Receptor, ACE2, and TMPRSS2 Is Lower in Children Than Adults and Increases with Smoking and COPD. Mol Ther Methods Clin Dev. 2020; 18: 1-6.

3. Bourgonje AR, Abdulle AE, Timens W, Hillebrands JL, Navis GJ, Gordijn SJ, Bolling MC, Dijkstra G, Voors AA, Osterhaus AD, van der Voort PH, Mulder DJ, van Goor H. Angiotensin- converting enzyme 2 (ACE2), SARS-CoV-2 and the pathophysiology of coronavirus disease 2019 (COVID-19). J Pathol. 2020 Jul;251(3):228-248.

4. Heurich A, Hofmann- Winkler H, Gierer S, Liepold T, Jahn O, Pohlmann S. TMPRSS2 and AD AM 17 cleave ACE2 differentially and only proteolysis by TMPRSS2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein. J Virol 2014 Jan;88(2): 1293-307.

5. Qiao Y, Wang XM, Mannan R, Pitchiaya S, Zhang Y, Wotring JW, Xiao L, Robinson DR, Wu YM, Tien JC, Cao X, Simko SA, Apel lJ, Bawa P, Kregel S, Narayanan SP, Raskind G, Ellison SJ, Parolia A, Zelenka-Wang S, McMurry L, Su F, Wang R, Cheng Y, Delekta AD, Mei Z, Pretto CD, Wang S, Mehra R, Sexton JZ, Chinnaiyan AM. Targeting transcriptional regulation of SARS-CoV-2 entry factors ACE2 and TMPRSS2. Proc Natl Acad Sci USA. 2020 Dec 11 :202021450.

6. Chikhale, R. V., Gupta, V. K., Eldesoky, G. E., Wabaidur, S. M., Patil, S. A., & Islam, M. A. (2020). identification of potential anti-TMPRSS2 natural products through homology modelling, virtual screening and molecular dynamics simulation studies. Journal ofBiomolecular Structure and Dynamics, 1-16.

7. Andersen, K. G., Rambaut, A., Lipkin, W. 1., Holmes, E. C, & Garry R. F. (2020). The proximal origin of SARS-CoV- 2. Nature medicine, 26(4), 450-452.

8. Stopsack, K. H., Mucci, L. A., Antonarakis, E. S., Nelson, P. S., & Kantoff, P. W. (2020). TMPRSS2 and COVID-19: serendipity or opportunity for intervention?. Cancer discovery, 10(6), 779-782.

9. Choudhary, S., Sreenivasulu, K., Mitra, P., Misra, S., & Sharma, P. (2021). Role of genetic variants and gene expression in the susceptibility and seventy of COVID-19. Annals of laboratory medicine, 41(2), 129-138.

10. Kuba, K., Imai, Y., & Penninger, J. M. (2006). Angiotensin-converting enzyme 2 in lung diseases. Current opinion in pharmacology. ·, 6(3), 271-276.

11. Tikellis, C., & Thomas, M. C. (2012). Angiotensin-converting enzyme 2 (ACE2) is a key modulator of the renin angiotensin system in health and disease. International journal of peptides, 2012. 12. Gopallawa, L, & Uhal, B. D. (2014). Molecular and cellular mechanisms of the inhibitory effects of ACE-2/ANG1- 7/Mas axis on lung injury. Current topics in pharmacology, 18(1), 71.

13. Baughn, L. B., Sharma, N., Elhaik, E., Sekulie, A., Bryce, A. H., & Fonseca, R. (2020, September). Targeting TMPRSS2 in SARS-CoV-2 infection. In Mayo Clinic Proceedings (Vol. 95, No. 9, pp. 1989-1999). Elsevier.

14. Wang, X., Dhindsa, R., Povysil, G., Zoghbi, A., Motelow, J., Hostyk, J., & Goldstein, D. (2020). Transcriptional inhibition of host viral entry proteins as a therapeutic strategy for SARS-CoV-2.

15. Piva, F., Sabanovic, B., Cecati, M., & Giulietti, M. (2021). Expression and co-expression analyses of TMPRSS2, a key element in COVID-19. European Journal of Clinical Microbiology & Infectious Diseases, 40(2), 451-455.

16. Iwata-Yoshikawa, N., Qkamura, T., Shimizu, Y., Hasegawa, H., Takeda, M., & Nagata, N. (2019). TMPRSS2 contributes to virus spread and immunopathology in the airways of murine models after coronavirus infection. Journal of virology, 93(6), e01815- 18.

17. Shirato, K., Kawase, M., & Matsuyama, S. (2018). Wild-type human coronaviruses prefer cell-surface TMPRSS2 to endosomal cathepsins for cell entry. Virology, 517, 9-15.

18. Sharif-Askari, N. S., Sharif- Askari, F. S., Alabed, M., Temsah, M. H., A1 Heialy, S., Hamid, Q., & Halwani, R. (2020). Airways expression of SARS-CoV-2 receptor, ACE2, and TMPRSS2 is lower in children than adults and increases with smoking and COPD. Molecular Therapy-Methods & Clinical Development, 18, 1-6. 19. Zarubin, A., Stepanov, V., Markov, A., Kolesnikov, N,, Marusin, A., Khitrinskaya, I., et al. (2021). Structural variability , expression profile, and pharmacogenetic properties of TMPRSS2 gene as a potential target for COVID-19 therapy. Genes, 12(1), 19.

All publications, patent applications, patents, and other references mentioned in the specification are indicative of the level of those skilled in the art to which the presently disclosed subject matter pertains. All publications, patent applications, patents, and other references are herein incorporated by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. It will be understood that, although a number of patent applications, patents, and other references are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.

Claims

1. A method of determining a prognosis of a subject with a COVID-19 infection comprising the steps of: a) measuring the expression level of two or more target nucleic acids and one or more control nucleic acids in a test sample from the subject, wherein the two or more target nucleic acids comprise ACE2 and TMPRSS2 nucleic acids or fragments thereof, and wherein the one or more control nucleic acids comprise GAPDH, ACTB, and/or RPL13A nucleic acids or fragments thereof; b) comparing the expression level of the two or more target nucleic acids to the expression level of the one or more control nucleic acids; c) determining the prognosis of COVID-19 infection in the subject based on altered expression of the two or more target nucleic acids as compared to the expression level of the one or more control nucleic acids, wherein an increase in expression is indicative that the COVID-19 infection is likely to be severe; and d) when the COVID-19 infection is likely to be severe, treating the subject with a therapeutically effective amount of a corticosteroid, a monoclonal antibody- based therapy, and/or an antiviral agent.

2. The method of claim 1, wherein: step (a) further comprises the steps of: i) contacting the sample with a primer that specifically hybridizes to the ACE2, TMPRSS2 , GAPDH, ACTB, and/or RPL13A nucleic acids; ii ) amplifying the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL 13A nucleic acids or fragments thereof to produce an ACE2 , TMPRSS2 , GAPDH, ACTB, and/or RPL13A nucleic acid amplification product; iii) sequencing the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid amplification product; and iv) detecting the expression level of the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acids.

3. The method of claim 2, wherein the ACE2, IMPREST, GAPDH, ACTB, and/or RPL13A nucleic acid is mRNA.

4. The method of claim 3, wherein the sample is selected from the group consisting of blood, serum, plasma, urine, and saliva.

5. A diagnostic kit for determining the prognosis of a subject with a COVID- 19 infection, comprising means for carrying out the method of any one of claims 1 to 4 along with instructions for use.

6. A point-of-care device for determining a prognosis of a subject with a COVID-19 infection, comprising: a) a housing; b) a power supply disposed within the housing; c) a memory disposed within the housing; d) a user interface attached to or integrated into the housing; e) one or more communication interfaces disposed within, attached to, or integrated into the housing; and f) a test cartridge interface disposed within, attached to, or integrated into the housing, wherein the test cartridge is configured to: i) measure the expression level of two or more target nucleic acids and one or more control nucleic acids in a test sample from the subject, wherein the two or more target nucleic acids comprise ACE2 and TMPRSS2 nucleic acids or fragments thereof, and wherein the one or more control nucleic acids comprise GAPDH, ACTB , and/or RPL13A nucleic acids or fragments thereof; ii) compare the expression level of the two or more target nucleic acids to the expression level of the one or more control nucleic acids; and iii) produce a report via the user interface that indicates: aa) the expression level of the two or more target nucleic acids or fragments thereof compared to the expression level of the control nucleic acids or fragments thereof, w herein an increase in expression is indicative that the COVID-19 infection is likely to be severe; and bb) a treatment plan, wherein when the COVID-19 infection is likely to be severe, the treatment plan comprises treating the subject with a therapeutically effecti ve amount of a corticosteroid, a monoclonal antibody -based therapy, and/or an antiviral agent.

7. The point-of-care device of claim 6, wherein: step (f)(i) further comprises the steps of: aa) contacting the sample with a primer that specifically hybridizes to the ACE2, TMPRSS2 , GAPDH, ACTB, and/or RPL13A nucleic acids or fragments thereof; bb) amplifying the ACE2 , TMPRSS2, GAPDH, ACTB , and/or RPL13A nucleic acids or fragments thereof to produce an ACE2 , TMPRSS2 , GAPDH, ACTB, and/or RPL13A nucleic acid amplification product; cc) sequencing the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid amplification product; and dd) detecting the expression level of the ACF.2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid.

8. The point-of-care device of claim 7, wherein the ACE2, TMPRSS2, GAPDH, ACTB, and/or RPL13A nucleic acid is mRNA.

9. The point-of-care device of claim 8, wherein the sample is selected from the group consisting of blood, serum, plasma, urine, and saliva.