Biomarkers of allergic asthma and methods of treatment

Abstract

Provided herein are method for diagnosis and treatment of severe and moderate allergic asthma using skin surface biomarkers.

Description

PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center BIOMARKERS OF ALLERGIC ASTHMA AND METHODS OF TREATMENT CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Application No. 63 / 620,338, filed January 12, 2024, the entire contents of which are incorporated herein by reference in their entirety. FIELD

[0002] The present disclosure relates to biomarker-based diagnostics and treatment methods for Allergic Asthma. BACKGROUND

[0003] Asthma, a chronic inflammatory lung disease with a complex etiology and pathogenesis, has attained a global prevalence of 11.5% and is currently the most frequent chronic disease in children. Its occurrence is closely linked to other allergic diseases, such as atopic dermatitis (AD). In 2019, there were 262 million cases of asthma and 171 million cases of AD globally. Pediatric asthma is often misdiagnosed, and treatment frequently does not adequately improve quality of life, symptoms or prevent exacerbations, motivating research into specific diagnostic tools and therapies targeted to asthma endotypes. In addition, children with asthma experience non- reversible airway remodeling when proper treatment is not initiated, potentially leading to permanent lung damage. Non-invasive biomarkers such as blood eosinophils or fractional exhaled nitric oxide (FeNO) are frequently unreliable as no clear cut-offs are validated for the pediatric population and high levels of false positives due to parasitic infections or the atopic background. Sputum cytology is not performed routinely due to safety and technical challenges. Bronchial biopsies and bronchial lavage, while the gold standard, are highly invasive and have potential side effects and thus are particularly impractical in children due to inaccessibility and discomfort. Given the distinctive biomechanisms of different asthma endotypes and age groups, disease- specific biomarkers are urgently needed for pediatric asthma. Validation of minimally invasive, reliable approaches for disease-specific biomarkers is critical for developing personalized pediatric asthma treatments. SUMMARY -1- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center

[0004] In one aspect, the current disclosure encompasses a method of treatment of allergic asthma in a subject, comprising: (a) providing a surface skin sample from the subject obtained from one or more tape-strips applied to a skin surface, wherein application of the tape-strip(s) to the skin surface obtains skin materials to the tape- strip; (b) determining a level of one or more biomarkers in the surface skin sample; (c) comparing the levels of the one or more biomarker in the surface skin sample to a reference level of the one or more biomarker to obtain a comparative level; (d) determining that the subject has allergic asthma based on the comparative level; and (e) administering to the subject an allergic asthma therapy. Also disclosed is a method of diagnosing allergic asthma in a subject, comprising: steps (a)-(d). In an aspect, the current disclosure also encompasses a method of detecting the efficacy of treatment of allergic asthma in a subject, comprising: (a) providing a surface skin sample from the subject obtained from one or more tape-strips applied to a skin surface, wherein application of the tape-strip(s) to the skin surface obtains skin materials to the tape- strip; and wherein the surface skin sample is obtained at a time after administering an allergic asthma therapy to the subject; (b) determining a level of one or more biomarkers in the surface skin sample; comparing the levels of the one or more biomarker in the surface skin sample to a reference level to obtain a comparative level; (c) determining the efficacy of allergic asthma therapy based on the comparative level. Also encompassed is a method of treatment of allergic asthma in a subject by administering an allergic asthma therapy; wherein the subject is diagnosed with allergic asthma based on the steps of: (a) providing a surface skin sample from the subject obtained from one or more tape-strips applied to a skin surface, wherein application of the tape-strip(s) to the skin surface obtains skin materials to the tape- strip; (b) determining a level of one or more biomarkers in the surface skin sample; (c) comparing the levels of the one or more biomarker in the surface skin sample to a reference level of the one or more biomarker to obtain a comparative level; (d) determining that the subject has allergic asthma based on the comparative level.

[0005] One or more than one tape strips may be applied to the skin surface of the subject for providing the skin sample. In some aspects, 5-25, or 5-10, or 10-15, or 15- 20, or 20-25 tape strips may be applied to the skin surface of the subject for providing the skin sample. In some aspects, levels of two or more biomarkers in the surface skin -2- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center sample may be determined. In an aspect, levels of two biomarkers may be determined in the surface skin sample. In some aspects, the one or more biomarker(s) may comprise a gene product of a Wnt-signaling / β-catenin pathway gene, a cell-adhesion gene, a lipid metabolism gene, NADH / respiratory chain gene, ATP synthase pathway gene, an oxidative stress gene, a immunomodulatory gene, a collagen gene, an extracellular matrix synthesis pathway gene, a gene related to COPD pathogenesis and airway inflammation / hyper-responsiveness, a transcription factor, a muco-ciliary activity related gene, a taste receptor related gene, a epithelial keratinization related gene, or combinations thereof. In an aspect, the one or more biomarker(s) may comprise a gene product of TSSC4, FAM212B, SCNM1, EPC2, ZNF235, EFN235, EFNA4, NOSTRIN, AREG, NACAD, or combinations thereof. In an aspect, the one or more biomarker(s) may comprise the gene product of FAM212B. In some aspects, the two biomarkers may comprise gene products of TSSC4 and FAM212B, SCNM1 and FAM212B, EPC2 and FAM212B, ZNF235 and FAM212B, EFN235 and FAM212B, EFNA4 and FAM212B, NOSTRIN and FAM212B, AREG and FAM212B, or NACAD and FAM212B. The gene-product may be down-regulated or upregulated in the subject. The gene product may be an RNA transcript, a protein, or both.

[0006] In an aspect, the level of one or more biomarkers in the surface skin sample may be determined by using any method known in the field including RNA-sequencing analysis, a PCR analysis, a qPCR analysis, a proteomic analysis, or combinations thereof. In an aspect, the method may be RNA-sequencing analysis. In an aspect, the level may be determined by measuring mRNA levels of the biomarker or the cDNA levels of the biomarker. In an aspect, the reference level comprises one or more of the levels of the at least one biomarker from a subject not suffering for allergic asthma, an average of the level of the at least one biomarker from subjects not suffering for allergic asthma, a previously provided reference level from the subject.

[0007] In an aspect, the one or more tape-strip(s) is applied to the same skin surface one or more times. In an aspect, the skin surface is non-lesioned skin. In an aspect, the subject does not have atopic dermatitis.

[0008] In an aspect, the current disclosure also encompasses administering an allergic asthma therapy comprising a topical treatment, phototherapy, inhalation -3- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center therapy, oral medication, a biologic, or combinations thereof based on the diagnostic methods provided herein. In an aspect, the allergic asthma therapy comprises administering a long-acting beta-agonist, a long-acting anticholinergic agent, leukotriene modifier, mast cell stabilizer, theophylline, an inhaled corticosteroid, a combination inhaler, a biologic, an immunomodulator; a short-acting beta-agonist, systemic corticosteroids, or combinations thereof. The inhaled corticosteroid may comprises fluticasone propionate (Flovent HFA, Flovent Diskus, Xhance), budesonide (Pulmicort Flexhaler, Pulmicort Respules, Rhinocort), ciclesonide (Alvesco), beclomethasone (Qvar Redihaler), mometasone (Asmanex HFA, Asmanex Twisthaler) or fluticasone furoate (Arnuity Ellipta); the leukotriene modifier comprises montelukast (Singulair), zafirlukast (Accolate) or zileuton (Zyflo); the short-acting beta- agonist comprises albuterol or levalbuterol; the anticholinergic agent comprises ipratropium or tiotropium; the systemic corticosteroid comprises prednisone, methyl prednisone; biologics comprise omalizumab (Xolair), mepolizumab (Nucala), dupilumab (Dupixent), reslizumab (Cinqair), or benralizumab (Fasenra).

[0009] In an aspect of the disclosure, the subject does not require a lung biopsy, blood tests, or nasal brushing for determining that the subject has allergic asthma. In an aspect, the subject is a human.

[0010] In an aspect, the current disclosure also encompasses a method of differentially treating moderate allergic asthma (MAA) or severe allergic asthma (SAA) in a subject, comprising: (a) providing a surface skin sample from the subject obtained from one or more tape-strips applied to a skin surface, wherein application of the tape- strip(s) to the skin surface obtains skin materials to the tape-strip; (b) determining a level of at least one biomarker in the surface skin sample; (c) comparing the levels of the at least one biomarker in the surface skin sample to a reference level of the at least one biomarker in a reference sample to obtain a comparative level; (d) determining that the subject has MAA or SAA based on the comparative level; and (e) administering to the subject an allergic asthma therapy suitable for MAA or SAA.

[0011] In an aspect, the current disclosure also encompasses a method of determining the efficacy of treatment of allergic asthma in a subject, comprising: (a) providing a first level of one or more biomarker in a first surface skin sample from the -4- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center subject; (b) administering to the subject an allergic asthma therapy suitable for MAA or SAA; (c) providing a second level of one or more biomarker in a second surface skin sample from the subject obtained at a later time; (d) comparing the first level of the at least one biomarker to the second level of the at least one biomarker to obtain a comparative level; and (e) determining the efficacy of the treatment based on the comparative level; wherein the first skin sample is obtained from a first set of one or more tape-strips applied to a skin surface, and the second skin sample is obtained from a second set of one or more tape-strips applied to a skin surface; and wherein application of the tape-strip(s) to the skin surface obtains skin materials to the tape- strip. In an aspect, the method further comprises changing the treatment modality if the treatment is determined to be ineffective based on the comparative level. In an aspect, the subject is less than 5 years, or less than 10 year, or less than 13 years of age.

[0012] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Aspects of the present disclosure are illustrated by way of example in which like reference numerals indicate similar elements and in which:

[0014] FIGs.1A-1D provide an overview of differentially expressed genes. FIG.1A: Venn diagram of shared and unique differentially expressed genes (DEGs) of children with MAA versus healthy HC, SAA versus HCs, and SAA versus MAA. DEGs were defined by fold change / FCH>2 and false discovery rate / FDR< 0.05. Red numbers represent upregulated genes, blue numbers represent downregulated genes. FIG.1B: Principal component analysis (PCA) based on RNA-sequencing data of tape-strips from MAA, SAA, and HCs. FIG.1C: Summary heatmap of barrier-associated genes in MAA. and SAA versus HC, and SAA versus MAA. Differentially expressed genes (DEGs) were defined by criteria of fold change (|FCH|)>2 and false discovery rate (FDR)<0.05. Samples are sorted by hierarchical clustering. MAA, moderate allergic asthma; SAA, severe allergic asthma; HC, healthy controls; PC, principal component. **FDR<0.01, *FDR<0.05, +FDR<0.1. FIG.1D: Heatmap of Lung Extracellular Milieu- associated markers. Comparisons of moderate allergic asthma (MAA) and severe -5- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center allergic asthma (SAA) versus healthy controls (HCs), and across groups using RNA- seq, by criteria of fold change (|FCH|)>2 and false discovery rate (FDR)<0.05. Samples are sorted by hierarchical clustering. MAA, moderate allergic asthma; SAA, severe allergic asthma; N, normal. **FDR < 0.01, *FDR < 0.05, +FDR < 0.1.

[0015] FIGs. 2A-2F provide results from a gene-set variation analysis (GSVA). Boxplots of mean gene set z-scores for epidermal-related (FIG. 1A) and major T- helper-related pathways (FIG.1B-1F) are shown. Red bars represent means. MAA, moderate allergic asthma; SAA, severe allergic asthma; HC, healthy controls. ****p<0.0001 ***p<0.001, **p<0.01, *p<0.05, +p<0.1.

[0016] FIG.3 is a heatmap of the top 100 upregulated genes ordered by SAA fold changes. Summary heatmap of the top upregulated genes in MAA. and SAA versus HC, and SAA versus MAA. Differentially expressed genes (DEGs) were defined by criteria of fold change (|FCH|)>2 and false discovery rate (FDR)<0.05. Samples are sorted by hierarchical clustering. MAA, moderate allergic asthma; SAA, severe allergic asthma; HC, healthy controls. **FDR<0.01, *FDR<0.05, +FDR<0.1.

[0017] FIGs.4A-4B are heatmaps of oxidative phosphorylation and aging pathway genes. Heatmap of (FIG.4A) oxidative phosphorylation and (FIG.4B) aging pathway comparing MAA and SAA versus HC, and SAA versus MAA. DEGs were defined as fold change (|FCH|)>2 and false discovery rate (FDR)<0.05. MAA, moderate allergic asthma; SAA, severe allergic asthma; HC, healthy controls. **FDR< 0.01, *FDR< 0.05, +FDR< 0.1.

[0018] FIG. 5. provides enrichment analysis usingcanonical / KEGG / REACTOME / BioCarta gene ontologies for top downregulated and upregulated genes for moderate allergic asthma (MAA) and severe allergic asthma (SAA) performed with XGR software. MAA, moderate allergic asthma; SAA, severe allergic asthma; N, normal. Dashed line denotes significant threshold (FDR <0.05)

[0019] FIGs.6A-D provide gene classifiers discriminating asthma. Two-gene (FIG. 6A) and one-gene classifiers (FIG.6B-6D) determined by area under the receiving operating characteristic (ROC) curves for transcriptomic changes found by tape- -6- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center stripping healthy appearing skin of moderate allergic asthma (MAA) and severe allergic asthma (SAA) children. AUC, area under curve.

[0020] FIG.7 provides an overview of the study and results. DETAILED DESCRIPTION

[0021] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects and aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.

[0022] The present disclosure is based, in part, on the surprising discovery that analysis of biomarkers in skin samples obtained from tape stripping are sufficient and can provide accurate diagnosis of allergic asthma without the need for more invasive techniques. These biomarkers were determined after extensive experimentation to be suitable for making a diagnosis from skin samples. FIG.7 provides an overview of the study design and the accuracy levels that can be achieved by the method provided herein with minimal discomfort to the patient.

[0023] I. Terminology

[0024] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present disclosure or the appended claims.

[0025] Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” -7- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1%, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1%, such as less than or equal to ± 0.05%.

[0026] The terms “comprising,” “including” and “having” are used interchangeably in this disclosure. The terms “comprising,” “including” and “having” mean to include, but not necessarily be limited to the things so described.

[0027] The terms “or” and “and / or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B or C” or “A, B and / or C” mean any of the following: “A,” “B” or “C”; “A and B”; “A and C”; “B and C”; “A, B and C.” An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.

[0028] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991), all of which are incorporated by reference herein. As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0029] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and -8- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center techniques of, pharmacology, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, genetics and protein and nucleic acid chemistry, described herein, are those well- known and commonly used in the art. In case of conflict, the present specification, including definitions, will control.

[0030] The practice of the present application will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al., Short Protocols in Protein Science, John Wiley & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999).

[0031] The nomenclatures used in connection with, and the laboratory procedures and techniques of biochemistry, immunology, microbiology, molecular biology, and virology described herein are those well-known and commonly used in the art. The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. -9- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center

[0032] When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles “a”, “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Wherever the terms “comprising” or “including” are used, it should be understood the disclosure also expressly contemplates and encompasses additional aspects “consisting of” the disclosed elements, in which additional elements other than the listed elements are not included.

[0033] The term “about” or “approximately,” as used herein, can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” can mean an acceptable error range for the particular value, such as 10% of the value modified by the term “about.” As used herein, the term “about,” can mean relative to the recited value, e.g., amount, dose, temperature, time, percentage, etc., ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0034] Further, as the present disclosure is susceptible to aspects of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present disclosure and not intended to limit the present disclosure to the specific aspects shown and described. Any one of the features of the present disclosure may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and / or the like in the description mean that the feature and / or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect,” “aspects,” and / or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present disclosure may include a variety of combinations and / or integrations of the aspects described herein. Additionally, all -10- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present disclosure will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be encompassed by the claims.

[0035] Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.” Numeric ranges are inclusive of the numbers defining the range. As used herein, the term “about” permits a variation of ±10% within the range of the significant digit.

[0036] Notwithstanding that the disclosed numerical ranges and parameters are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein. For example, a stated range of “1 to 10” should be considered to include any and all subranges between (and inclusive of) the minimum value of 1 and the maximum value of 10; that is, all subranges beginning with a minimum value of 1 or more, e.g., 1 to 6.1, and ending with a maximum value of 10 or less, e.g., 5.5 to 10.

[0037] Where aspects or embodiments are described in terms of a Markush group or other grouping of alternatives, the present application encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members. The present application also envisages the explicit exclusion of one or more of any of the group members in the Markush group or other grouping of alternatives.

[0038] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the -11- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center practice or testing of the various aspects and embodiments. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0039] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double- stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues See, e.g., Batzer et al., Nucleic Acid Res.19:5081 (1991), the disclosure of which is incorporated in its entirety herein.

[0040] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0041] As used herein, the term “allergic asthma” has its usual meaning in the art and encompasses both severe allergic asthma (SAA) and moderate allergic asthma -12- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center (MAA) unless specified otherwise. Allergic asthma is a type of asthma characterized by an inflammatory response in the airways that is triggered by exposure to allergens. Asthma itself is a chronic respiratory condition marked by inflammation and narrowing of the airways, leading to symptoms such as wheezing, shortness of breath, coughing, and chest tightness. In the case of allergic asthma, the immune system reacts excessively to certain substances known as allergens, which are typically harmless to most people. Common allergens that can trigger allergic asthma include pollen, mold spores, pet dander, dust mites, and certain foods. When a person with allergic asthma is exposed to these allergens, their immune system responds by releasing chemicals that cause inflammation in the airways, making breathing more difficult. Symptoms of allergic asthma can vary in severity and may be triggered by specific allergens or environmental factors.

[0042] As used herein, and unless otherwise specified, the terms “treat”, “treating” and “treatment” and variations thereof refer to an action that occurs in a subject with allergic asthma, which reduces the severity of at least one discernible symptom of asthma, or retards or slows the progression of at least one discernible symptom of asthma. In some embodiments, “treat” and its variations refers to an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient. In some embodiments, “treat” and its variations refers to inhibiting or reducing or slowing the progression of allergic asthma, either physically (e.g., stabilization of a discernible symptom), physiologically (e.g., stabilization of a physical parameter), or both, relative to an untreated control. In certain embodiments, “treat” and its variations refers to slowing the progression or reversing the progression of allergic asthma relative to an untreated control.

[0043] The term "likelihood" generally refers to an increase in the probability of an event. The term "likelihood" when used in reference to the effectiveness of a patient response generally contemplates an increased probability that the symptoms of allergic asthma will be lessened or decreased.

[0044] As used herein, the term “predict” refers to determine or foresee in advance. When used to “predict” the effectiveness of allergic asthma treatment, for example, the term “predict” can mean that the likelihood of the outcome of the treatment can be -13- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center determined at the outset, before the treatment has begun, or before the treatment period has progressed substantially.

[0045] As used herein, the term “monitor” refers to regularly observing and tracking the progress or quality over a period of time. In the context of this disclosure, “monitoring” may refer to tracking the effectiveness in treating allergic asthma in a subject, or a cell or tissue obtained from a subject. Similarly, the term "monitoring," when used in connection with subject compliance, either individually, or within a clinical trial, refers to the tracking or confirming that the subject is actually following the treatment regimen being tested as prescribed.

[0046] As used herein, the term “discriminate” refers to the ability to distinguish allergic asthma from other conditions, based on the biomarker profile obtained from the surface skin samples. Suitable biomarkers and combinations thereof are provided herein.

[0047] In the “tape-stripping” approach, serial adhesive films are used to capture the stratum corneum and the upper part of the granular layer as described previously. See Helen He et al.2020 Front. Immunol.; vol 11, Kim BE, et al. Journal of Investigative Dermatology 2019; 139:2387; Dyjack N, et al. J Allergy Clin Immunol 2018; 141:1298- 309. In some instances, at least 5-25 tape-strips are obtained from the same skin surface and pooled to obtain a single sample. “Tape-stripping” or equivalent methods are described in U.S. Patent 7183057, and U.S. patent application US 18 / 155,702 incorporated herein in their entirety.

[0048] As used herein, the term “control” refers to a tape-stripped surface skin sample taken from a healthy subject.

[0049] A “reference level” of a biomarker in a control refers to the baseline amount of that biomarker in the tape-stripped normal skin sample. In an aspect, the reference level may be an average level of a biomarker obtained from subjects without allergic asthma. In some aspects, the reference level may correspond to the biomarker level obtained from the subject for whom a diagnosis is being made or a treatment efficacy is being determined, but at a time prior to current. Thus, in some aspects, the reference level may comprise one or more of the levels of the at least one biomarker from a -14- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center subject not suffering for allergic asthma, an average of the level of the at least one biomarker from subjects not suffering for allergic asthma, a previously provided reference level from the subject. The amount of the biomarker that is measured in the sample may be relative or absolute. In some embodiments the relative expression of mRNA or cDNA is measured in the test sample versus the control sample. In other embodiments, the absolute amount of protein biomarker is measured in the test sample versus the control sample.

[0050] As used herein, the term “expressed” or “expression” refers to the transcription from a gene to a ribonucleic acid (RNA) molecule at least complementary in part to a region of one of the two nucleic acid strands of the gene. Alternatively, the term “expressed” or “expression” may refer to the translation from the RNA molecule to give a protein, a polypeptide or a portion thereof.

[0051] The level of mRNA or protein expression may “decrease” in a subject administered an allergic asthma treatment. Alternatively, the level of mRNA, cDNA or protein may “increase” following allergic asthma treatment. In some situations, the mRNA, cDNA or protein level may remain unchanged upon a given treatment., an mRNA, cDNA or protein level can be “downregulated”, i.e., the level of mRNA or protein may be decreased, for example, by about 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 1% or less of the comparative control mRNA, cDNA or protein level. Alternatively, an mRNA, cDNA or protein level from a subject sample can be “upregulated”, i.e., the level of mRNA, cDNA or protein may be increased, for example, by about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 100%, 200%, 300%, 500%, 1,000%, 5,000%, 10,000%, 15,000%, 20,000% or more of the comparative control mRNA, cDNA or protein level.

[0052] An “individual” or “subject,” as used interchangeably herein, is a mammal. In certain aspects, the individual or subject is a human.

[0053] The term “administration” and variants thereof (e.g., “administering” a composition) in reference to a treatment composition of the disclosure means introducing the composition or a prodrug of the composition into the system of the subject in need of treatment. When a composition of the disclosure or prodrug thereof -15- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center is provided in combination with one or more other active agents (e.g., a cytotoxic agent, etc.), “administration” and its variants are each understood to include concurrent and sequential introduction of the composition or prodrug thereof and other agents. The present disclosure includes within its scope prodrugs of the compositions of this disclosure. In general, such prodrugs will be functional derivatives of the compositions of this disclosure which are readily convertible in vivo into the required composition. Thus, in the methods of treatment of the present disclosure, the term “administering” shall encompass the treatment of the various conditions described with the composition specifically disclosed or with a composition which may not be specifically disclosed, but which converts to the specified composition in vivo after administration to the patient.

[0054] The term “therapeutically effective amount” as used herein means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.

[0055] As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who is in need of the treatment, for example, having a target disease or disorder, a symptom of the disease / disorder, or a predisposition toward the disease / disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease or disorder. Alleviating a target disease / disorder includes delaying the development or progression of the disease or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, “delaying” the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and / or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and / or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically -16- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[0056] “Development” or “progression” of a disease means initial manifestations and / or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a target disease or disorder includes initial onset and / or recurrence. II. Methods of diagnosis and treatment

[0057] In an aspect, the current disclosure encompasses methods of diagnosing and / or treatment of allergic asthma in a subject, comprising: (a) providing a surface skin sample from the subject obtained from one or more tape-strips applied to a skin surface, wherein application of the tape-strip(s) to the skin surface obtains skin materials to the tape-strip; (b) determining a level of one or more biomarkers in the surface skin sample; (c) comparing the levels of the one or more biomarker in the surface skin sample to a reference level of the one or more biomarker to obtain a comparative level; (d) determining that the subject has allergic asthma based on the comparative level. In an aspect, the method further includes treatment of allergic asthma in the subject by administering to the subject an allergic asthma therapy. Thus, in an aspect, the current disclosure encompasses a method of treatment of allergic asthma in a subject by administering an allergic asthma therapy; wherein the subject is diagnosed with allergic asthma based on the steps (a)-(d).

[0058] In an aspect, the current disclosure further provides a method of detecting the efficacy of treatment of allergic asthma in a subject previously treated with an asthma medication, comprising comparing the levels of the one or more biomarker in the surface skin sample obtained as provided herein, before and at a time after treatment to obtain a comparative level of said biomarker, and making a determination of the efficacy of allergic asthma therapy based on the comparative level. In an aspect, the -17- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center method may further comprise changing the treatment modality if the treatment is determined to be ineffective based on the comparative level.

[0059] In an aspect, the current disclosure also encompasses methods of differentially diagnosing and treating moderate allergic asthma (MAA) and severe allergic asthma (SAA) in a subject, using the levels of suitable biomarkers to make a differential diagnosis as determined by using steps (a)-(d), and administering a treatment regimen effective against MAA or SAA.

[0060] The steps of the methods provided herein are described in detail below: (a) Tape Stripping

[0061] Tape-stripping is a minimally invasive method using adhesive tapes to assess skin inflammation and barrier defects in patients with inflammatory skin diseases. The method of tape-stripping involves applying adhesive tape to the skin in a manner sufficient to isolate and remove an epidermal sample adhering to the tape that includes nucleic acid and / or protein molecules. The tape stripping method of the disclosure does not bare the viable epidermis unlike a biopsy which includes cells of the epidermis as well as the dermis resulting in permanent scarring and possible risk of infection. Factors such as the flexibility, softness, and composition of the adhesive tape used, the time the tape is allowed to adhere to the skin before it is removed, the force applied to the tape as it is applied to the skin, the prevalence of a gene product being analyzed, the disease status of the skin, and patient / patient variability are typically taken into account in deciding on a protocol useful for a particular tape stripping method in order to assure that sufficient nucleic acids are present in the epidermal sample. A tape stripped sample includes, but may not be limited to, tissues that are restricted to the surface of skin and preferentially recovers vellus hair follicles and cells lining sebaceous, eccrine, and sweat ducts (i.e. the adnexal structures associated with the stratum corneum and epidermis), as well as corneocytes. Tape stripping is stopped before viable epidermis is exposed by ceasing tape stripping before the tissue glistens. Therefore, the tape stripping method is considered a “noninvasive” method. Tape stripping sufficient to isolate an epidermal sample is tape stripping that is performed on the skin sufficient times to obtain an RNA sample, -18- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center wherein the tape stripping is stopped before the tissue glistens (i.e. becomes shiny, appears “moistened” or reflective).

[0062] Adhesive strips useful for tape-stripping, which need not specifically be in a tape format, include adhesive tapes such as D-squame® and Sebutape™ (polyacrylate ester adhesives; CuDerm Corporation, Dallas, Tex.) or Blenderm™, Tegaderm™, Duct tape (333 Duct Tape, Nashua tape products), Scotch® Tape (3M Scotch 810, St. Paul, Minn.), Diamond™ (The Sellotape Company; Eindhoven, the Netherlands), Sentega™ (polypropylene tape, Sentega Eiketten BV, Utrecht, The Netherlands), hydrogels such as Hypan™ (Hymedix International, Inc., Dayton, N.J.), or SequentialBioTM, may be used. The adhesive may be any of the commonly used pressure-sensitive-type adhesives or those which solidify quickly upon skin contact (such as cynaoacrylates). The adhesives may be on flexible or solid backings to make sampling easier. A constant pressure device (e.g. Desquame Pressure Instrument, CuDerm; Dallas, Tex.) can be used to apply pressure to the adhesive device during sampling. Other types of materials with adhesive properties such as glues, gums, and resins may also be used. In some instances, minimally invasive methods equivalent to tape-stripping that do not cause scarring may also be used, such as, mechanical scraping, swabbing and / or direct elution, pressure blotting, electric transfer, or the like.

[0063] Virtually any size and / or shape of adhesive tape and target skin site size and shape can be used and analyzed, respectively, by the methods of the present invention. For example, adhesive tape can be fabricated into circular discs of diameter between 10 millimeters and 100 millimeters, for example between 15 and 25 millimeters in diameter. The adhesive tape can have a surface area of between about 50 mm2and 1000 mm2, between about 100 mm2to 500 mm2or about 250 mm2.

[0064] The Examples illustrate tape stripping methods provided herein. Generally, before contacting a skin site with adhesive tape, a skin site to be stripped is cleaned, for example using an antiseptic cleanser such as alcohol. Next, tape is applied to a skin site with pressure. Pressure can be applied for a fraction of a second, but can be applied for between 1 second and 5 minutes, typically between 10 seconds and 45 seconds. In certain illustrative examples, tape is applied for 30 seconds for each tape stripping. It will be understood that the amount of pressure applied to a skin site and -19- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center the length of time for stripping can be varied to identify ideal pressures and times for a particular application. Generally, pressure is applied by manually pressing down the adhesive tape on the skin, however, objects, such as blunt, standardized pressurizer, flat objects can be used to assist in applying the tape to the skin, especially for areas of the skin from which it is more difficult to obtain nucleic acid samples from skin. The tape stripping methods provided herein typically involve applying an adhesive tape to the skin of a subject and removing the adhesive tape from the skin of the subject one or more times. In certain examples, the adhesive tape is applied to the skin and removed from the skin about one to ten times. Alternatively, about 1-25 adhesive tapes can be applied to the skin and removed from the skin. These adhesive tapes are then combined for further analysis. Accordingly, an adhesive tape can be applied to and removed from a target site 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 time, and / or 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 adhesive tape can be applied to and removed from the target site. In one illustrative example, the adhesive tape is applied to the skin between about one and eight times, in another example, between one and five times, and in another illustrative example the tape is applied and removed from the skin four times. One or more sites on the skin of the subject may be tape stripped and pooled or processed independently. (b) Biomarkers

[0065] In order to diagnose subjects with allergic asthma, identify subjects who would benefit from treatment with an allergic asthma therapy (such as the one or more of the therapies described below), differentially diagnose MAA and SAA, or to determine if a therapy is working, it is helpful to have one or more biomarker(s). A biomarker is a characteristic that can be objectively measured and evaluated as an indicator of normal biologic processes, pathologic processes, or pharmacological responses to a therapeutic intervention. Biomarkers can be biological (e.g., small molecules, metabolites, peptides, proteins, RNA, DNA), physiological (e.g., blood pressure, electromyography, respiratory function), or structural measures (e.g., ultrasound, magnetic resonance imaging, or histological assessment). The biomarkers may be prognostic biomarkers that predict a future clinical outcome; disease progression biomarkers that are indicative of the severity of disease impact; predictive biomarkers that predict a future clinical response to therapy and helps stratify therapies; -20- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center pharmacodynamics biomarkers that monitor or quantify a therapeutic effect; and surrogate end point biomarkers that predict a future clinical response to therapy wherein a change in the end point is associated with a future clinical response.

[0066] A biomarker for use in the current method can be any suitable biomarker that shows increased or decreased levels in a subject with allergic asthma in comparison to a subject who does not have allergic asthma. The level of mRNA or protein expression may “decrease” in a subject suffering from, or suspected of having allergic asthma compared to a subject who does not have allergic asthma. Alternatively, the level of mRNA, cDNA or protein may “increase” in a subject suffering from or suspected of having allergic asthma compared to a subject who does not have allergic asthma. Further, the level of mRNA or protein expression may “decrease” in a subject administered an allergic asthma treatment. Alternatively, the level of mRNA, cDNA or protein may “increase” following allergic asthma treatment.

[0067] In an aspect, a suitable biomarker is a gene product, for example an RNA or a protein. In as aspect, a suitable biomarker may be discerned using the methods provided in the examples herein. For example, a suitable biomarker can be discerned based on the data provided herein and / or available on the Gene Expression Omnibus (GSE245608).

[0068] Examples of suitable biomarkers may include biomarkers associated with epidermal barrier molecular pathways, including epidermal differentiation complex / cornified envelope (EDC-CE), lipid metabolism, tight-junction genes and epithelial hyperplasia markers. For examples, studies provided here show that subjects with allergic asthma show significant attenuation of terminal-differentiation- (LCE1C) and cell-adhesion- (CDH19, CDHR1, JAM2, GJA3) related markers. SAA showed more significantly pronounced downregulation of terminal differentiation (FLG, FLG2, CDSN, LCE1B / C / D / F, LCE2A), cornified envelope (LCE2D, NR2F2), cell- adhesion (CDHR1, CDH11 / 19, GJA3), keratin (e.g. KRT23 / KRT77), and lipid- biosynthesis / metabolism (ACOT2, FADS2, LOXL2, ALOXE3) products (all FDR<0.05). Significant downregulation of the cytoskeletal-organization-related genes (e.g., ACTA1, ACTB, ACTC1) and CLDN18, a marker that may mediate epithelial barrier permeability in the lung, was also observed in both MAA and SAA. Significant -21- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center downregulation of the combined epithelial barrier, lipid, and tight junction gene-set in SAA compared to healthy controls, with a similar trend for MAA was observed. Enrichment of the Th1 / IFN-γ pathway and genes upregulated by IFN-γ in keratinocytes in both MAA and SAA compared to controls was also observed herein. Thus, dysregulation of levels of gene products linked to any of these pathways and genes provided herein may be used in the methods disclosed herein.

[0069] Examples of suitable biomarkers may include biomarkers associated with tissue pathways contributory to asthma pathogenesis, including bronchial epithelium barrier, airway smooth muscle hypertrophy, and TGF-β signaling, and noted broader and stronger changes within the severe allergic asthma phenotype. As provided in the examples herein, tape-strips are able to capture upregulation of DEGs in MAA and SAA involved in the smooth-muscle-associated mTOR pathway (e.g., PTEN, RHEB, LAMTOR3), mechanosensory-associated Wnt signaling (e.g., IQGAP1, VANGL2), ErbB pathway (e.g., ERBB3, EGFR), which has been implicated in bronchial epithelium repair, and TGF-β signaling pathway (e.g., ACVR1B, ID1 / 2). Tape strip analysis may also capture upregulation of genes related to cell-stress-response pathways such as ubiquitin-dependent catabolism (e.g., CUL4B, ARRDC3, CANX) and mitochondrial structure and function (e.g., HIGD2A / hypoxia inducible domain, VDAC3, NDUFB9). Additionally, tape-stripped skin from MAA versus HCs showed upregulated DEGs involved in the necroptosis pathway (e.g., CHMP4B, CHMP5), while SAA versus HCs showed upregulated DEGs involves in the ferroptosis-related (e.g., ACSL3, SLC39A8, SLC3A2) pathways. Thus, dysregulation of levels of gene products linked to any of these pathways and genes provided herein may be used in the methods disclosed herein.

[0070] Additionally, gene products of genes that show significant expression in asthma patients’ endobronchial biopsies may be used as allergic asthma associated biomarkers. Non-limiting examples of such genes include smooth-muscle- (MBNL1 / muscleblind-like splicing regulator 1), RNA-metabolism- (RNASET2, CTR9), and cilia-function-related (SPEF2) markers, as well as ITGAV, an integrin gene contributory to Wnt pathway modulation, and HBB, which encodes hemoglobin subunit beta. Differential diagnosis of MAA vs SAA can be based on the observation herein that tape strips from MAA vs healthy controls (HC) capture dysregulation related to -22- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center RNA metabolism (DHX15) and fibroblast signaling (FGF13 / fibroblast growth factor 13), while SAA versus HCs show dysregulation of markers related to apoptosis (BCL2), host antimicrobial defense (LYZ), and cilia activity (STRBP / spermatid perinuclear RNA-binding protein). Levels of gene products linked to any of these pathways and genes provided herein may be used in the methods disclosed herein.

[0071] Further, levels of gene products linked with lung airway remodeling and epithelial barrier disruption may be used as allergic asthma associated biomarkers. ECM-related gene significantly upregulated in SAA patients compared to HCs, with similar trends in MAA, include genes linked to remodeling, including matrix metalloproteinase inhibitor TIMP3, and TGF-β co-receptor CD109, and mediators of cellular processing (EFEMP1, HCFC1, TNFSF10, PLXNB2). Downregulation of extracellular matrix regulatory genes (e.g., C1QTNF2, SEMA3B / E, PLOD3, LOXL1 / 2), core-matrisome-associated genes (e.g., COMP, FBLN2, TNXB), collagens (COL4A1 / 5A1 / 6A3 / 14A1), and secreted ECM factors (e.g., CHRDL1, INHBE) in SAA versus HCs, with similar trends observed in MAA were observed and are disclosed herein. Thus, dysregulation of levels of gene products linked to any of these pathways and genes provided herein may be used in the methods disclosed herein.

[0072] Levels of gene products linked with to oxidative phosphorylation and aging- associated changes in asthma may also be used as allergic asthma associated biomarkers. ATP-synthase- (ATP5G2 / 6V0B / 5H) and NADH / respiratory-chain-related (NDUFA7 / A8 / AB1 / B8 / S3 / S5 / V1) genes were found in this study to be significantly upregulated in SAA versus HCs, and mitochondrial-respiratory-chain- (COX6B1 / 7B), ATP synthase- (ATP5C1 / 5F1 / 5G3 / 5L / 6V1G1 / 6V0E1), beta-amyloid- (APPBP2), NADH / respiratory-chain-related (NDUFA10 / B4 / B5 / B10 / S2) and oxidative-stress- related (GPNMB) genes were significantly upregulated in both MAA and SAA. In SAA versus HCs downregulation of MPEG1, reported to increase susceptibility to microbialinfection was also observed. Thus, dysregulation of levels of gene products linked toany of these pathways and genes provided herein may be used in the methods disclosed herein.

[0073] Broadly, gene products of one or more of a Wnt-signaling / β-catenin pathway gene, a cell-adhesion gene, a lipid metabolism gene, NADH / respiratory chain gene, -23- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center ATP synthase pathway gene, an oxidative stress gene, a immunomodulatory gene, a collagen gene, an extracellular matrix synthesis pathway gene, a gene related to COPD pathogenesis and airway inflammation / hyper-responsiveness, a transcription factor, a muco-ciliary activity related gene, a taste receptor related gene, a epithelial keratinization related gene, or any combinations thereof may be used in the methods described herein. In an aspect, the one or more biomarker(s) comprises a gene product of TSSC4, FAM212B, SCNM1, EPC2, ZNF235, EFN235, EFNA4, NOSTRIN, AREG, NACAD, or combinations thereof.

[0074] In an aspect, levels of any one of the biomarkers provided herein can be used in the diagnostic and treatment methods disclosed herein. In an aspect, the levels of more that one biomarker may be used in the diagnostic and treatment methods provided herein. In an aspect, the level of at least two, or at least 3, or at least 4, or at least 5, or at least 6, or at least 7, or at least 8, or at least 9, or at least 10 or more biomarkers may be used in the diagnostic and treatment methods provided herein.

[0075] In an aspect, the one or more biomarker(s) comprises the gene product of FAM212B. In exemplary aspects, the two or more biomarkers may comprise gene products of TSSC4, FAM212B, SCNM1, EPC2, ZNF235, EFN235, EFNA4, NOSTRIN, AREG, NACAD, or combinations thereof, for example TSSC4 and FAM212B, SCNM1 and FAM212B, EPC2 and FAM212B, ZNF235 and FAM212B, EFN235 and FAM212B, EFNA4 and FAM212B, NOSTRIN and FAM212B, AREG and FAM212B, or NACAD and FAM212B. In an aspect, any of the gene products of genes listed in Table 1-4 or any combination thereof may be used as biomarkers for the allergic asthma diagnostic and treatment methods disclosed herein. (c) Determining Expression Levels / Concentrations of Biomarkers

[0076] Gene expression can be detected as RNA or protein expression of one or more target genes (gene product). That is, the presence or expression level (amount) of a gene product can be determined by detecting and / or measuring the level of mRNA or protein expression of the gene. In some aspects, gene expression can be detected as the activity of the gene product. -24- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center

[0077] In another embodiment, the expression level of a gene product of interest is determined by measuring RNA levels. A variety of suitable methods can be employed to detect and / or measure the level of mRNA expression of a gene. For example, mRNA expression can be determined using Northern blot or dot blot analysis, reverse transcriptase-PCR (RT-PCR; e.g., quantitative RT-PCR), in situ hybridization (e.g., quantitative in situ hybridization) or nucleic acid array (e.g., oligonucleotide arrays, gene chips) analysis, advanced transcriptomics methods such as RNA sequencing (RNA-seq). Details of such methods are described below and in, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual Second Edition vol. 1, 2 and 3. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, USA, Nov. 1989; Gibson et al. (1999) Genome Res., 6(10):995-1001; and Zhang et al. (2005) Environ. Sci. Technol., 39(8):2777-2785; U.S. Publication No.2004086915; European Patent No. 0543942; and U.S. Patent No. 7,101,663; the disclosures of each of which are incorporated herein by reference in their entirety.

[0078] In one example, the presence or amount of one or more discrete mRNA populations in a surface skin sample can be determined by isolating total mRNA from the surface skin sample (see, e.g., Sambrook et al. (supra) and U.S. Patent No. 6,812,341) and subjecting the isolated mRNA to agarose gel electrophoresis to separate the mRNA by size. The size-separated mRNAs are then transferred (e.g., by diffusion) to a solid support such as a nitrocellulose membrane. The presence or amount of one or more mRNA populations in the surface skin sample can then be determined using one or more detectably-labeled-polynucleotide probes, complementary to the mRNA sequence of interest, which bind to and thus render detectable their corresponding mRNA populations. Detectable-labels include, e.g., fluorescent (e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, allophycocyanin (APC), or phycoerythrin), luminescent (e.g., europium, terbium, Qdot™ nanoparticles supplied by the Quantum Dot Corporation, Palo Alto, CA), radiological (e.g., 125I, 131I, 35S, 32P, 33P, or 3H), and enzymatic (horseradish peroxidase, alkaline phosphatase, beta- galactosidase, or acetylcholinesterase) labels.

[0079] In another example, the presence or amount of discrete populations of mRNA (e.g., mRNA for biomarkers provided herein) in a surface skin sample can be -25- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center determined using nucleic acid (or oligonucleotide) arrays. For example, isolated mRNA from a surface skin sample can be amplified using RT-PCR with, e.g., random hexamer or oligo(dT)-primer mediated first strand synthesis. The amplicons can be fragmented into shorter segments. The RT-PCR step can be used to detectably-label the amplicons, or, optionally, the amplicons can be detectably-labeled subsequent to the RT-PCR step. For example, the detectable-label can be enzymatically (e.g., by nick-translation or kinase such as T4 polynucleotide kinase) or chemically conjugated to the amplicons using any of a variety of suitable techniques (see, e.g., Sambrook et al., supra). The detectably-labeled-amplicons are then contacted with a plurality of polynucleotide probe sets, each set containing one or more of a polynucleotide (e.g., an oligonucleotide) probe specific for (and capable of binding to) a corresponding amplicon, and where the plurality contains many probe sets each corresponding to a different amplicon. Generally, the probe sets are bound to a solid support and the position of each probe set is predetermined on the solid support. The binding of a detectably-labeled amplicon to a corresponding probe of a probe set indicates the presence or amount of a target mRNA in the surface skin sample. Additional methods for detecting mRNA expression using nucleic acid arrays are described in, e.g., U.S. Patent Nos.5,445,934; 6,027,880; 6,057,100; 6,156,501; 6,261,776; and 6,576,424; the disclosures of each of which are incorporated herein by reference in their entirety.

[0080] In an aspect, expression of more than one nucleic acid molecule can be detected. As illustrated in the examples, expression of about multiple genes is altered in the skin of an allergic asthma patient. Therefore, changes of skin state from normal to a state corresponding to allergic asthma, are accompanied by changes in at least these gene products. Therefore, in certain examples, methods provided herein characterize skin by analyzing expression of 2 or more, 5 or more, 10 or more, 25 or more, 50 or more, 100 or more, or all of the biomarkers provided herein. In certain examples, expression is detected for a gene listed in Tables 2-4, which lists genes / biomarkers identified in the studies disclosed herein, with the most dramatic expression changes in skin. For example, a detected nucleic acid can be an expression product of a Wnt-signaling / β-catenin pathway gene, a cell-adhesion gene, a lipid metabolism gene, NADH / respiratory chain gene, ATP synthase pathway gene, an oxidative stress gene, a immunomodulatory gene, a collagen gene, an extracellular -26- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center matrix synthesis pathway gene, a gene related to COPD pathogenesis and airway inflammation / hyper-responsiveness, a transcription factor, a muco-ciliary activity related gene, a taste receptor related gene, a epithelial keratinization related gene, or combinations thereof. In an aspect, the one or more biomarker(s) comprises a gene product of TSSC4, FAM212B, SCNM1, EPC2, ZNF235, EFN235, EFNA4, NOSTRIN, AREG, NACAD, or combinations thereof. In an aspect, the one or more biomarker(s) comprises the gene product of FAM212B. In an aspect, two or more biomarkers may be used. Here, the two or more biomarkers may comprise gene products of TSSC4, FAM212B, SCNM1, EPC2, ZNF235, EFN235, EFNA4, NOSTRIN, AREG, NACAD, or combinations thereof, for example TSSC4 and FAM212B, SCNM1 and FAM212B, EPC2 and FAM212B, ZNF235 and FAM212B, EFN235 and FAM212B, EFNA4 and FAM212B, NOSTRIN and FAM212B, AREG and FAM212B, or NACAD and FAM212B.

[0081] In another embodiment, the present invention provides a method for identifying an expression profile indicative of allergic asthma in a subject, including applying an adhesive tape to an area of skin afflicted with the disease or pathological state in a manner sufficient to isolate an epidermal sample adhering to the adhesive tape, wherein the epidermal sample includes nucleic acid molecules, and applying the nucleic acid molecules to a microarray or a related advanced sequencing technique (for example, RNAseq). Nucleic acid molecules can optionally be isolated from the epidermal sample before being applied to the microarray. Expression levels of at least 10 genes is then determined using the microarray; wherein an altered expression level for at least 2, 3, 4, 5, 6, 7, 8, 9, or each of the at least 10 genes as compared with expression in an epidermal sample from a normal skin sample identifies a subject is afflicted with MAA or SAA, thereby identifying the expression profile indicative of the disease.

[0082] Thus, in embodiments where expression of more than 1 gene is analyzed, the detection can be performed using a microarray. For example, the microarray can include an array of probes, for example, directed to 2 or more, 10 or more, 25 or more, 50 or more, 100 or more, 500 or more, 1000 or more genes. The microarrays form another embodiment of the invention. For microarray expression analysis, approximately 0.1 to 1 milligram, typically 1 to 10 nanograms of RNA are isolated from -27- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center an epidermal sample, for example obtained using a tape stripping method disclosed herein. Isolated RNA is then amplified and used for hybridization to probes on a biochip. Amplification typically results in a total of at least 1 microgram, and more typically at least 20 micrograms of amplified nucleic acid. For example, amplification can be performed using a MessageAMp™ RNA kit (Ambion Inc.). Isolated RNA can be biotin labeled before contacting the biochip such that binding to the target array can be detected using streptavidin. The probes may bind specifically to one or more of the genes listed in Tables 2-4, or a complement thereof.

[0083] Methods of detecting and / or for quantifying a detectable label depend on the nature of the label. The products of reactions catalyzed by appropriate enzymes (where the detectable label is an enzyme; see above) can be, without limitation, fluorescent, luminescent, or radioactive or they may absorb visible or ultraviolet light. Examples of detectors suitable for detecting such detectable labels include, without limitation, x-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers, and densitometers.

[0084] Methods for detecting or measuring gene expression (e.g., protein or mRNA expression) can optionally be performed in formats that allow for rapid preparation, processing, and analysis of multiple samples. This can be, for example, in multi-welled assay plates (e.g., 96 wells or 386 wells) or arrays (e.g., nucleic acid chips or protein chips). Stock solutions for various reagents can be provided manually or robotically, and subsequent sample preparation (e.g., RT-PCR, labeling, or cell fixation), pipetting, diluting, mixing, distribution, washing, incubating (e.g., hybridization), sample readout, data collection (optical data) and / or analysis (computer aided image analysis) can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting the signal generated from the assay. Examples of such detectors include, but are not limited to, spectrophotometers, luminometers, fluorimeters, and devices that measure radioisotope decay. Exemplary high-throughput cell-based assays (e.g., detecting the presence or level of a target protein in a cell) can utilize ArrayScan® VTI HCS Reader or KineticScan® HCS Reader technology (Cellomics Inc., Pittsburg, PA). -28- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center

[0085] In some embodiments, the expression level of the biomarkers of this disclosure can be assessed and / or measured. To aid in detecting the presence or level of expression of the biomarker genes of interest, any part of the nucleic acid sequence of the genes can be used, e.g., as hybridization polynucleotide probes or primers (e.g., for amplification or reverse transcription). The probes and primers can be oligonucleotides of sufficient length to provide specific hybridization to an RNA, DNA, cDNA, or fragments thereof isolated from a surface skin sample. Depending on the specific application, varying hybridization conditions can be employed to achieve varying degrees of selectivity of a probe or primer towards target sequence. The primers and probes can be detectably-labeled with reagents that facilitate detection (e.g., fluorescent labels, chemical labels (see, e.g., U.S. Patent Nos.4,582,789 and 4,563,417), or modified bases).

[0086] Standard stringency conditions are described by Sambrook, et al. (supra) and Haymes, et al. Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985). In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular hybridization conditions (e.g., solvent and salt concentrations) employed.

[0087] Hybridization can be used to assess homology between two nucleic acid sequences. A nucleic acid sequence described herein, or a fragment thereof, can be used as a hybridization probe according to standard hybridization techniques. The hybridization of a probe of interest (e.g., a probe containing a portion of a nucleotide sequence described herein or its complement) to DNA, RNA, cDNA, or fragments thereof from a test source is an indication of the presence of DNA or RNA corresponding to the probe in the test source. Hybridization conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6, 1991. Moderate hybridization conditions are defined as hybridization in 2X sodium chloride / sodium citrate (SSC) at 30ºC, followed by a wash in 1 X SSC, 0.1% SDS at 50ºC. Highly stringent conditions are defined as hybridization in 6X SSC at 45ºC, followed by a wash in 0.2 X SSC, 0.1% SDS at 65°C. -29- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center

[0088] Primers can be used in a variety of PCR-type methods. For example, polymerase chain reaction (PCR) techniques can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. The PCR primers are designed to flank the region that one is interested in amplifying. Primers can be located near the 5' end, the 3' end or anywhere within the nucleotide sequence that is to be amplified. The amplicon length is dictated by the experimental goals. For qPCR, the target length is closer to 100 base pairs and for standard PCR, it is near 500 base pairs. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. PCR primers can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3’ to 5’ direction using phosphoramidite technology) or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair.

[0089] In addition, the nucleic acid sequences or fragments thereof (e.g., oligonucleotide probes) can be used in nucleic acid arrays for detection and / or quantitation of gene expression.

[0090] In one embodiment, the expression of a gene can be determined by detecting and / or measuring expression or concentration of a protein encoded by the gene. Methods of determining protein expression / concentration are well known in the art. A generally used method involves the use of antibodies specific for the target protein of interest. For example, methods of determining protein expression include, but are not limited to, western blot or dot blot analysis, immunohistochemistry (e.g., quantitative immunohistochemistry), immunocytochemistry, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent spot (ELISPOT; Coligan, J. E., et al., eds. (1995) Current Protocols in Immunology. Wiley, New York), radioimmunoassay, chemiluminescent immunoassay, electrochemiluminescence immunoassay, latex -30- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center turbidimetric immunoassay, latex photometric immunoassay, immuno- chromatographic assay, and antibody array analysis (see, e.g., U.S. Publication Nos. 2003 / 0013208 and 2004 / 171068, the disclosures of each of which are incorporated herein by reference in their entirety). Further description of many of the methods above and additional methods for detecting protein expression can be found in, e.g., Sambrook et al. (supra).

[0091] In one example, the presence or amount of a protein gene product can be determined using a western blotting technique. For example, a lysate can be prepared from a surface skin sample, or the surface skin sample itself, can be contacted with Laemmli buffer and subjected to sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE-resolved proteins, separated by size, can then be transferred to a filter membrane (e.g., nitrocellulose) and subjected to immunoblotting techniques using a detectably-labeled antibody specific to the protein of interest. The presence or amount of bound detectably-labeled antibody indicates the presence or amount of protein in the surface skin sample.

[0092] In another example, an immunoassay can be used for detecting and / or measuring the protein expression of a biomarker disclosed herein, including but not restricted to OLINK Proteomics, Luminex, Elisa, Singulex Erenna or other proteomic platforms. As above, for the purposes of detection, an immunoassay can be performed with an antibody that bears a detection moiety (e.g., a fluorescent agent or enzyme). Proteins from a surface skin sample can be conjugated directly to a solid-phase matrix (e.g., a multi-well assay plate, nitrocellulose, agarose, sepharose, encoded particles, or magnetic beads) or it can be conjugated to a first member of a specific binding pair (e.g., biotin or streptavidin) that attaches to a solid-phase matrix upon binding to a second member of the specific binding pair (e.g., streptavidin or biotin). Such attachment to a solid-phase matrix allows the proteins to be purified away from other interfering or irrelevant components of the surface skin sample prior to contact with the detection antibody and also allows for subsequent washing of unbound antibody. Here, as above, the presence or amount of bound detectably labeled antibody indicates the presence or amount of protein in the surface skin sample. -31- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center

[0093] There is no particular restriction as to the form of the antibody and the present disclosure includes polyclonal antibodies, as well as monoclonal antibodies. The antiserum obtained by immunizing animals, such as rabbits with a protein or fragment thereof of the invention, as well polyclonal and monoclonal antibodies of all classes, human antibodies, and humanized antibodies produced by genetic recombination, are also included.

[0094] The antibodies may be conjugated to various molecules, such as fluorescent substances, radioactive substances, and luminescent substances. Methods to attach such moieties to an antibody are already established and conventional in the field (see, e.g., US 5,057,313 and 5,156,840).

[0095] Examples of methods that assay the antigen-binding activity of the antibodies include, for example, measurement of absorbance, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and / or immunofluorescence, and or Proximity Extension Assay (PEA) technology that is used by OLINK platform. For example, when using ELISA, a protein encoded by a biomarker of the invention is added to a plate coated with the antibodies of the present disclosure, and then, the antibody sample, for example, culture supernatants of antibody-producing cells, or purified antibodies are added. Then, secondary antibody recognizing the primary antibody, which is labeled by alkaline phosphatase and such enzymes, is added, the plate is incubated and washed, and the absorbance is measured to evaluate the antigen-binding activity after adding an enzyme substrate such as p-nitrophenyl phosphate. As the protein, a protein fragment, for example, a fragment comprising a C-terminus, or a fragment comprising an N-terminus may be used. To evaluate the activity of the antibody of the invention, BIAcore (Pharmacia) may be used.

[0096] By using these methods, the antibody and a sample presumed to contain a protein of the disclosure are contacted, and the protein encoded by a biomarker of the disclosure is detected or assayed by detecting or assaying the immune complex formed between the above-mentioned antibody and the protein. -32- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center

[0097] Mass spectrometry-based quantitation assay methods, for example, but not limited to, multiple reaction monitoring (MRM)-based approaches in combination with stable-isotope labeled internal standards, are an alternative to immunoassays for quantitative measurement of proteins. These approaches do not require the use of antibodies and so the analysis can be performed in a cost- and time- efficient manner (see, for example, Addona et al., Nat. Biotechnol., 27:633–641, 2009; Kuzyk et al., Mol. Cell Proteomics, 8:1860–1877, 2009; Paulovich et al., Proteomics Clin. Appl., 2:1386-1402, 2008). In addition, MRM offers superior multiplexing capabilities, allowing for the simultaneous quantification of numerous proteins in parallel. The basic theory of these methods has been well-established and widely utilized for drug metabolism and pharmacokinetics analysis of small molecules. (d) Reference and comparative levels

[0098] In an aspect, the current disclosure also encompasses comparing the levels of the one or more biomarker in the surface skin sample of a subject to the reference levels of the one or more biomarker. The reference level for use herein will depend on the application and can be determined by a person of ordinary skill in the art. The reference level may be the level of the corresponding biomarker in a person not suffering from allergic asthma. The reference level may be an average or other statistical aggregate of the level of the corresponding biomarker or biomarkers in a population of healthy individuals without any history of atopic conditions, including allergic asthma, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, food allergies, and / or eosinophilic esophagitis. Healthy is defined in relation to these aforementioned conditions and cannot be confined to an absolute evaluation or status. Healthy individuals may therefore present with other diseases, including other respiratory conditions. The reference level may also be the subject’s own biomarker level at a time other than the current level. (e) Methods of treatment

[0099] The methods disclosed herein enable the diagnosis of allergic asthma based on biomarker levels in a subject’s surface skin, followed by treatment of said subject with an appropriate asthma therapy. The methods disclosed herein enable the -33- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center assessment whether or not a subject having or suspected of having allergic asthma is likely to respond to a therapy. A subject having or suspected of having allergic asthma who is likely to respond to the asthma therapy can be administered the therapy. Conversely, a subject having or suspected of having allergic asthma who is not likely to respond to a therapy can be administered a different therapy that is suitable for treatment of allergic asthma.

[0100] The methods of this disclosure also enable the stratification of subjects having or suspected of having allergic asthma into groups of subjects that are more likely to benefit, and groups of subjects that are less likely to benefit, from treatment comprising a asthma therapy. The ability to select such subjects from a pool of subjects who are being considered for treatment with an asthma therapy is beneficial for administering an effective treatment to the subject. For example, the biomarkers may be used to classify patients based the severity of allergic asthma (MAA or SAA) and a treatment regimen can be decided accordingly.

[0101] The subjects who are considered for treatment comprising a therapy include, but are not limited to, subjects having, suspected of having, or likely to develop allergic asthma. In one aspect, the subject to be treated with a asthma therapy has, is suspected of having, or is likely to develop mild allergic asthma. In one embodiment, the subject to be treated with a therapy has, is suspected of having, or is likely to develop MAA. In one embodiment, the subject to be treated with a therapy has, is suspected of having, or is likely to develop SAA.

[0102] If the subject having allergic asthma is more likely to respond to a therapy (based on levels of biomarkers of this disclosure), the subject can then be administered an effective amount of the therapy. An effective amount of the compound can suitably be determined by a health care practitioner taking into account, for example, the characteristics of the patient (age, sex, weight, race, etc.), the progression of the disease, and prior exposure to the drug. If the subject is less likely to respond to one asthma therapy, the subject can then be optionally administered a different therapy. -34- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center

[0103] In addition to the prediction of the likelihood of treatment effectiveness in a patient with allergic asthma, the progress of a treatment modality can be followed by monitoring the levels of the biomarkers described above. In some embodiments, a method of assessing or monitoring the effectiveness of a treatment in a subject is provided. A surface skin sample is obtained from the subject, and the levels of one or more of the above-described biomarkers, are measured to determine whether the biomarker levels are increased or decreased compared to the levels prior to the initiation of the treatment.

[0104] In one embodiment, provided herein is a method of monitoring subject response to a allergic asthma treatment comprising: obtaining a surface skin sample from the subject; measuring the level of any of the biomarkers or combinations thereof as listed in Tables 2-4, in the first sample; administering a treatment compound to the subject; thereafter obtaining a second surface skin sample from the subject; measuring the level of any of the biomarkers or combinations thereof as listed in Tables 2-4, in the second sample; and comparing the levels of the biomarkers obtained from first and second samples; wherein a decreased level of the biomarkers in the second sample indicates an effective response. In one embodiment, provided herein is a method of monitoring subject response to a allergic asthma treatment comprising: obtaining a surface skin sample from the subject; measuring the level of any of the biomarkers or combinations thereof as listed in Tables 2-4, in the first sample; administering a treatment compound to the subject; thereafter obtaining a second surface skin sample from the subject; measuring the level of any of the biomarkers or combinations thereof as listed in Tables 2-4, in the second sample; and comparing the levels of the biomarkers obtained from first and second samples; wherein a increased level of the biomarkers in the second sample indicates an effective response.

[0105] The biomarkers can also be used to track and adjust individual patient treatment effectiveness. The biomarkers can be used to gather information needed to make adjustments in a patient's treatment, increasing or decreasing the dose of an agent as needed. For example, a patient receiving a treatment compound can be tested using a biomarker to see if the dosage is becoming effective, or if a more aggressive treatment plan may be needed. -35- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center

[0106] Subjects of all ages can be affected by allergic asthma. Therefore, a surface skin sample used in a method described herein can be obtained from a human subject of any age, including a fetus, an infant, a child, an adolescent, or an adult, such as an adult having, or suspected of having, allergic asthma.

[0107] The methods can also be applied to individuals at risk of developing allergic asthma treatable by an asthma therapy. Such individuals include those who have (i) a family history of (a genetic predisposition for) such disorders or (ii) one or more risk factors for developing such disorders.

[0108] After stratifying or selecting a subject based on whether the subject will be more likely or less likely to respond to a therapy, a medical practitioner (e.g., a doctor) can administer the appropriate therapeutic modality to the subject. Methods of administering allergic asthma therapies are known in the art.

[0109] Suitable allergic asthma therapies may comprise a topical treatment, phototherapy, inhalation therapy, oral medication, a biologic, or combinations thereof.

[0110] In an aspect, the allergic asthma therapy may comprise administering a long- acting beta-agonist, a long-acting anticholinergic agent, leukotriene modifier, mast cell stabilizer, theophylline, an inhaled corticosteroid, a combination inhaler, a biologic, an immunomodulator; a short-acting beta-agonist, systemic corticosteroids, or combinations thereof.

[0111] In an aspect, the inhaled corticosteroid comprises fluticasone propionate (Flovent HFA, Flovent Diskus, Xhance), budesonide (Pulmicort Flexhaler, Pulmicort Respules, Rhinocort), ciclesonide (Alvesco), beclomethasone (Qvar Redihaler), mometasone (Asmanex HFA, Asmanex Twisthaler) or fluticasone furoate (Arnuity Ellipta); the leukotriene modifier comprises montelukast (Singulair), zafirlukast (Accolate) or zileuton (Zyflo); the short-acting beta-agonist comprises albuterol or levalbuterol; the anticholinergic agent comprises ipratropium or tiotropium; the systemic corticosteroid comprises prednisone, methyl prednisone; biologics comprise omalizumab (Xolair), mepolizumab (Nucala), dupilumab (Dupixent), reslizumab (Cinqair), or benralizumab (Fasenra). -36- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center

[0112] Combination therapies (e.g., co-administration of a allergic asthma therapy and one or more additional therapies or additional therapeutic agents) can be, e.g., simultaneous or successive. For example, a allergic asthma therapy and the additional therapeutic agent(s) can be administered at the same time or at different times. In some embodiments, the one or more additional therapeutic agents can be administered first in time and the astma therapy can be administered second in time.

[0113] In an aspect, the current disclosure further encompasses methods of treating allergic asthma in a subject diagnosed using the disclosed methods. (f) Kits

[0114] This disclosure also provides kits. In certain embodiments, the kit can include one or more means for obtaining skin samples. In some embodiments, the kit includes probes that can be used to identify or detect any of the biomarkers disclosed herein. In some embodiments, the kit includes any of the nucleic acid arrays. In some embodiments, the kit includes probes and antibodies that can be used to identify or detect any of the biomarkers disclosed herein or their expression or expression levels. The kits can, optionally, contain instructions for detecting and / or measuring the concentration of one or more proteins or the levels of mRNA in a surface skin sample.

[0115] In some embodiments, the kits can include one or more reagents for processing a surface skin sample (e.g., calibration reagents, buffers, diluents, color reagents, reagents to stop a reaction). For example, a kit can include reagents for isolating nucleic acid or protein from a surface skin sample and / or reagents for detecting the presence and / or amount of one or more biomarkers in a surface skin sample (e.g., probes for detection of a biomarker RNA, an antibody that binds to the protein that is the subject of the detection assay and / or an antibody that binds the antibody that binds to the protein).

[0116] The kits can optionally include, e.g., a control (e.g., a concentration standard for the protein being assessed) or control labeled-amplicon set containing known amounts of one or more amplicons recognized by nucleic acid probes of the array. In some instances, the control can be an insert (e.g., a paper insert or electronic medium such as a CD, DVD, or floppy disk) containing an expression level or expression level -37- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center ranges of one or more proteins or RNAs predictive of allergic asthma, or of responsiveness to an allergic asthma therapy.

[0117] In certain embodiments, the kit includes at least one microplate (e.g., a 96 well plate; i.e., 12 strips of 8 wells). The microplate can be provided with its corresponding plate cover. The microplate can be polystyrene or of any other suitable material. The microplate can have the antibody that is used to identify the presence of a particular biomarker coated inside each well. The antibody may be conjugated to a detectable label. The kit may also include at least one adhesive strip.

[0118] In some embodiments, the kits can include a software package for analyzing the results of, e.g., expression profile or a microarray analysis.

[0119] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art can develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention. EXAMPLES

[0120] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0121] The examples provided herein demonstrate that tape-strips, a minimally invasive method, may help identify asthma-specific biomarkers in the skin of children with allergic asthma. Methods provided herein can be optimized using routine techniques and be applied to patients of any age group. Allergic asthma is usually -38- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center associated with other atopic diseases, including food allergy, AD, and allergic rhinitis (AR), collectively called the "atopic march". Typically, AD and food allergy occur in infancy, followed by AR and allergic asthma development after three years of age. A compromised epithelial barrier may facilitate sensitization to environmental factors, triggering innate and adaptive type 2 (T2) immune responses; thus, non-invasive evaluation of the epithelial barrier may be useful as a diagnostic.

[0122] Tape-stripping is a minimally invasive method using adhesive tapes to assess skin inflammation and barrier defects in patients with inflammatory skin diseases.

[0123] It was hypothesized that tape-stripping skin from children with allergic asthma could identify asthma-specific biomarkers through a minimally invasive method. Using RNA-seq profiling, normal-appearing skin of children with moderate allergic asthma (MAA) and severe allergic asthma (SAA) was characterized in comparison to healthy controls (HCs). The aim was to identify mechanistic processes described as the shared dysregulation of genes, potentially associated with asthma and the atopic march. A further goal was to develop non-invasive diagnostics and treatment solutions. Example 1: Methods Study Population

[0124] A group of 32 children (age ≤12 years) with MAA (n=11) and SAA (n=9) without concomitant AD and 12 HCs were enrolled at the Asthma and Allergy Department at Theramed Healthcare (Brasov, Romania). Participants’ parents provided informed, written consent approved by the institutional review board. Asthma severity was determined by GINA 2020 guidelines: frequency of asthma symptoms, night-time awakenings, rescue medication use, activity limitation, lung function, and level of asthma controller medication. According to personal history, allergic asthma diagnosis was defined as sensibilization to aeroallergens (serum-specific IgE) as key drivers of asthma symptoms. A nasal allergen provocation test was performed when the patient's history was unclear to ensure a correct diagnosis of allergic asthma. History and other measures of asthma profile included family and personal account of atopic comorbidities, age at asthma onset, annualized asthma exacerbation rate, -39- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center asthma treatment, total serum IgE levels (IU / mL), fractional excretion of nitric oxide (FeNO), forced expiratory volume in 1 second (FEV1) (% predicted), impulse oscillometry (IOS) R5-20 Hz, as well as BMI of study participants (Table 1). Table 1: Demographics and asthma visible properties (Mean ± SE if not specified, BMI, ICS, body mass index; MAA, moderate allergic asthma; SAA, severe allergic asthma. FEV1, forced expiratory volume in 1 second; FeNO, fractional excretion of nitric oxide; ICS, inhaled corticosteroid; LABA: long-acting β2-agonist, LTRA, leukotriene receptor antagonists; R5-R20, peripheral airway resistance) Healthy Controls MAA SAA p- n=12 n=11 n=9 value Age (years) Mean (Standard Deviation) 7.36 (± 2.37) 7.0 (± 1.67) 9.11 (± 2.03) 0.071 Range 4-11 5-10 6-11 Sex n % n % n % Female 3 21.4% 4 36.4% 1 11.1% 0.53 Male9 78.6% 7 63.6% 8 88.9%Age of Asthma Onset (years) Mean (Standard Deviation) N / A 5.23 (± 1.62) 4.56 (±1.67) 0.35 Range N / A 4-9 3-8 FEV1 (% predicted) Mean (Standard Deviation) N / A 97.88 (± 4.87) 86.28 (± 3.77) Range N / A 90.8-105.7 81-90.8 IgE Levels (UI / mL) Value (Standard Deviation) N / A 389.41 (± 395.58) 1056.3 (± 1102.40) Range N / A 99.5-1388 197-3079 FeNO (ppb) Value (Standard Deviation) N / A 30.28 (± 14.87) 56.44 (± 25.27) Range N / A 8-43 12-100 R5-R20 (Hz) Value (Standard Deviation) N / A 4.27 (± 1.54) 3.2 (± 1.41) Range N / A 1.96-6.04 0.99-5.55 Exacerbation Rate (exacerbations / yr) Value (Standard Deviation) N / A 1.36 (± 0.5) 4.22 (± 0.67) Range N / A 1-2 4-5 BMI Value (Standard Deviation) 20.56 (± 1.48) 21.48 (± 2.51) 23.71 (± 4.21) > 0.05 Range 18.6-22.4 18.5-23.70 18.10-29.3 Concomitant AD Number (n) N / A 0 0Concomitant Food Allergy Number (n) N / A 1 0Family History of Atopy Number (n) N / A 7 7-40- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center Current Medication Regimen ICS Number (n) N / A 11 0 ICS / LABA Number (n) N / A 0 8 ICS / LABA / LTRA Number (n)N / A 0 1 Race n % n % n % White 12 100.0% 11 100.0% 9 100.0% Asian 0 0.0% 0 0.0% 0 0.0% 1.0 Black 0 0.0% 0 0.0% 0 0.0% Other 0 0.0% 0 0.0% 0 0.0% Tape-strip collection

[0125] A total of 20 consecutive D-Squame (CuDerm, Dallas, Texas) circumferential tape-strips were taken from healthy-appearing skin in the antecubital fossa. Tape- strips were placed on the skin, and pressure was applied evenly with a pressurizer of equal diameter for approximately 5 seconds. The tapes were placed in individual sterile bags, frozen on dry ice, and stored at –80oC. RNA-Seq

[0126] RNA-seq was performed using known techniques described. Briefly, RNAwas extracted using miRNeasy Mini Kits (Qiagen, Hilden, Germany), and RNA-seqlibraries were constructed with Ion AmpliSeq Transcriptome Human Gene Expression Kits (ThermoFisher, Waltham, MA) using 5ng of RNA per sample. RNA-seq libraries were pooled and sequenced on the Ion S5 XL System Sequencer with Ion 550 Chips (ThermoFisher, Waltham, MA), screening more than 20,000 genes per reaction. Statistical Analysis

[0127] Statistical analyses were performed using R Software (www.R-project.org) and packages available through Bioconductor (www.bioconductor.org). Sample quality was assessed with FastQC (Cambridge, United Kingdom) and MultiQC algorithms. Samples were aligned to the human reference genome using STAR (open- source aligner). Mapped sequencing reads were assigned to genomic features by using the featureCounts function. Counts were transformed to log2-scale with voom, and fold-change (FCH) estimation and hypothesis testing were conducted using contrasts under the general framework for linear models with the limma package. For RNAseq, p-values were adjusted for multiple hypotheses with the Benjamini-Hochberg procedure, controlling for false discovery rate (FDR). Differentially expressed genes -41- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center (DEGs) were defined with |FCH|>2 and FDR<0.05. Unsupervised clustering in heatmaps of estimated marker expression means was performed with a McQuitty agglomeration algorithm.

[0128] Gene set variation scores were estimated with the z-score method using the GSVA package. Pathway enrichment analyses were performed with XGR software using the canonical / KEGG / REACTOME / BioCarta gene ontologies. Spearman correlations were used to evaluate the association between inflammatory markers and asthma-related outcomes (annualized exacerbation rate, lung function - FEV1, IOS R5-R20, and inflammation - FeNO).

[0129] To evaluate the performance of asthma classifiers with tape-strip biomarkers, Area Under the Receiver Operating Characteristic Curves (AUROCs) were generated, in which the true positive rate (sensitivity) is plotted against the false-positive rate (1- specificity), and each point represents the sensitivity / specificity corresponding to a decision threshold. The area under the curve (AUC) measures the classifier’s discriminative ability, where a perfect classifier would have an AUC of 1 corresponding to 100% sensitivity and specificity. Example 2: Children with moderate and severe asthma have a distinct transcriptomic profile in the healthy skin

[0130] Tape-strips were analyzed from normal-appearing skin of children with MAA (n=11), SAA (n=9), and age-matched HCs (n=12), with a 100% sample recovery rate (32 of 32 individuals). The mean ages (years) of the MAA, SAA, and HC cohorts were 7.36, 7.0, and 9.11, respectively, and the mean ages of asthma onset (years) for MAA and SAA patients were 5.23 and 4.56, respectively. None of the patients included in the study had AD or a personal history of AD (Table 1).

[0131] Using criteria of |FCH|>2 and FDR<0.05 to define DEGs, the study identified 1113 DEGs (523 upregulated; 590 downregulated) in MAA versus HCs, and 2117 DEGs (967 upregulated; 1150 downregulated) in SAA versus HCs (results summarized in a Venn diagram in (FIG.1A).319 DEGs were unique to children with MAA (181 upregulated; 138 downregulated) and 1322 to children with SAA (625 upregulated; 697 downregulated). 794 DEGs (342 upregulated and 452 -42- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center downregulated) were shared. Comparison between SAA vs MAA showed only one DEG. The principal component analysis (PCA) showed that MAA and SAA patients largely clustered together and separated from controls (FIG.1B). Example 3: Tape-strips highlight epithelial barrier dysregulation in normal- appearing skin from pediatric asthma

[0132] A curated list combining previously published epidermal barrier molecular pathways, including epidermal differentiation complex / cornified envelope (EDC-CE), lipid metabolism and tight-junction genes was evaluated (FIG. 1C). Significant (FDR<0.05) upregulation of epithelial hyperplasia markers (e.g. S100A4 / 10 / 11; ANXA3 / 7) in MAA and / or SAA versus HCs (FIG. 1C-1D) was seen. MAA showed significant attenuation of terminal-differentiation- (LCE1C) and cell-adhesion- (CDH19, CDHR1, JAM2, GJA3) related markers. SAA showed more significantly pronounced downregulation of terminal differentiation (FLG, FLG2, CDSN, LCE1B / C / D / F, LCE2A), cornified envelope (LCE2D, NR2F2), cell-adhesion (CDHR1, CDH11 / 19, GJA3), keratin (e.g. KRT23 / KRT77), and lipid-biosynthesis / metabolism (ACOT2, FADS2, LOXL2, ALOXE3) products (all FDR<0.05). Significant downregulation of the cytoskeletal-organization-related genes (e.g., ACTA1, ACTB, ACTC1) and CLDN18, a marker that may mediate epithelial barrier permeability in the lung, was observed in both MAA and SAA.

[0133] To understand changes in MAA and SAA at the pathway-level, a gene-set- variation-analysis (GSVA) using published immune response and barrier gene-setswas performed. The analysis revealed significant downregulation of the combinedepithelial barrier, lipid, and tight junction gene-set in SAA compared to healthycontrols, with a similar trend for MAA (P<0.05; FIG.2A). Significant enrichment of theTh1 / IFN-γ pathway and genes upregulated by IFN-γ in keratinocytes in both MAA andSAA compared to controls (P<0.05; FIG. 2B-C) was found. No significant differenceswere found in Th2-, Th17-, and Th22-mediated pathways between either asthma group and HCs (FIG 2D-F). Example 4: Tape-strips capture molecular processes relevant to asthma tissue remodeling and other pathogenetic pathways -43- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center

[0134] Common signature across the allergic asthma spectrum related to various tissue pathways contributory to asthma pathogenesis, including bronchial epithelium barrier, airway smooth muscle hypertrophy, and TGF-β signaling, and noted broader and stronger changes within the severe allergic asthma phenotype was observed (FIG.3). Tape-strips captured upregulation of DEGs in MAA and SAA involved in the smooth-muscle-associated mTOR pathway (e.g., PTEN, RHEB, LAMTOR3), mechanosensory-associated Wnt signaling (e.g., IQGAP1, VANGL2), ErbB pathway (e.g., ERBB3, EGFR), which has been implicated in bronchial epithelium repair, and TGF-β signaling pathway (e.g., ACVR1B, ID1 / 2). Upregulation of genes related to cell- stress-response pathways such as ubiquitin-dependent catabolism (e.g., CUL4B, ARRDC3, CANX) and mitochondrial structure and function (e.g., HIGD2A / hypoxia inducible domain, VDAC3, NDUFB9) were also captured. Additionally, tape-stripped skin from MAA versus HCs showed upregulated DEGs involved in the necroptosis pathway (e.g., CHMP4B, CHMP5), while SAA versus HCs showed upregulated DEGs involves in the ferroptosis-related (e.g., ACSL3, SLC39A8, SLC3A2) pathways (FIG. 3; |FCH|>2, FDR<0.05).

[0135] The study revealed upregulation DEGs of the type 1 immune response pathway (IFNGR1 / 2, STAT1, JAK1, STAT2) with concomitant downregulation of T2- related markers (CCL7 / CCL17 CCL18 / CCL22) and T-cell-activation (CD40LG) in MAA and / or SAA children versus HCs, with limited modulation of Th17 / Th22 related markers (IL32) in SAA (|FCH|>2, FDR<0.05). Additionally, common downregulation in MAA and SAA of genes encoding olfactory GPCRs (e.g., OR4A16, OR5AR1, OR10AG1) systems was observed, which may serve in protective mechanisms for the remodeling of the respiratory tract (|FCH|>2, FDR<0.05). Example 5: Comparison between tape strips biomarkers and endobronchial biopsies biomarkers

[0136] The tape-strip data was compared to 33 significantly expressed genes derived from a RNA-seq study on asthma patients’ endobronchial biopsies (Table 2). With tape-strips, dysregulation of 8 and 12 of the genes found in biopsies, in MAA and SAA groups, respectively (P<0.05) were captured. These genes include smooth- muscle- (MBNL1 / muscleblind-like splicing regulator 1), RNA-metabolism- (RNASET2, -44- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center CTR9), and cilia-function-related (SPEF2) markers, as well as ITGAV, an integrin gene contributory to Wnt pathway modulation, and HBB, which encodes hemoglobin subunit beta. Uniquely, MAA versus HCs captured gene dysregulation related to RNA metabolism (DHX15) and fibroblast signaling (FGF13 / fibroblast growth factor 13), while SAA versus HCs showed dysregulation of markers related to apoptosis (BCL2), host antimicrobial defense (LYZ), and cilia activity (STRBP / spermatid perinuclear RNA-binding protein). Table 2: Overlapping genes from the tape-strip data-set and previously published lung biopsy transcriptome showing comparisons of MAA and SAA vs Healthy control children MAA vs. Healthy Controls SAA vs. Healthy Controls SAA vs. MAA log2 Fold Fold p- DEG log2 Fold Fold DEG log2 Fold Fold DEG Marker Change Changevalue FDRStatus ChangeChange p-value FDRStatus ChangeChange p-value FDRStatus ACAP2 0.16 1.12 0.903 0.954 0 1.13 2.18 0.448 0.636 0 0.97 1.96 0.529 0.962 0APCDD1 1.16 2.24 0.436 0.666 0 0.94 1.92 0.567 0.730 0 -0.22 -1.16 0.899 0.99. 0BICC1 0.34 1.27 0.678 0.829 0 -1.04 -2.06 0.207 0.394 0 -1.39 -2.62 0.128 0.852 0C9orf84 -1.16 -2.23 0.103 0.295 0 0.06 1.04 0.946 0.973 0 1.22 2.33 0.170 0.877 0DCP1B 0.56 1.48 0.331 0.577 0 1.11 2.15 0.122 0.278 0 0.54 1.46 0.446 0.950 0DHX15 2.32 4.98 0.032 0.150 0 0.44 1.36 0.717 0.835 0 -1.87 -3.67 0.137 0.854 0seiDMXL1 1.92 3.79 0.087 0.268 0 2.73 6.64 0.035 0.123 0 0.81 1.75 0.536 0.962 0spFAM83B 0.63 1.55 0.635 0.803 0 1.58 2.99 0.302 0.499 0 0.95 1.93 0.547 0.962 0oiBFGF13 -1.73 -3.31 0.017 0.106 0 -1.49 -2.81 0.067 0.187 0 0.24 1.18 0.774 0.987 0niFKTN -0.21 -1.15 0.785 0.891 0 0.04 1.03 0.964 0.983 0 0.25 1.19 0.784 0.987 0dGCC2 0.74 1.67 0.367 0.607 0 0.38 1.3 0.687 0.816 0 -0.37 -1.29 0.704 0.982 0etal HBS1L 2.1 4.29 0.084 0.261 0 1.21 2.31 0.371 0.566 0 -0.89 -1.86 0.523 0.961 0ugITGAV 3.17 9.02 0.025 0.132 0 3.49 11.21 0.031 0.114 0 0.31 1.24 0.846 0.992 0er LRIG3 1.56 2.95 0.205 0.438 0 2.91 7.5 0.049 0.152 0 1.35 2.55 0.360 0.924 0p UMBNL1 3.39 10.47 0.006 0.058 0 3.46 11.02 0.013 0.065 0 0.07 1.05 0.958 0.996 0PARP14 0.39 1.31 0.778 0.888 0 -0.52 -1.43 0.732 0.843 0 -0.91 -1.87 0.568 0.967 0POSTN -1.27 -2.42 0.349 0.592 0 -0.97 -1.96 0.535 0.706 0 0.3 1.23 0.849 0.992 0SPEF2 -2.59 -6.01 0.027 0.137 0 -3.31 -9.9 0.006 0.041 -1 -0.72 -1.65 0.568 0.967 0STRBP 0.99 1.98 0.164 0.387 0 2.12 4.35 0.019 0.086 0 1.14 2.2 0.199 0.866 0UBP1 -0.86 -1.81 0.443 0.671 0 -1.08 -2.12 0.380 0.576 0 -0.23 -1.17 0.859 0.992 0WARS 0.79 1.73 0.497 0.711 0 0.77 1.7 0.554 0.720 0 -0.03 -1.02 0.984 0.997 0BCL2 -1.1 -2.14 0.189 0.419 0 -2.19 -4.57 0.011 0.059 0 -1.09 -2.13 0.232 0.889 0CTR9 2.18 4.53 0.046 0.184 0 3.65 12.54 0.005 0.034 1 1.47 2.77 0.248 0.890 0nDDB2 0.15 1.11 0.879 0.942 0 -0.02 -1.01 0.987 0.994 0 -0.17 -1.13 0.884 0.992 0i dDYX1C1 -0.9 -1.87 0.162 0.385 0 -2.04 -4.12 0.004 0.033 -1 -1.14 -2.21 0.119 0.841 0etsHBB -3.31 -9.93 0.017 0.107 0 -5.08 -33.71 0.001 0.015 -1 -1.76 -3.4 0.260 0.896 0al euis ITPR2 0.6 1.52 0.450 0.676 0 1.61 3.06 0.092 0.231 0 1.01 2.01 0.295 0.909 0g e p roi LAMC1 0.54 1.45 0.550 0.749 0 0.11 1.08 0.908 0.952 0 -0.43 -1.34 0.680 0.979 0n BLYZ -1.09 -2.13 0.350 0.593 0 -3.88 -14.72 0.007 0.046 -1 -2.79 -6.9 0.053 0.784 0woNUS1 1.42 2.67 0.299 0.545 0 1.64 3.12 0.290 0.487 0 0.23 1.17 0.887 0.992 0DRNASET2 3.87 14.62 0.003 0.036 1 3.83 14.27 0.004 0.034 1 -0.03 -1.02 0.980 0.997 0RNF180 -0.29 -1.22 0.613 0.791 1 -0.15 -1.11 0.825 0.901 0 0.14 1.1 0.845 0.992 0SLC9A8 -0.4 -1.32 0.715 0.850 0 2.52 5.75 0.084 0.217 0 2.92 7.56 0.043 0.761 0Example 6: Tape-strips capture dysregulation of the extracellular milieu in the lung parenchima of children with asthma -45- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center

[0137] The impaired turnover of the extracellular milieu (ECM) in the asthmatic lungs is associated with airway remodeling and epithelial barrier disruption. To understand how the skin may reflect dysregulation in the lung ECM, the overlap tape-strip DEGs with a curated gene list combining published datasets defining the asthmatic lung ECMwas analyzed (FIG. 1D). ECM-related genes were significantly upregulated in SAApatients compared to HCs, with similar trends in MAA, including genes linked to remodeling, including matrix metalloproteinase inhibitor TIMP3, and TGF-β co- receptor CD109, and mediators of cellular processing (EFEMP1, HCFC1, TNFSF10, PLXNB2) (all FCH>2, FDR<0.05; FIG.1D).

[0138] Downregulation of extracellular matrix regulatory genes (e.g., C1QTNF2, SEMA3B / E, PLOD3, LOXL1 / 2), core-matrisome-associated genes (e.g., COMP, FBLN2, TNXB), collagens (COL4A1 / 5A1 / 6A3 / 14A1), and secreted ECM factors (e.g., CHRDL1, INHBE) in SAA versus HCs, with similar trends observed in MAA (all |FCH|>2, FDR<0.05; FIG.1D) was observed. Example 7: Skin tape-strips inform on dysregulation of oxidative phosphorylation and aging mechanisms in the lung

[0139] Given the potential role of oxidative phosphorylation and aging-associated changes in asthma, two previously published datasets related to these processes (FIGs. 4A-B) against the tape-strips biomarkers were evaluated. ATP-synthase- (ATP5G2 / 6V0B / 5H) and NADH / respiratory-chain-related (NDUFA7 / A8 / AB1 / B8 / S3 / S5 / V1) genes were significantly upregulated in SAA versus HCs, and mitochondrial-respiratory-chain- (COX6B1 / 7B), ATP synthase- (ATP5C1 / 5F1 / 5G3 / 5L / 6V1G1 / 6V0E1), beta-amyloid- (APPBP2), NADH / respiratory- chain-related (NDUFA10 / B4 / B5 / B10 / S2) and oxidative-stress-related (GPNMB) genes were significantly upregulated in both MAA and SAA. In SAA versus HCs downregulation of MPEG1, reported to increase susceptibility to microbial infection was also observed (FIGs.4A-B; |FCH| ≥2, FDR<0.05 for all). Example 8: Pathway analysis highlights enrichment of disease-relevant gene ontologies -46- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center

[0140] For a broader perspective on the transcriptomic biomarkers diagnostic value, all DEGs were analyzed using function-based pathway databases (canonical / KEGG / REACTOME / BioCarta pathways; FDR<0.05). In both MAA and SAA versus HCs significant enrichment of oxidative phosphorylation, ER-Phagosome, RAC1, and Wnt-signaling pathways was observed. ErbB1 downstream signaling and PDGFR-beta signaling pathways were significantly enriched in MAA compared to HCs, and auto-degradation of CDH1 and interferon-signaling pathways were significantly enriched in SAA. In MAA genes from pathways involving autophagy and IFN-α signaling were significantly downregulated, while in SAA pathways involving the extracellular matrix and associated proteins and secreted factors were downregulated. Both MAA and SAA had significant downregulation of genes involved in pathways of olfactory transduction and GPCR signaling (FIG.5). Example 9: Skin tape transcriptomic biomarkers correlate with asthma relevant outcomes

[0141] Spearman correlations was used to assess the relationship between skin tape-strip transcriptomic biomarkers and asthma relevant outcomes: annualized exacerbation rate, lung function (large airways - FEV1, small airways - IOS R5-R20 and lung inflammation - FeNO). (Table 3). Significant negative correlations were found between FEV1 and transcriptomic biomarkers related to cytoskeleton and ciliary function (SYNE, C11ORF74), RNA transcription (NAGK, REXO1), as well as markers implicated in COPD pathogenesis and airway inflammation / hyper-responsiveness (ACER2, MCRS1, NOD1, CANT1, LEPREL1) (|r|>0.49; P<0.05 for all; Table 3). IOS R5-R20, a measure of peripheral airway resistance, was strongly and positively associated with lung-surfactant-production- (LPCAT1), ciliary-function- (e.g., C2CD3), EGFR- (LRRFIP1), and mitochondria-related (e.g., SLC25A37) genes (|r|>0.5; P<0.05 for all; Table 3). FeNO (fractional exhaled nitric oxide) correlated strongly and negatively with markers related to epithelial keratinization (KRTAP12-1 / 2,KRTAP5- 1 / 5, KRT73) and muco-ciliary activity (SCNM1, NEK10), as well as with taste receptor TAS2R43, regulatory marker CTLA4, and AKAP12, a gene found to be dysregulated in bronchial epithelial cells of asthma patients (|r|>0.5; P<0.05 for all; Table 3). Of note, many of the skin transcriptomic biomarkers correlated strongly and positively with the -47- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center annualized exacerbation rate (ACER2, NAGK, MCRS1, CANT1, MOSPD2, REXO1) (r >0.5, P<0.05 for all). Table 3: Top 25 correlations of tape strips transcriptomic biomarkers asthma relevant outcomes Asthma relevant outcomes Exacerbation x YR FEV1 IOS R5-R20 (Hz) FeNo Marker R p-value Marker R p-value Marker R p-value Marker R p-value RPTN -0.66 0.00 ZDHHC12 -0.74 0.00 DHPS -0.74 0.00 FAM227A -0.68 0.00EID2B -0.65 0.00 SLC44A2 -0.72 0.00 RXRB -0.70 0.00 STK17B -0.62 0.01TPM2 -0.63 0.00 CANT1 -0.72 0.00 MAPRE2 -0.70 0.00 C6orf123 -0.61 0.00PMCHL1 -0.63 0.01 REXO1 -0.71 0.00 OCLN -0.68 0.00 SMG7 -0.61 0.01ZNF670 -0.63 0.00 NOD1 -0.71 0.00 IGFL4 -0.68 0.00 CD38 -0.60 0.01DPH1 -0.63 0.00 NAGK -0.71 0.00 SLC38A1 -0.67 0.00 KRTAP12-2 -0.59 0.00LIPA -0.61 0.00 TASP1 -0.71 0.00 GSTO2 -0.67 0.00 SCNM1 -0.58 0.01PNO1 -0.60 0.01 XPC -0.70 0.00 USP28 -0.67 0.00 CTLA4 -0.58 0.00MALT1 -0.60 0.00 ZNF330 -0.70 0.00 FOXO1 -0.66 0.00 WDR66 -0.57 0.04MMP12 -0.59 0.01 LEPREL1 -0.69 0.00 SGK223 -0.66 0.00 KRT73 -0.57 0.01HLA-DQA2 -0.59 0.00 BLVRA -0.68 0.00 AHNAK -0.66 0.00 AKAP12 -0.57 0.00CLMP -0.59 0.01 HERC6 -0.68 0.00 C1orf112 -0.66 0.00 ZNF516 -0.56 0.02FOPNL -0.59 0.02 C11orf74 -0.68 0.00 ERBB3 -0.65 0.00 KRTAP5-5 -0.56 0.02DLX5 -0.59 0.04 MOSPD2 -0.68 0.00 TBRG1 -0.65 0.00 SIRPB2 -0.55 0.03CRISPLD1 -0.59 0.01 APLP2 -0.68 0.01 LRP11 -0.65 0.00 OR7C1 -0.55 0.02DOCK10 -0.59 0.00 LRRC41 -0.67 0.00 GGT6 -0.65 0.00 ADD3 -0.55 0.04PCDH 18 -0.59 0.00 SYNE2 -0.67 0.00 RNF152 -0.65 0.00 GRIN3B -0.55 0.01KLHL33 -0.59 0.01 TMUB2 -0.67 0.01 HECTD3 -0.63 0.00 KRTAP12-1 -0.55 0.02DDHD1 -0.58 0.01 PDPR -0.67 0.00 PPP1R8 -0.62 0.00 KRTAP5-1 -0.55 0.05PRMT7 -0.57 0.01 HSPA13 -0.67 0.02 RPL37A -0.62 0.00 FKBP5 -0.54 0.03ZNF320 -0.57 0.00 CMAS -0.67 0.00 ATL2 -0.61 0.00 SLC6A20 -0.54 0.03RFTN2 -0.57 0.01 TSPYL2 -0.66 0.01 MPZL2 -0.59 0.00 TAS2R43 -0.54 0.01CTDSPL2 -0.56 0.01 WRB -0.66 0.00 SEC24C -0.59 0.00 CLEC4M -0.54 0.02CMAS 0.59 0.00 OR1K1 0.80 0.00 GALNS 0.52 0.00 ACAT1 0.76 0.00C6orf136 0.59 0.01 TK1 0.73 0.00 ZNF259 0.52 0.00 SIK1 0.66 0.00U2AF2 0.60 0.01 SIM2 0.71 0.00 TSPY26P 0.52 0.00 TMED3 0.65 0.00REXO1 0.60 0.01 GPT2 0.71 0.00 EMR2 0.55 0.00 KRT8 0.63 0.01MOSPD2 0.61 0.02 DENND5B 0.70 0.01 BEST1 0.56 0.00 POMGNT1 0.63 0.01PPP1R12C 0.61 0.00 DPH1 0.70 0.00 SAT1 0.58 0.00 OBSL1 0.62 0.01PAQR6 0.61 0.00 TPM2 0.69 0.00 RNASET2 0.59 0.00 LRRC14 0.62 0.01TSR3 0.61 0.01 LINC00675 0.69 0.02 LPCAT1 0.59 0.00 TRMU 0.60 0.00IRF2BPL 0.62 0.00 LINC00494 0.69 0.01 ZNF165 0.59 0.00 WDR34 0.59 0.00CANT1 0.62 0.00 RABL2A 0.69 0.00 DOC2B 0.61 0.00 NRBP2 0.59 0.01LPCAT4 0.62 0.00 PTPDC1 0.68 0.00 LHFPL2 0.61 0.00 DGKA 0.59 0.00PON3 0.64 0.00 PPP2RC 0.67 0.01 PTPRE 0.61 0.00 POP5 0.58 0.01TMEM161B 0.64 0.00 RNF157 0.67 0.02 OLFML3 0.62 0.00 LZTS2 0.57 0.02FOXO4 0.64 0.00 FADS1 0.67 0.01 USP36 0.6. 0.00 MUS81 0.57 0.01CROCCP2 0.65 0.00 KLHL33 0.67 0.00 KCNJ15 0.65 0.00 KRT6A 0.57 0.00RCE1 0.66 0.00 RPTN 0.67 0.00 MT1E 0.66 0.00 A2ML1 0.57 0.01VPS8 0.67 0.00 GABARAPL2 0.66 0.01 GPCPD1 0.67 0.00 MCRS1 0.56 0.00MCRS1 0.68 0.00 ADAMTS10 0.66 0.00 RAB30 0.69 0.00 DAPK3 0.56 0.00SLC35A2 0.68 0.00 CLCF1 0.66 0.01 SMCHD1 0.69 0.00 ARHGEF26 0.56 0.01-48- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center NAGK 0.71 0.00 SLURP1 0.66 0.00 ANKS3 0.70 0.00 ATP10D 0.56 0.01BCOR 0.71 0.00 C16orf45 0.66 0.01 SLC25A37 0.71 0.00 LZIC 0.56 0.00RRNAD1 0.72 0.00 PRKRIP1 0.65 0.00 NEIL1 0.71 0.00 AES 0.56 0.03IKBKE 0.83 0.00 C1QL2 0.65 0.01 RFFL 0.79 0.00 CASZ1 0.56 0.01Example 9: Skin tape-strips biomarkers distinguish children with asthma from healthy controls

[0142] Using receiver operator characteristic (ROC) curves, tape strips biomarkers were evaluated for use to accurately discriminate between allergic asthma and healthy controls (FIGs. 6A-6D; Table 4). TSSC4, a tumor-suppressor gene linked toemphysema, and FAM212B, a negative regulator of catalytic activity that isdysregulated in fibroblasts in idiopathic lung fibrosis, comprised the best two-gene classifier, discriminating allergic asthma from HCs with 100% accuracy (area under the curve / AUC, 1.0) when both genes were detected (FIG.6A). TAS1R3, a nasal cilia sensor for microbial glucose metabolism, and ZNF416, a regulator of fibroblast mechano-activation, were the best single-gene classifiers, discriminating allergic asthma from HCs with an AUC of 0.975 (FIGs.6B-C). SCNM1, a regulator of length and functioning of cilia in COPD, had an AUC of 0.95 for asthma diagnosis (FIG.6D). Other top genes discriminating asthma from HC are shown in Table 4. Table 4: List of top 50 two-gene and one-gene classifiers determined by area under the receiving operating characteristic (ROC) curves for transcriptomic changes found by tape-stripping healthy appearing skin of moderate allergic asthma (MAA) and severe allergic asthma (SAA) children. Paired Genes Paired AUC Score Gene AUC ScoreTSSC4 and FAM212B 1.000 TAS1R3 0.975SCNM1 and FAM212B 0.975 ZNF416 0.975EPC2 and FAM212B 0.975 SCNM1 0.950ZNF235 and FAM212B 0.975 LRRC71 0.950EFNA4 and FAM212B 0.975 EPC2 0.950 NOSTRIN and0.975 SMC2 0.950FAM212B AREG and FAM212B 0.975 TINF2 0.950FAM212B and NACAD 0.975 ZNF235 0.950FAM212B and ALKBH1 0.975 RRP7B 0.950 SLC47A2 and 0.975 YARS 0.933 FAM212B SEPW1 and FAM212B 0.975 EFNA4 0.933FAM212B and LZIC 0.975 NOSTRIN 0.933 SNRPC and FAM212B 0.975 AREG 0.933PLAC9 and FAM212B 0.975 FBXO4 0.933 -49- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center SLC39A9 and 0.975 NACAD 0.933 FAM212B GABPB1 and FAM212B 0.975 SH3BGRL 0.933FAM212B and SMC2 0.950 ALKBH1 0.933ZNF416 and FAM212B 0.933 SLC47A2 0.933LRRC71 and FAM212B 0.933 SEPW1 0.933TINF2 and FAM212B 0.933 EIF1AY 0.933YARS and FAM212B 0.933 LZIC 0.925FBXO4 and FAM212B 0.933 FAM212B 0.925SH3BGRL and 0.933 DAB2 0.925 FAM212B EIF1AY and FAM212B 0.933 SNRPC 0.925 DAB2 and FAM212B 0.933 IFNA16 0.925IFNA16 and FAM212B 0.933 PLAC9 0.925TOMM40 and 0.933 TSSC4 0.925 FAM212B TAS1R3 and FAM212B 0.908 SLC39A9 0.925FAM212B and RRP7B 0.908 GABPB1 0.925SNRPCand YARS 0.867 TOMM40 0.925IFNA16 and SCNM1 0.867 ZNF138 0.917SLC39A9 and YARS 0.867 ARPC3 0.917 SNRPC and YARS 0.858 NUCB1 0.917YARS and TAS1R3 0.850 LRRC3 0.917YARS and EPC2 0.850 MUL1 0.908YARS and ZNF235 0.850 DNAJC8 0.908EFNA4 and YARS 0.850 KCNN3 0.908 YARS and NOSTRIN 0.850 CACNA1E 0.908AREG and YARS 0.850 SOS 1 0.908 YARS and NACAD 0.850 SLC22A14 0.908YARS and ALKBH1 0.850 CD80 0.908YARS and LZIC 0.850 MCM2 0.908PLAC9 and YARS 0.850 STAG1 0.908YARS and SMC2 0.842 NME9 0.908YARS and ZNF416 0.825 ZNF518B 0.908YARS and SCNM1 0.825 FBXO8 0.908 YARS and LRRC71 0.825 GPM6A 0.908SLC47A2 and YARS 0.825 TMEM174 0.908YARS and SEPW1 0.825 BTNL9 0.908 Example 10: DISCUSSION

[0143] This study evaluated the value of exploring the normal-appearing skin of children with allergic asthma using non-invasive tape-strips. A transcriptomic signature was captured that characterizes asthma pathogenetic pathways (immune response, ECM dysregulation, smooth muscle hypertrophy, epithelium barrier defect, TGF-β signaling, and oxidative stress), significantly correlates with asthma-relevant outcome, and distinguishes with high accuracy asthmatic children from healthy controls. -50- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center

[0144] A dysregulated respiratory epithelial barrier has been described as an essential pathogenetic mechanism in asthma. Systemic, shared molecular / pathological processes among epithelial barriers interacting with the environment were hypothesized. Significant downregulation of the skin epithelial barrier in normal-appearing skin in MAA, which was more pronounced in SAA than in healthy children, was observed. The dysregulation included decreased terminal differentiation products (e.g., FLG / filaggrin, CDSN), keratins (e.g., KRT23 / KRT77), and cell-adhesion markers (e.g., CLDN18, CDH19, JAM2). This observation is aligned with prior observations on barrier abnormalities in normal-appearing (non-lesional) skin in patients with AD.

[0145] This study revealed a significant upregulation of the Th1 / IFN-γ pathway (IFNGR1 / 2, STAT1, JAK1, STAT2) in the tape-stripped skin, with the most remarkable upregulation in the skin from SAA. This finding suggests a distinct inflammatory profile in the skin of asthma patients, different from the T2-driven inflammation reported in the primarily affected organ, the respiratory tract. However, local and systemic T1 immune response upregulation has been reported in patients with severe asthma and found to be related to increased disease severity. Th1 / IFN-γ upregulation may be associated with epithelial barrier abnormalities, as suggested by the results. In addition, the absence of T2 inflammation in the skin may explain the normal appearance of skin in asthma.

[0146] Given the well-established role of oxidative stress and ageing pathways in the asthmatic respiratory tract, relevant biomarkers were studied and it was observed that there is significant dysregulation in apoptosis- (BCL2), respiratory-chain- (COX56B1, COX7B), ATP-metabolism- (e.g., ATP5C1, ATP6V1G1), and ubiquitination-pathway- related (e.g., CUL4B, CANX) genes in MAA versus HCs, with greater dysregulation in SAA versus HCs. Barrier dysfunction is key to the skin and lung oxidative process and has been specifically linked to mitochondrial damage by producing reactive oxygen species (ROS) and hypoxia-inducible factor 1-alpha (HIF1α). In the respiratory tract, oxidative stress and Wnt signaling influence mitochondrial morphogenesis, mechano- activation, myofibroblast proliferation, and further cellular reprogramming. In skin, catabolic changes through increased upregulation of Wnt / β-catenin- (e.g., IQGAP1, VANGL2), mTOR- (e.g., PTEN, RHEB, RRAGA), ErbB- (e.g., ERBB2 / 3, CRK, EGFR), -51- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center TGF-β-signaling markers, and smooth muscle-Rac1-related genes in MAA were observed, and even more significantly in SAA, versus HCs.

[0147] Using previously published gene-sets characterizing the ECM of the lung dysregulation of matrix-related markers were identified, as well as genes involved in smooth muscle activity and EGFR / TGF-β signaling in MAA and SAA.

[0148] Moreover, tape-strips were able to detect dysregulation in genes associated with lung surfactant homeostasis (LPCAT1).

[0149] Downregulations in GPCRs was identified in MAA and SAA related to the olfactory and taste-receptor family (e.g., OR4A16, TAS2R50, TAS2R43). Olfactory receptor gene transcripts have been reported dysregulated in other atopic diseases such as AD, especially on S. aureus-colonized lesions. In the respiratory tract, taste and olfactory receptors are known to sense bacterial products, regulate chemosensory systems, and mediate ciliary beating and smooth muscle growth. These findings suggest that taste / olfactory receptor dysregulation may be shared in atopic diseases and detectable even in normal-appearing skin.

[0150] Multiple transcriptomic skin biomarkers were identified to strongly correlate with asthma-related outcomes (annualized exacerbation rate, lung function - FEV1 and IOS R5-R20, and lung inflammation - FeNO). Additionally, two lung-related genes (TSSC4 and FAM212B) were also identified that achieve 100% accuracy in distinguishing children with allergic asthma from healthy children. These results support the potential role of skin tape-strips as minimally invasive tools to evaluate pediatric asthma endotypes.

[0151] It is envisaged that this data, supported by the strong clinical correlations and ability to distinguish children with allergic asthma from healthy children, accurately provides a solid foundation for more extensive prospective investigations that would include and compare biomarkers across all compartments (lung, blood, and skin) in different pediatric populations and age groups.

[0152] In conclusion, tape-strips of normal-appearing skin from children with moderate and severe allergic asthma reveal a unique transcriptomic profile -52- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center distinguishing asthma from healthy controls with excellent accuracy, highlighting asthma severity-dependent dysregulation of the epithelial barrier, T1 inflammation, and biomarkers associated with several asthma pathogenetic processes and with relevant asthma-related outcomes such as exacerbations, lung function, and lung inflammation. Key findings provided herein include: tape-strips of normal appearing skin from children with moderate allergic asthma (MAA) and severe allergic asthma (SAA) depict dysregulation of genes associated with epithelial barrier (e.g., FLG, KRT23); (b) tape-strips from MAA and SAA children capture transcriptomic biomarkers that characterize the lung extracellular milieu such as Wnt signaling / β-catenin (e.g., IQGAP1, VANGL2), and GPCRs (e.g., TAS1R3, OR4A16); (c) the transcriptomic biomarkers correlate with asthma related outcomes and a two-gene classifier, for example TSSC4 and FAM212B, differentiate asthmatic children from HCs with 100% accuracy. These data validate the role of tape strips as a minimally invasive tool to study molecular mechanisms underlying asthma endotypes, identify new therapeutic targets, and monitor changes in disease activity over time. Example 11: SUMMARY

[0153] Introduction: Tape-strips, a minimally invasive method validated for the evaluation of several skin diseases, can identify asthma-specific biomarkers in the skin of children with allergic asthma and can be used to differentiate patients with allergic asthma from healthy controls.

[0154] Methods: Skin tape-strips are obtained and analyzed with RNA-seq / qPCR method from children with allergic asthma and healthy controls (HCs). Differentially expressed genes (DEGs) are identified by fold change ≥2 with a false discovery rate <0.05. Transcriptomic biomarkers are analyzed for their accuracy in distinguishing asthma from HCs, their relationships with asthma-related outcomes (exacerbation rate, lung function - FEV1, IOS-R5-20, and lung inflammation - FeNO), and their links to skin (barrier and immune response) and lung (remodeling, metabolism, aging) pathogenetic pathways.

[0155] Results: Significant downregulation of epidermal transcriptomic biomarkers could be seen for terminal differentiation (e.g., FLG / filaggrin), cell adhesion (e.g., -53- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center CDH19, JAM2), lipid biosynthesis / metabolism (e.g., ACOT2, LOXL2) in patients with asthma as compared to controls. Skin transcriptomic biomarkers are expected to correlate with the annualized exacerbation rate and with lung function parameters. A two-gene classifier (TSSC4-FAM212B) will be used to differentiate asthma from HCs with 100% accuracy.

[0156] Conclusion: Tape-strips are able to detect epithelial barrier and asthma- associated signatures in normal-appearing skin from children with allergic asthma and may serve as an alternative to invasive approaches for evaluating asthma endotypes and monitor response to treatment. -54- 101054975

Claims

PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center CLAIMS What is claimed is:

1. A method of treatment of allergic asthma in a subject, comprising: (a) providing a surface skin sample from the subject obtained from one or more tape-strips applied to a skin surface, wherein application of the one or more tape-strips to the skin surface obtains skin materials to the tape- strip; (b) determining a level of one or more biomarkers in the surface skin sample; (c) comparing the levels of the one or more biomarkers in the surface skin sample to a reference level of the one or more biomarkers to obtain a comparative level; (d) determining that the subject has allergic asthma based on the comparative level; and (e) administering to the subject an allergic asthma therapy.

2. A method of diagnosing allergic asthma in a subject, comprising: (a) providing a surface skin sample from the subject obtained from one or more tape-strips applied to a skin surface, wherein application of the one or more tape-strips to the skin surface obtains skin materials to the tape- strip; (b) determining a level of one or more biomarkers in the surface skin sample; (c) comparing the levels of the one or more biomarkers in the surface skin sample to a reference level of the one or more biomarkers to obtain a comparative level; and -55- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center (d) determining that the subject has allergic asthma based on the comparative level.

3. A method of detecting the efficacy of treatment of allergic asthma in a subject, comprising: (a) providing a surface skin sample from the subject obtained from one or more tape-strips applied to a skin surface, wherein application of the one or more tape-strips to the skin surface obtains skin materials to the tape- strip; and wherein the surface skin sample is obtained at a time after administering an allergic asthma therapy to the subject; (b) determining a level of one or more biomarkers in the surface skin sample; (c) comparing the levels of the one or more biomarkers in the surface skin sample to a reference level to obtain a comparative level; and (d) determining the efficacy of allergic asthma therapy based on the comparative level.

4. A method of treatment of allergic asthma in a subject by administering an allergic asthma therapy; wherein the subject is diagnosed with allergic asthma based on the steps of: (a) providing a surface skin sample from the subject obtained from one or more tape-strips applied to a skin surface, wherein application of the one or more tape-strips to the skin surface obtains skin materials to the tape- strip; (b) determining a level of one or more biomarkers in the surface skin sample; (c) comparing the levels of the one or more biomarkers in the surface skin sample to a reference level of the one or more biomarker to obtain a comparative level; and -56- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center (d) determining that the subject has allergic asthma based on the comparative level.

5. The method of any one of claims 1-4, wherein more than one tape strips are applied to the skin surface of the subject for providing the skin sample.

6. The method of claim 5, wherein 5-25, or 5-10, or 10-15, or 15-20, or 20-25 tape strips are applied to the skin surface of the subject for providing the skin sample.

7. The method of any one of claims 1-4, comprising determining the levels of two or more biomarkers in the surface skin sample.

8. The method of claim 7, comprising determining the levels of two biomarkers in the surface skin sample.

9. The method of any one of claim 1-4, 7 or 8, wherein the one or more biomarkers comprises a gene product of a Wnt-signaling / β-catenin pathway gene, a cell- adhesion gene, a lipid metabolism gene, NADH / respiratory chain gene, ATP synthase pathway gene, an oxidative stress gene, a immunomodulatory gene, a collagen gene, an extracellular matrix synthesis pathway gene, a gene related to COPD pathogenesis and airway inflammation / hyper-responsiveness, a transcription factor, a muco-ciliary activity related gene, a taste receptor related gene, a epithelial keratinization related gene, or combinations thereof.

10. The method of claim 9, wherein the one or more biomarkers comprises a gene product of TSSC4, FAM212B, SCNM1, EPC2, ZNF235, EFN235, EFNA4, NOSTRIN, AREG, NACAD, or combinations thereof.

11. The method of claim 10, wherein the one or more biomarkers comprises the gene product of FAM212B.

12. The method of claim 8, wherein the two biomarkers comprise gene products of TSSC4 and FAM212B, SCNM1 and FAM212B, EPC2 and FAM212B, ZNF235 and FAM212B, EFN235 and FAM212B, EFNA4 and FAM212B, NOSTRIN and FAM212B, AREG and FAM212B, or NACAD and FAM212B. -57- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center 13. The method of any one of claims 9-12, wherein the gene product is down- regulated in the subject.

14. The method of any one of claims 9-12, wherein the gene product is up- regulated in the subject.

15. The method of any one of claims 9-14, wherein the gene product is an RNA transcript, a protein, or both.

16. The method of any one of claims 1-4, wherein step (b) uses one or more of an RNA-sequencing analysis, a PCR analysis, a qPCR analysis, a proteomic analysis, or combinations thereof.

17. The method of claim 16, wherein step (b) uses an RNA-sequencing analysis.

18. The method of any one of claims 1-4, wherein the level of the one or more biomarkers is determined by measuring mRNA levels of the biomarker.

19. The method of any one of claims 1-4, wherein the level of the one or more biomarkers is determined by measuring cDNA levels of the biomarker.

20. The method of any one of claim 1-4, wherein the reference level comprises one or more of the levels of the one or more biomarkers from a subject not suffering for allergic asthma, an average of the level of the one or more biomarkers from subjects not suffering for allergic asthma, a previously provided reference level from the subject.

21. The method of any one of claims 1-6, wherein the one or more tape-strips is applied to the same skin surface one or more times.

22. The method of any one of claims 1-6, and 21, wherein the skin surface is non- lesioned skin.

23. The method of claim 1, wherein the allergic asthma therapy comprises a topical treatment, phototherapy, inhalation therapy, oral medication, a biologic, or combinations thereof. -58- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center 24. The method of claim 1, wherein the allergic asthma therapy comprises administering a long-acting beta-agonist, a long-acting anticholinergic agent, leukotriene modifier, mast cell stabilizer, theophylline, an inhaled corticosteroid, a combination inhaler, a biologic, an immunomodulator; a short-acting beta- agonist, systemic corticosteroids, or combinations thereof.

25. The method of claim 24, wherein the inhaled corticosteroid comprises fluticasone propionate (Flovent HFA, Flovent Diskus, Xhance), budesonide (Pulmicort Flexhaler, Pulmicort Respules, Rhinocort), ciclesonide (Alvesco), beclomethasone (Qvar Redihaler), mometasone (Asmanex HFA, Asmanex Twisthaler) or fluticasone furoate (Arnuity Ellipta); the leukotriene modifier comprises montelukast (Singulair), zafirlukast (Accolate) or zileuton (Zyflo); the short-acting beta-agonist comprises albuterol or levalbuterol; the anticholinergic agent comprises ipratropium or tiotropium; the systemic corticosteroid comprises prednisone, methyl prednisone; biologics comprise omalizumab (Xolair), mepolizumab (Nucala), dupilumab (Dupixent), reslizumab (Cinqair), or benralizumab (Fasenra).

26. The method of claim 1, wherein the subject does not have atopic dermatitis.

27. The method of claim 1, wherein the subject does not require a lung biopsy, blood tests, or nasal brushing for determining that the subject has allergic asthma.

28. The method of claim 1, wherein the subject is a human.

29. A method of differentially treating moderate allergic asthma (MAA) or severe allergic asthma (SAA) in a subject, comprising: (a) providing a surface skin sample from the subject obtained from one or more tape-strips applied to a skin surface, wherein application of the one or more tape-strips to the skin surface obtains skin materials to the tape- strip; (b) determining a level one or more biomarkers in the surface skin sample; -59- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center (c) comparing the levels of the one or more biomarkers in the surface skin sample to a reference level of the one or more biomarkers in a reference sample to obtain a comparative level; (d) determining that the subject has MAA or SAA based on the comparative level; and (e) administering to the subject an allergic asthma therapy suitable for MAA or SAA.

30. A method of determining the efficacy of treatment of allergic asthma in a subject, comprising: (a) providing a first level of one or more biomarkers in a first surface skin sample from the subject; (b) administering to the subject an allergic asthma therapy suitable for MAA or SAA; (c) providing a second level of one or more biomarkers in a second surface skin sample from the subject obtained at a later time; (d) comparing the first level of the one or more biomarkers to the second level of the one or more biomarkers to obtain a comparative level; and (e) determining the efficacy of the treatment based on the comparative level; wherein the first skin sample is obtained from a first set of one or more tape-strips applied to a skin surface, and the second skin sample is obtained from a second set of one or more tape-strips applied to a skin surface; and wherein application of the tape-strips to the skin surface obtains skin materials to the tape-strip.

31. The method of claim 30, further comprising changing the treatment modality if the treatment is determined to be ineffective based on the comparative level. -60- 101054975PATENT-PCT Attorney Docket No.093698-831013 Via Patent Center 32. The method of any one of claims 1-4 and 29-30, wherein the subject is less than 5 years, or less than 10 year, or less than 13 years of age. -61- 101054975

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