Adropin as a biomarker for cardiometabolic disease

Abstract

Methods for diagnosing cardiometabolic disease are disclosed herein. The methods utilize analysis of adropin as a biomarker. The presently disclosed subject matter further relates to the use of adropin as a biomarker for the detection of heart failure with preserved ejection fraction (HFpEF).

Description

[0001] ADROPIN AS A BIOMARKER FOR CARDIOMETABOLIC DISEASE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 710,806 filed October 23, 2024, and U.S. Provisional Application No. 63 / 797,411 filed April 30, 2025, priority to each of which is claimed, and the contents of each of which are incorporated by reference in their entireties.

[0004] SEQUENCE LISTING A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on October 17, 2025, is entitled “072396.1104_ST26. xml”, and is 5,948 bytes in size.

[0005] GRANT INFORMATION

[0006] This invention was made with government support under HL147861 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] INTRODUCTION

[0008] The presently disclosed subject matter relates to the use of adropin as a biomarker for cardiometabolic disease. The presently disclosed subject matter further relates to the use of adropin as a biomarker for the detection of heart failure with preserved ejection fraction (HFpEF).

[0009] BACKGROUND

[0010] Heart failure with preserved ejection fraction (HFpEF) is a metabolic disease that affects approximately three million Americans each year and has an expected mortality of about 50% within five years of diagnosis. Current diagnosis protocols rely on echocardiography or invasive hemodynamic measurements of cardiac diastolic dysfunction.

[0011] There are currently no single diagnostic or prognostic biomarkers for HFpEF in humans. Typical heart failure markers (e.g., natriuretic peptides) give confounding results in HFpEF, and are not as useful as in other heart failure etiologies. Alternative measures of diagnosing or prognosticating HFpEF are expensive or invasive. As such, there is an ongoing need for non-invasive or minimally-invasive biomarkers (diagnostic or prognostic) for HFpEF.

[0012] SUMMARY OF THE INVENTION

[0013] The presently disclosed subject matter provides methods for diagnosing a cardiometabolic disease, comprising a) determining the level of adropin in one or more samples from a subject; b) comparing the level of adropin to a reference level; and c) diagnosing the subject as having a high risk of cardiometabolic disease if the level of adropin in the one or more samples is less than the reference level. In certain embodiments, the method further comprises administering a therapeutically effective amount of one or more therapeutic agent. In certain embodiments, the one or more therapeutic agent comprises an anti-inflammatory agent, an anti-fibrotic agent, a diuretic, an angiotensin receptor-neprilysin inhibitor (ARNI), an angiotensin receptor blockers (ARB), a mineralocorticoid antagonist (MRA), a sodium-glucose cotransporter-2 (SGLT2) inhibitor, metformin, a glucagon-like peptide-1 (GLP-1) agonist, a gastric inhibitory polypeptide (GIP) antagonist, an angiotensinconverting enzyme (ACE) inhibitor, a beta-blocker, a nondihydropyridine calcium channel blocker, or a combination thereof. In certain embodiments, the one or more therapeutic agent comprises an adropin-based therapy. In certain embodiments, the method further comprises providing surgical intervention or a pacemaker to the subject.

[0014] In certain embodiments, the one or more samples are collected from at least one selected from the group consisting of whole blood, blood plasma, and combinations thereof. In certain embodiments, the reference level is the amount of adropin in one or more reference samples collected from a healthy individual. In certain embodiments, the reference level is the level of adropin in the same subject at an earlier timepoint. In certain embodiments, the level of adropin in the one or more samples is determined using ELISA or mass spectrometry. In certain embodiments, the cardiometabolic disease is selected from the group consisting of heart failure with preserved ejection fraction (HFpEF), hypertension, or non-alcoholic fatty liver disease (NAFLD). In certain embodiments, the cardiometabolic disease is heart failure with preserved ejection fraction (HFpEF). In certain embodiments, the adropin-based therapy is administered intravenously, intraperitoneally, or subcutaneously. In certain embodiments, the methods comprise diagnosing the subject as having a high risk of cardiometabolic disease if the level of adropin in the one or more samples is decreased by at least about 20% with respect to the reference level. In certain embodiments, the adropin-based therapy is a recombinant adropin peptide. In certain embodiments, the recombinant adropin peptide comprises SEQ ID NO.: 1 or SEQ ID NO.: 2. In certain embodiments, the recombinant adropin peptide is administered in an amount between about 1 ng / kg to about 200 mg / kg. In certain embodiments, the adropin-based therapy is a composition comprising a virus, wherein the virus comprises a heterologous nucleic acid encoding an Energy Homeostasis-associated (ENHO) gene. In certain embodiments, the virus is administered into the liver of the subject. In certain embodiments, the virus is administered to hepatocytes of the subject. In certain embodiments, the heterologous nucleic acid is operably linked to a promoter selected from the group consisting of thyroxine binding globulin (TBG), albumin (ALB), hepatitis virus (HBV), alpha- 1 antitrypsin (AAT), and human cytomegalovirus (CMV). In certain embodiments, the virus is an adenoviral associated virus (AAV). In certain embodiments, the AAV isotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV / DJ, and AAV / DJ8. In certain embodiments, the ENHO gene comprises SEQ ID NO.: 3.

[0015] The presently disclosed subject matter further provides methods for treating a cardiometabolic disease in a subject in need thereof, comprising a) determining the level of adropin in one or more samples from a subject; b) comparing the level of adropin to a reference level; and c) determining that the level of adropin in the one or more samples is decreased with respect to the reference level. In certain embodiments, the method further comprises administering a therapeutically effective amount of one or more therapeutic agent. In certain embodiments, the one or more therapeutic agent comprises an anti-inflammatory agent, an anti-fibrotic agent, a diuretic, an angiotensin receptor-neprilysin inhibitor (ARNI), an angiotensin receptor blockers (ARB), a mineralocorticoid antagonist (MRA), a sodiumglucose cotransporter-2 (SGLT2) inhibitor, metformin, a glucagon-like peptide- 1 (GLP-1) agonist, a gastric inhibitory polypeptide (GIP) antagonist, an angiotensin-converting enzyme (ACE) inhibitor, a beta-blocker, a nondihydropyridine calcium channel blocker, or a combination thereof. In certain embodiments, the one or more therapeutic agent comprises an adropin-based therapy. In certain embodiments, the method further comprises providing surgical intervention or a pacemaker to the subject.

[0016] In certain embodiments, the one or more samples are collected from at least one selected from the group consisting of whole blood, blood plasma, and combinations thereof. In certain embodiments, the reference level is the amount of adropin in one or more reference samples collected from a healthy individual. In certain embodiments, the reference level is the level of adropin in the same subject at an earlier timepoint. In certain embodiments, the level of adropin in the one or more samples is determined using ELISA or mass spectrometry. In certain embodiments, the cardiometabolic disease is selected from the group consisting of heart failure with preserved ejection fraction (HFpEF), hypertension, or non-alcoholic fatty liver disease (NAFLD). In certain embodiments, the cardiometabolic disease is heart failure with preserved ejection fraction (HFpEF). In certain embodiments, the adropin-based therapy is administered intravenously, intraperitoneally, or subcutaneously. In certain embodiments, the methods comprise determining that the level of adropin in the one or more samples is decreased by at least about 20% with respect to the reference level.

[0017] In certain embodiments, the adropin-based therapy is a recombinant adropin peptide. In certain embodiments, the recombinant adropin peptide comprises SEQ ID NO.: 1 or SEQ ID NO.: 2. In certain embodiments, the recombinant adropin peptide is administered in an amount between about 1 ng / kg to about 200 mg / kg. In certain embodiments, the adropin-based therapy is a composition comprising a virus, wherein the virus comprises a heterologous nucleic acid encoding an Energy Homeostasis-associated (ENHO) gene. In certain embodiments, the virus is administered into the liver of the subject. In certain embodiments, the virus is administered to hepatocytes of the subject. In certain embodiments, the heterologous nucleic acid is operably linked to a promoter selected from the group consisting of thyroxine binding globulin (TBG), albumin (ALB), hepatitis virus (HBV), alpha- 1 antitrypsin (AAT), and human cytomegalovirus (CMV). In certain embodiments, the virus is an adenoviral associated virus (AAV). In certain embodiments, the AAV isotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV / DJ, and AAV / DJ8. In certain embodiments, the ENHO gene comprises SEQ ID NO.: 3.

[0018] BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The following figures are included to illustrate certain aspects of the present disclosure and should not be viewed as exclusive embodiments. The subject matter disclosed is capable of considerable modifications, alterations, combinations, and equivalents in form and function, without departing from the scope of this disclosure.

[0020] FIGS. 1A-1B demonstrate adropin levels in human plasma. FIG. 1A shows adropin plasma concentration in age- and gender-matched human control and HFpEF patients, N = 19-20, unpaired students T-test. ** = p-value < 0.01. FIG. IB shows the receiver operator curve (ROC) of the sensitivity and specificity of adropin plasma concentration for the detection of HFpEF in human patients, N = 39 age- and gender-matched human control and HFpEF patients.

[0021] FIGS. 2A-2K demonstrates that recombinant adropin treatment restricts HFpEF progression. FIG. 2A shows adropin plasma concentration in age- and gender-matched human control and HFpEF patients, N = 19-20, unpaired students T-test. FIG. 2B shows study protocol using male C57BL / 6J chow (ProLab IsoPro RMH 3000), HFpEF (60% high fat diet, D12492, Research Diets; and 0.5 g / L-NAME in drinking water, Thermo Scientific), and HFpEF + adropin (Adr.; 450 nmol / kg / day i.p.) treated mice. FIGS. 2C-2D show intraperitoneal glucose tolerance test (IPGTT) (FIG. 2C) and glucose area under the curve (AUC) (FIG. 2D), N = 8-12, one-way ANOVA with Tukey’s post-hoc test. FIG. 2E shows representative M-mode echocardiography, pulsed-wave Doppler, Masson’s Trichrome stain, and Wheat Germ Agglutinin stain images. FIGS. 2F-2K show left ventricle ejection fraction (LVEF) (FIG. 2F), isovolumetric relaxation time (IVRT) (FIG. 2G), left ventricle myocardial performance index (LV MPI) (FIG. 2H), fibrosis (FIG. 21), cardiomyocyte cross sectional area (FIG. 2J), and isovolumetric contraction time (systolic function), N = 8-12, one-way ANOVA with Tukey’s post-hoc test.

[0022] FIGS. 3A-3D show pathway analyses following adropin treatment. FIG. 3A shows ShinyGO gene ontology pathway enrichment analysis of bulk RNA-seq from HFpEF+Adr. vs. HFpEF hearts, N = 4. FIG. 3B shows relative expression of cardiac fibrosis and extracellular matrix (ECM) genes in HFpEF+Adr. vs. HFpEF hearts, N = 4. FIG. 3C shows BioCyc pathway enrichment analysis of untargeted cardiac metabolites from HFpEF+Adr. vs. HFpEF hearts, N = 6. FIG. 3D shows Western blot analysis of total cardiac protein O-GlcNAcylation, N = 8-12, one-way ANOVA with Tukey’s post-hoc test.

[0023] FIG. 4 shows hexosamine biosynthesis / O-GlcNAcylation pathway. Regulatory enzymes measured by western blot (GF AT, OGT, OGA) are shown in black, N = 8-12, oneway ANOVA with Tukey’s post-hoc test. Pathway metabolites are shown in pink, N = 6, one-way ANOVA with Tukey’s post-hoc test.

[0024] FIGS. 5A-5G demonstrate that recombinant adropin treatment restricts HFpEF progression by modulating cardiac energy metabolism and protein O-GlcNAcylation. FIG.

[0025] 5A shows schematic of long chain acyl-CoA dehydrogenase (LCAD) enzymatic function. FIG. 5B shows immunoprecipitation analysis of LCAD O-GlcNAcylation status, N = 4. FIG.

[0026] 5C shows LCAD enzymatic activity from bulk cardiac tissue, N = 4, one-way ANOVA with Tukey’s post-hoc test. FIG. 5D shows simple linear regression analysis of relationship between LCAD O-GlcNAcylation status and enzymatic activity. FIG. 5E shows in vitro enzymatic activity of recombinant LCAD after exposure to O-GlcNAcylation substrate (UDP-GlcNAc) and / or recombinant O-GlcNAc transferase (OGT), N = 3, Kruskal-Wallace with Dunn’s post-hoc test. FIGS. 5F-5G show Western blot (FIG. 5G) and analysis (FIG. 5F) of cardiac PLIN5 abundance, N = 8-12, one-way ANOVA with Tukey’s post-hoc test.

[0027] FIGS. 6A-6C show that adropin functions in vivo through GPR19. FIG. 6A shows study protocol using female C57BL / 6N wildtype or GPR19 KO chow, HFpEF, and HFpEF + adropin (Adr.) treated mice. FIG. 6B shows left ventricle mass (LV Mass), left ventricle ejection fraction (LVEF), and isovolumetric relaxation time (IVRT), N = 6-10, two-way ANOVA with Tukey’s post-hoc test. FIG. 6C shows schematic of proposed GPR19-dependent adropin function in an experimental mouse HFpEF model. Use of parametric statistical tests was defined a priori based on sample sizes, and were carried out after meeting post-hoc normality testing outcomes. Non-parametric statistical tests were carried out when N < 4, or when data did not meet post-hoc normality testing outcomes.

[0028] FIGS. 7A-7B show body weight data. FIG. 7A shows final body weight of mice. FIG.

[0029] 7B shows body weight timecourse data. N = 8-12.

[0030] FIGS. 8A-8B show final heart weight normalized to tibia length (FIG. 8A) and cardiac output (FIG. 8B). N = 8-12.

[0031] FIGS. 9A-9L show bulk RNA-seq measurements of ECM / Fibrosis genes, specifically, Acta (FIG. 9A), Postn (FIG. 9B), Thyl (FIG. 9C), Pdgfra (FIG. 9D), Tcf21 (FIG.

[0032] 9E), Comp (FIG. 9F), Fnl (FIG. 9G), Vim (FIG. 9H), Collal (FIG. 91), Col3al (FIG. 9J), Col5al (FIG. 9K), and AYOT / U (FIG. 9L). N=4.

[0033] FIG. 10 shows correlation between LCAD activity and enzyme abundance. N = 4 per group. Circles = chow mice, squares = HFpEF mice and triangles = HFpEF+Adr. mice.

[0034] FIGS. 11A-11B show Western blot (FIG. 11 A) and analysis (FIG. 1 IB) of cardiac CD36 protein abundance. N = 8-12.

[0035] DETAILED DESCRIPTION

[0036] The presently disclosed subject matter relates to the use of adropin as a biomarker for cardiometabolic disease, e.g., heart failure with preserved ejection fraction (HFpEF). In certain embodiments, adropin is provided as recombinant protein. In certain embodiments, adropin is provided using gene therapy methods for inducing endogenous production of adropin. In certain embodiments, adropin is endogenously produced in liver hepatocytes.

[0037] For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections: 1. Definitions;

[0038] 2. Biomarkers for Non-invasive Detection;

[0039] 3. Methods of Treatment; and

[0040] 4. Pharmaceutical Compositions

[0041] 1. Definitions

[0042] The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the disclosure and how to make and use them.

[0043] As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Still further, the terms “having,” “including,” “containing” and “comprising” are interchangeable and one of skill in the art is cognizant that these terms are open ended terms.

[0044] As used herein, the terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of’, and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0045] As used herein, the term “about” or “approximately” means 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, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

[0046] As used herein, the term “culturing” refers to contacting a cell with a cell culture medium under conditions suitable to the survival, growth and / or proliferation of the cell. As used herein, the term “culture medium” refers to a nutrient solution used for growing cells, e.g., prokaryotic or eukaryotic cells, that typically provides at least one component from one or more of the following categories:

[0047] 1) an energy source, usually in the form of a carbohydrate such as glucose;

[0048] 2) all essential amino acids, and usually the basic set of twenty amino acids plus cysteine;

[0049] 3) vitamins and / or other organic compounds required at low concentrations;

[0050] 4) free fatty acids; and

[0051] 5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.

[0052] As used herein, the term “cell” refers to any suitable cell for use in the present disclosure, e.g., eukaryotic cells. For example, but not by way of limitation, suitable eukaryotic cells include animal cells, e.g., mammalian cells. In certain embodiments, suitable cells are cultured cells. In certain embodiments, suitable cells are host cells, recombinant cells, and recombinant host cells. In certain embodiments, suitable cells are cell lines obtained or derived from mammalian tissues which are able to grow and survive when placed in media containing appropriate nutrients and / or growth factors.

[0053] As used herein, the terms “host cell,” “host cell line” and “host cell culture” are used interchangeably and refer to cells and their progeny into which exogenous nucleic acid can be subsequently introduced to create recombinant cells. In certain embodiments, these host cells can also be modified (i.e., engineered) to alter or delete the expression of certain endogenous host cell proteins. Host cells can include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny does not need to be completely identical in nucleic acid content to a parent cell, but can contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. The introduction of exogenous nucleic acid (e.g., by transfection) to these host cells would create recombinant cells that are derived from the original “host cell,” “host cell line” or “host cell line”. The terms “host cell,” “host cell line” and “host cell culture” can also refer to such recombinant cells and their progeny.

[0054] As used herein, the terms “expression” or “expresses,” as used herein, refer to transcription and translation occurring within a cell, e.g., mammalian cell. In certain embodiments, the level of expression of a gene and / or nucleic acid in a cell can be determined on the basis of either the amount of corresponding mRNA that is present in the cell or the amount of the protein encoded by the gene and / or nucleic acid that is produced by the cell. For example, mRNA transcribed from a gene and / or nucleic acid is desirably quantitated by northern hybridization. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring Harbor Laboratory Press, 1989). Protein encoded by a gene and / or nucleic acid can be quantitated either by assaying for the biological activity of the protein or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay using antibodies that are capable of reacting with the protein. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989).

[0055] As used herein, the terms “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein or vector, indicate that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. For example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed, overexpressed or not expressed at all.

[0056] As used herein, the terms “vector” or “plasmid”, which can be used interchangeably, as used herein, refer to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The terms includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

[0057] As used herein, the terms “nucleic acid molecule” and “nucleotide sequence,” as used herein, refer to a single or double-stranded covalently-linked sequence of nucleotides in which the 3' and 5' ends on each nucleotide are joined by phosphodiester bonds. The nucleic acid molecule can include deoxyribonucleotide bases or ribonucleotide bases, and can be manufactured synthetically in vitro or isolated from natural sources.

[0058] As used herein, the terms “polypeptide,” “peptide,” “amino acid sequence” and “protein,” used interchangeably herein, refer to a molecule formed from the linking of at least two amino acids. The link between one amino acid residue and the next is an amide bond and is sometimes referred to as a peptide bond. A polypeptide can be obtained by a suitable method known in the art, including isolation from natural sources, expression in a recombinant expression system, chemical synthesis or enzymatic synthesis. The terms can apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. The term “protein” can refer to a sequence of amino acids for which the chain length is sufficient to produce the higher levels of tertiary and / or quaternary structure. This is to distinguish from “peptides” or other small molecular weight polypeptides that do not have such structure. In certain embodiments, the protein herein will have a molecular weight of at least about 15-20 kDa, e.g., about 20 kDa or greater. Examples of proteins encompassed within the definition herein include host cell proteins as well as all mammalian proteins, in particular, therapeutic and diagnostic proteins, such as therapeutic and diagnostic antibodies, and, in general proteins that contain one or more disulfide bonds, including multi-chain polypeptides comprising one or more inter- and / or intrachain disulfide bonds.

[0059] As used herein, the terms “protein variant” or “polypeptide variant”, refer to a protein or polypeptide that comprise modifications and / or truncations compared to a parent or wild type protein or polypeptide. In certain embodiments, a protein variant can differ from the parent protein or wild type protein by at least one amino acid modification, e.g., from about one to about ten amino acid modifications. In certain embodiments, the sequence of a protein variant sequence has at least about 80%, at least about 90%, at least about 95% or at least about at least about 99% identity to a parent or wild type protein sequence. In certain embodiments, a protein variant can differ from another variant of the protein by at least one amino acid modification, e.g., from about one to about ten amino acid modifications. In certain embodiments, the sequence of a protein variant sequence has at least about 80%, at least about 90%, at least about 95% or at least about at least about 99% identity to a different variant of the protein.

[0060] As used herein, the term “mutation” refers to a mutation in an amino acid sequence or in a nucleic acid sequence. In certain embodiments, a mutation in an amino acid sequence can be a substitution (replacement), an insertion (addition), or a deletion (truncation) of at least one amino acid in the amino acid sequence. In certain embodiments, a mutation in a nucleic acid sequence can be a substitution (replacement), an insertion (addition), or a deletion (truncation) of at least nucleotide of the nucleic acid sequence.

[0061] As used herein, the term “endogenous,” refers to a nucleic acid molecule or polypeptide that is normally expressed in a cell or tissue.

[0062] As used herein, the term “exogenous,” refers to a nucleic acid molecule or polypeptide that is not endogenously present in a cell. The term “exogenous” would therefore encompass any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as foreign, heterologous, and over-expressed nucleic acid molecules and polypeptides. The term “exogenous” nucleic acid, as used herein, refers to a nucleic acid not present in a native wild-type cell; for example, an exogenous nucleic acid can vary from an endogenous counterpart by sequence, by position / location, or both. For clarity, an exogenous nucleic acid can have the same or different sequence relative to its native endogenous counterpart; it can be introduced by genetic engineering into the cell itself or a progenitor thereof, and can optionally be linked to alternative control sequences, such as a non-native promoter or secretory sequence.

[0063] As used herein, the term “increase,” refers to altering positively by at least about 5%. An alteration can be an increase of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.

[0064] As used herein, the terms “reduce” and “decrease”, used interchangeably herein, refer to altering negatively by at least about 5%. An alteration can be a decrease of about 5%, about 10%, about 25%, about 30%, about 50%, about 75% or more, even by about 100%.

[0065] As used herein, “a functional fragment” of a molecule or polypeptide includes a fragment of the molecule or polypeptide that retains at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary function of the molecule or polypeptide.

[0066] As used herein, the terms “substantially identical” or “substantially homologous” refer to a polypeptide or a nucleic acid molecule exhibiting at least about 50% identical or homologous to a reference amino acid sequence (for example, any of the amino acid sequences described herein) or a reference nucleic acid sequence (for example, any of the nucleic acid sequences described herein). In certain embodiments, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% identical or homologous to the amino acid sequence or the nucleic acid sequence used for comparison.

[0067] As used herein, an “individual” or “subject” is a vertebrate, such as a human or nonhuman animal, for example, a mammal. Mammals include, but are not limited to, humans, non-human primates, and rodents.

[0068] As used herein, the term “disease” refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. As used herein, the term “therapeutically effective amount” or “effective amount” refers to an amount that is sufficient to improve cardiac function, e.g., improve diastolic function, improve cardiac output, improve ejection fraction, improve isovolumetric relaxation time (IVRT), improve left ventricle myocardial performance index (LV MPI), improve cardiac morphology (e.g., left ventricle mass, volume, and / or wall thickness), reduces body weight, and / or reduce glucose intolerance. In certain embodiments, the therapeutically effective amount refers to an amount of a therapeutic agent, e.g., a virus composition or a recombinant adropin polypeptide. The amount of a therapeutic agent that is therapeutically effective or effective can vary depending on the context. An effective amount can be administered in one or more administrations.

[0069] As used herein, and as well -understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this subject matter, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more sign or symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, prevention of disease, delay or slowing of disease progression, and / or amelioration or palliation of the disease state. The decrease can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% decrease in severity of complications or symptoms. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0070] “In combination with,” as used herein, means that a virus disclosed herein, and one or more agents are administered to a subject as part of a treatment regimen or plan.

[0071] 2. Adropin as a Biomarker for Non-invasive Detection

[0072] Non-invasive methods for assessing HFpEF are not currently available. Establishing biomarkers for detecting HFpEF through non-invasive methods is an unmet clinical need. The present disclosure demonstrates that circulating (blood) adropin levels are significantly decreased in HFpEF patients. Thus, circulating adropin levels are useful as a biomarker for HFpEF.

[0073] The present disclosure provides methods for diagnosing a cardiometabolic disease, e.g., HFpEF, in a subject or determining whether a subject is at risk or has high risk for cardiometabolic disease. The methods comprising determining the level of adropin in one or more samples from the subject; comparing the level of adropin to a reference level; and diagnosing the subject as having a high risk of a cardiometabolic disease, diagnosing the subject as being at risk of cardiometabolic disease, or diagnosing the subject as having high risk of cardiometabolic disease if the level of adropin in the one or more samples is decreased with respect to the reference level. In certain embodiments, the method further comprises diagnosing the subject as not having a high risk of cardiometabolic disease or as having low risk of cardiometabolic disease if the level of adropin in the one or more samples is not decreased with respect to the reference level. In certain embodiments, the method further comprises diagnosing the subject as not having a high risk of cardiometabolic disease or not having a high risk of a cardiometabolic disease if the level of adropin in the one or more samples is increased with respect to the reference level.

[0074] Adropin levels can be assessed from one or more samples obtained from a subject through non-invasive methods. In certain embodiments, the one or more samples are collected from at least one selected from the group consisting of whole blood, blood plasma, urine, and combinations thereof. In certain embodiments, the one or more samples are collected from whole blood or blood plasma. In certain embodiments, the one or more samples are collected from urine.

[0075] According to the methods disclosed herein, the level of adropin in the one or more samples is compared to a reference level. The reference level refers to an amount of adropin that is relevant for comparison. In certain embodiments, the reference level is a predetermined level of adropin. In certain embodiments, the reference level is the level of adropin in a healthy individual free of cardiometabolic disease, e.g., HFpEF, or a population of healthy individuals free of cardiometabolic disease. In certain embodiments, the reference level is the level of adropin in the same subject at an earlier timepoint.

[0076] In certain embodiments, the subject is suspected of having HFpEF, is at risk of HFpEF, or is considered high risk of HFpEF if the level of adropin in the one or more samples is decreased by at least about 1 ng / mL, at least about 2 ng / mL, at least about 3 ng / mL, at least about 4 ng / mL, at least about 5 ng / mL, at least about 6 ng / mL, at least about 7 ng / mL, at least about 8 ng / mL, at least about 9 ng / mL, at least about 10 ng / mL, at least about 11 ng / mL, at least about 12 ng / mL, at least about 13 ng / mL, at least about 14 ng / mL, at least about 15 ng / mL, at least about 16 ng / mL, at least about 17 ng / mL, at least about 18 ng / mL, at least about 19 ng / mL, or at least about 20 ng / mL with respect to the reference level. In certain embodiments, the subject is suspected of having HFpEF, is at risk of HFpEF, or is considered high risk of HFpEF if the level of adropin in the one or more samples is decreased by about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, or about 20 ng / mL with respect to the reference level. In certain embodiments, the subject is suspected of having HFpEF, is at risk of HFpEF, or is considered high risk of HFpEF if the level of adropin in the one or more samples is decreased by between about 1 and 20 ng / mL, between about 1 and 15 ng / mL, between about 1 and 10 ng / mL, between about 1 and 5 ng / mL, between about 5 and 20 ng / mL, between about 5 and 15 ng / mL, between about 5 and 10 ng / mL, between about 10 and 20 ng / mL, between about 10 and 15 ng / mL, or between about 15 and 20 ng / mL with respect to the reference level.

[0077] Alternatively, the subject is suspected of having HFpEF, is at risk of HFpEF, or is considered high risk of HFpEF if the level of adropin in the one or more samples is decreased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% with respect to the reference level. In certain embodiments, the subject is suspected of having HFpEF, is at risk of HFpEF, or is considered high risk of HFpEF if the level of adropin in the one or more samples is decreased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% with respect to the reference level. In certain embodiments, the subject is suspected of having HFpEF, is at risk of HFpEF, or is considered high risk of HFpEF if the level of adropin in the one or more samples is decreased by between about 25% and about 90%, between about 25% and about 75%, between about 25% and about 50%, between about 50% and about 90%, between about 50% and about 75%, or between about 75% and about 90% with respect to the reference level.

[0078] In certain embodiments, the subject is suspected of having HFpEF, is at risk of HFpEF, or is considered high risk of HFpEF if the level of adropin in the one or more samples is less than about 10 ng / mL, less than about 9 ng / mL, less than about 8 ng / mL, less than about 7 ng / mL, less than about 6 ng / mL, less than about 5 ng / mL, less than about 4 ng / mL, less than about 3 ng / mL, less than about 2 ng / mL, or less than about 1 ng / mL. In certain embodiments, the subject is suspected of having HFpEF, is at risk of HFpEF, or is considered high risk of HFpEF if the level of adropin in the one or more samples is about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, or about 10 ng / mL. In certain embodiments, the subject is suspected of having HFpEF, is at risk of HFpEF, or is considered high risk of HFpEF if the level of adropin in the one or more samples is between about 1 and 10 ng / mL, between about 1 and 5 ng / mL, between about 5 and 10 ng / mL.

[0079] The level of adropin in a sample can be measured using methods known in the art. In certain embodiments, the level of adropin in the one or more samples is determined using a biochemical method or a mass spectrometry method. In certain embodiments, the biochemical method is an ELISA. In certain embodiments, the mass spectrometry method comprises mass spectrometry (MS), liquid chromatography-mass spectrometry (LC-MS) or liquid chromatography with tandem mass spectrometry (LC-MS / MS). In certain embodiments, the one or more samples are collected from at least one biological sample selected from the group consisting of whole blood, blood plasma, urine, and combinations thereof.

[0080] 3. Methods of Treatment

[0081] The present disclosure provides methods of treating a subject having a cardiometabolic disease, e.g., HFpEF, after determining that the subject is at risk of cardiometabolic disease or has high risk of cardiometabolic disease. In certain embodiments, the methods comprise determining the level of adropin in one or more samples from the subject; comparing the level of adropin to a reference level; and determining that the level of adropin in the one or more samples is decreased with respect to the reference level. In certain embodiments, the method further comprises administering a therapeutically effective amount of one or more therapeutic agent to the subject (e.g., an adropin-based therapy), providing surgical intervention to the subject, or providing a pacemaker to the subject.

[0082] In certain embodiments, the reference level is a predetermined level of adropin. In certain embodiments, the reference level is the level of adropin in a healthy individual free of cardiometabolic disease, e.g., HFpEF, or a population of healthy individuals free of cardiometabolic disease. In certain embodiments, the reference level is the level of adropin in the same subject at an earlier timepoint.

[0083] In certain embodiments, the therapeutically effective amount of the one or more therapeutic agent is administered to the subject after determining that the level of adropin in the one or more samples is decreased by at least about 1 ng / mL, at least about 2 ng / mL, at least about 3 ng / mL, at least about 4 ng / mL, at least about 5 ng / mL, at least about 6 ng / mL, at least about 7 ng / mL, at least about 8 ng / mL, at least about 9 ng / mL, at least about 10 ng / mL, at least about 11 ng / mL, at least about 12 ng / mL, at least about 13 ng / mL, at least about 14 ng / mL, at least about 15 ng / mL, at least about 16 ng / mL, at least about 17 ng / mL, at least about 18 ng / mL, at least about 19 ng / mL, or at least about 20 ng / mL with respect to the reference level. In certain embodiments, the therapeutically effective amount of the one or more therapeutic agent is administered to the subject after determining that the level of adropin in the one or more samples is decreased by about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, about 10 ng / mL, about 11 ng / mL, about 12 ng / mL, about 13 ng / mL, about 14 ng / mL, about 15 ng / mL, about 16 ng / mL, about 17 ng / mL, about 18 ng / mL, about 19 ng / mL, or about 20 ng / mL with respect to the reference level. In certain embodiments, the therapeutically effective amount of the one or more therapeutic agent is administered to the subject after determining that the level of adropin in the one or more samples is decreased by between about 1 and 20 ng / mL, between about 1 and 15 ng / mL, between about 1 and 10 ng / mL, between about 1 and 5 ng / mL, between about 5 and 20 ng / mL, between about 5 and 15 ng / mL, between about 5 and 10 ng / mL, between about 10 and 20 ng / mL, between about 10 and 15 ng / mL, or between about 15 and 20 ng / mL with respect to the reference level.

[0084] Alternatively, the therapeutically effective amount of the one or more therapeutic agent is administered to the subject after determining that the level of adropin in the one or more samples is decreased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% with respect to the reference level. In certain embodiments, the therapeutically effective amount of the one or more therapeutic agent is administered to the subject after determining that level of adropin in the one or more samples is decreased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% with respect to the reference level. In certain embodiments, the therapeutically effective amount of the one or more therapeutic agent is administered to the subject after determining that the level of adropin in the one or more samples is decreased by between about 25% and about 90%, between about 25% and about 75%, between about 25% and about 50%, between about 50% and about 90%, between about 50% and about 75%, or between about 75% and about 90% with respect to the reference level.

[0085] In certain embodiments, the therapeutically effective amount of the one or more therapeutic agent is administered to the subject after determining that the level of adropin in the one or more samples is less than about 10 ng / mL, less than about 9 ng / mL, less than about 8 ng / mL, less than about 7 ng / mL, less than about 6 ng / mL, less than about 5 ng / mL, less than about 4 ng / mL, less than about 3 ng / mL, less than about 2 ng / mL, or less than about 1 ng / mL. In certain embodiments, the therapeutically effective amount of the one or more therapeutic agent is administered to the subject after determining that the level of adropin in the one or more samples is about 1 ng / mL, about 2 ng / mL, about 3 ng / mL, about 4 ng / mL, about 5 ng / mL, about 6 ng / mL, about 7 ng / mL, about 8 ng / mL, about 9 ng / mL, or about 10 ng / mL. In certain embodiments, the therapeutically effective amount of the one or more therapeutic agent is administered to the subject after determining that the level of adropin in the one or more samples is between about 1 and 10 ng / mL, between about 1 and 5 ng / mL, between about 5 and 10 ng / mL.

[0086] In certain embodiments, the one or more therapeutic agent comprises an antiinflammatory agent, an anti-fibrotic agent, a diuretic, an angiotensin receptor-neprilysin inhibitor (ARNI), an angiotensin receptor blockers (ARB), a mineralocorticoid antagonist (MRA), a sodium-glucose cotransporter-2 (SGLT2) inhibitor, metformin, a glucagon-like peptide-1 (GLP-1) agonist, a gastric inhibitory polypeptide (GIP) antagonist, an angiotensinconverting enzyme (ACE) inhibitor, a beta-blocker, a nondihydropyridine calcium channel blocker, an adropin-based therapy, or a combination thereof. In certain embodiments, the one or more therapeutic agent is selected from the group consisting of anti-inflammatory agents, anti-fibrotic agents, diuretics, angiotensin receptor-neprilysin inhibitors (ARNIs), angiotensin receptor blockers (ARBs), and mineralocorticoid antagonists (MRAs), sodiumglucose cotransporter-2 (SGLT2) inhibitors, metformin, glucagon-like peptide-1 (GLP-1) agonists, gastric inhibitory polypeptide (GIP) antagonists, angiotensin-converting enzyme (ACE) inhibitors, beta-blockers, nondihydropyridine calcium channel blockers, an adropin-based therapy, and combinations thereof.

[0087] In certain embodiments, the adropin-based therapy comprises recombinant adropin peptide or a virus that comprises a nucleic acid that encodes adropin or a functional fragment thereof, and combinations thereof. In certain embodiments, the methods include administering to the subject a virus that expresses adropin or a functional fragment thereof. For example, but not by way of limitation, the methods can include administering to the subject a virus that comprises a nucleic acid that encodes adropin, e.g., human adropin, or a functional fragment thereof. Alternatively or in addition, the methods include administering to the subject recombinant adropin peptide. In certain embodiments, the abundance (circulating levels) of adropin is increased. In certain embodiments, the adropin-based therapy is administered intravenously, intraperitoneally, or subcutaneously.

[0088] 3.1 Adropin Therapy

[0089] The adropin-GPR19 pathway represents a novel therapeutic target, consisting of an endogenously produced ligand (adropin), and a membrane-bound receptor (GPR19). Previous work showed that: (i) adropin treatment restores glucose oxidation in the hearts of diabetic mice; (ii) adropin treatment decreases liver glucose production in diabetic mice; and (iii) that adropin function in cardiac cells is dependent on GPR19. The present disclosure shows that: (i) adropin treatment restores diastolic function in vivo in a mouse model of heart failure with preserved ejection fraction (HFpEF) - a model which combines obesity / diabetes and hypertension, two cardiometabolic disease features; and that (ii) adropin treatment reduces body weight and glucose intolerance in the same HFpEF model. Thus, adropin-GPR19 pathway is a novel, targetable pathway in the treatment of metabolic diseases. Non-limiting examples of metabolic diseases include HFpEF, type 2 diabetes, hypertension, and non-alcoholic fatty liver disease (NAFLD).

[0090] In certain embodiments, the cardiometabolic disease is selected from the group consisting of heart failure with preserved ejection fraction (HFpEF), hypertension, or nonalcoholic fatty liver disease (NAFLD). In certain embodiments, the cardiometabolic disease is HFpEF.

[0091] In certain embodiments, the method disclosed herein improves diastolic function, improves cardiac output, improves ejection fraction, improves isovolumetric relaxation time (IVRT), improves left ventricle myocardial performance index (LV MPI), improves cardiac morphology (e.g., left ventricle mass, volume, and / or wall thickness), reduces body weight, and / or reduces glucose intolerance. In certain embodiments, the method disclosed herein improves one or more feature selected from the group consisting of diastolic function, cardiac output, ejection fraction, IVRT, LV MPI, cardiac morphology (e.g., left ventricle mass, volume, and / or wall thickness), body weight, glucose intolerance, and combinations thereof. In certain embodiments, the method disclosed herein improves one or more features about 1 week post-treatment, about 2 weeks post-treatment, about 3 -weeks post-treatment, about 4 weeks post-treatment, about 5 weeks post-treatment, about 6 weeks post-treatment, about 7 weeks post-treatment, about 8 weeks post-treatment, about 9 weeks post-treatment, or about 10 weeks post-treatment.

[0092] 3,1,1 Recombinant Polypeptide

[0093] Full-length adropin is endogenously produced in liver hepatocytes, and a cleaved bioactive version of the peptide (adropin34-76) is secreted into the systemic circulation to act on other peripheral tissues. Recombinant adropin polypeptide can be produced using methods known in the art. Recombinant adropin polypeptide can be produced as the full-length peptide, or as the cleaved product of amino acids 34-76.

[0094] In certain embodiments, the polypeptide encodes a human adropin or a functional fragment thereof. In certain embodiments, the human adropin has an amino acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequence set forth in GenBank / NCBI database accession no. NP 940975.2. In certain embodiments, the adropin polypeptide can contain substitutions, insertions, or deletions relative to the amino acid sequence set forth in GenBank / NCBI database accession no. NP 940975.2, that do not significantly alter the function or activity of the human adropin.

[0095] Alternatively, the human adropin polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 1, which is provided below.

[0096] MGAAISQGALIAIVCNGLVGFLLLLLWVILCWACHSRSADVDSLSESSPNSSPGPCP EKAPPPQKPSHEGSYLLQP (SEQ ID NO. : 1)

[0097] Alternatively, the human adropin polypeptide comprises or consists of an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the amino acid sequence set forth in SEQ ID NO: 2, which is provided below.

[0098] CHSRSADVDSLSESSPNSSPGPCPEKAPPPQKPSHEGSYLLQP (SEQ ID NO.: 2)

[0099] In certain embodiments, recombinant adropin peptide is administered in an amount between about 1 to about 200 mg, between about 1 to about 100 mg, between about 10 to about 50 mg, about 10 mg, about 20 mg, about 25 mg, about 40 mg, about 50 mg, about 75 mg, or about 100 mg. In certain embodiments, recombinant adropin peptide is administered in an amount between about 10 to about 1000 ng, between 100 to about 1000 ng, between about 500 to about 1000 ng, about 100 ng, about 125 ng, about 150 ng, about 200 ng, about 300 ng, about 400 ng, about 500 ng, about 750 ng, or about 1000 ng. In certain embodiments, recombinant adropin peptide is administered in an amount that is at least about 100 ng, at least about 125 ng, at least about 150 ng, at least about 200 ng, at least about 300 ng, at least about 400 ng, at least about 500 ng, at least about 750 ng, at least about 1000 ng, at least about 50 mg, at least about 10 rag, at least about 20 mg, at least about 25 mg, at least about 40 mg, at least about 50 mg, at least about 75 mg, or at least about 100 mg. In certain embodiments, recombinant adropin peptide is administered in an amount between about 1 ng / kg to about 200 mg / kg. In certain embodiments, recombinant adropin peptide is administered in an amount between about 1 to about 200 mg / kg, between 1 to about 100 mg / kg, between about 1 to about 10 mg / kg, between about 10 to about 50 mg / kg, about 1 mg / kg, about 2 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, about 25 mg / kg, about 40 mg / kg, about 50 mg / kg, about 75 mg / kg, about 100 mg / kg, or about 200 mg / kg. In certain embodiments, recombinant adropin peptide is administered in an amount between about 1 to about 1000 ng / kg, between 1 to about 100 ng / kg, between about 1 to about 10 ng / kg, between about 10 to about 50 ng / kg, between about 50 to about 100 ng / kg, between about 100 to about 500 ng / kg, between about 500 to about 1000 ng / kg, about 1 ng / kg, about 2 ng / kg, about 5 ng / kg, about 10 ng / kg, about 20 ng / kg, about 25 ng / kg, about 40 ng / kg, about 50 ng / kg, about 75 ng / kg, about 100 ng / kg, about 200 ng / kg, about 300 ng / kg, about 400 ng / kg, about 500 ng / kg, about 600 ng / kg, about 700 ng / kg, about 800 ng / kg, or about 900 ng / kg. In certain embodiments, recombinant adropin peptide is administered in an amount that is at least about 1 ng / kg, at least about 2 ng / kg, at least about 5 ng / kg, at least about 10 ng / kg, at least about 20 ng / kg, at least about 25 ng / kg, at least about 40 ng / kg, at least about 50 ng / kg, at least about 75 ng / kg, at least about 100 ng / kg, at least about 200 ng / kg, at least about 300 ng / kg, at least about 400 ng / kg, at least about 500 ng / kg, at least about 600 ng / kg, at least about 700 ng / kg, at least about 800 ng / kg, at least about 900 ng / kg, at least about 1 mg / kg, at least about 2 mg / kg, at least about 5 mg / kg, at least about 10 mg / kg, at least about 20 mg / kg, at least about 25 mg / kg, at least about 40 mg / kg, at least about 50 mg / kg, at least about 75 mg / kg, at least about 100 mg / kg, or at least about 200 mg / kg.

[0100] In certain embodiments, recombinant adropin peptide is administered from about 1 week to about 4 weeks, about 1 week to about 2 weeks, about 2 weeks to about 4 weeks, about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the composition is administered daily. In some embodiments, the composition is administered once a week, twice a week, three times a week, or four times a week. In certain embodiments, the adropin peptide is administered intravenously, intraperitoneally, or subcutaneously.

[0101] 3,1,2 Gene Therapy Methods

[0102] Constructs encoding adropin, or constructs encoding related protein variants, as described herein, can be introduced into cells as one or more DNA molecules or constructs, in many cases in association with one or more markers to allow for selection of host cells which contain the construct(s). The constructs can be prepared in conventional ways, where the coding sequences and regulatory regions can be isolated, as appropriate, ligated, cloned in an appropriate cloning host, analyzed by restriction or sequencing, or other convenient means. Particularly, using PCR, individual fragments including all or portions of a functional unit can be isolated, where one or more mutations can be introduced using “primer repair”, ligation, in vitro mutagenesis, etc. as appropriate.

[0103] In certain embodiments, the expression construct encoding the polypeptide or protein of interest is integrated into one or more expression vectors. In certain embodiments, the expression vector is a nucleic acid and provides all required elements for the amplification of said vector in a mammalian cell. In certain embodiments, an expression vector is a vehicle for the introduction of an expression construct into a modified mammalian cell according to the subject matter of the present disclosure. In certain embodiments, a construct can be introduced as a single DNA molecule encoding multiple genes, or different DNA molecules having one or more genes. In certain embodiments, multiple constructs can be introduced simultaneously or consecutively, each with the same or different DNA molecule.

[0104] The construct(s) once completed and demonstrated to have the appropriate sequences can then be introduced into a host cell by any convenient means. The constructs can be integrated and packaged into non-replicating, defective viral genomes like Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others, including retroviral vectors, for infection or transduction into cells. In certain embodiments, the constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like. The host cells will in some cases be grown and expanded in culture before introduction of the construct(s), followed by the appropriate treatment for introduction of the construct(s) and integration of the construct s). The cells will then be expanded and screened by virtue of a marker present in the construct.

[0105] In certain embodiments, expressing one or more recombinant proteins of interest in a host cell includes culturing a cell comprising one or more nucleic acid(s) encoding the polypeptide or protein of interest, under conditions suitable for expression of the polypeptide or protein. Non-limiting examples of such cells are disclosed herein, e.g., mammalian cells can be used to express the polypeptide or protein. In certain embodiments, a host cell is transfected with a vector containing the nucleic acid sequence suitable for expression of said polypeptide or protein of interest.

[0106] Recombinant adropin provides a robust response in vivo, resulting in a significant increase in glucose tolerance and diastolic function in a mouse model of HFpEF. However, the in vivo stability of recombinant adropin is sub-optimal for therapeutic use, with 96% of the peptide cleared from circulation within 1 hour. The present disclosure relates to a gene therapy approach for providing more consistent therapeutic outcomes. The ENHO gene (which produces adropin) is encoded in an adenoviral-associated viral (AAV) vector, a standard, non-integrating virus that is routinely used for gene therapy in humans. In certain embodiments, the virus isotype is AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV / DJ, or AAV / DJ8.

[0107] In certain embodiments, the ENHO gene is expressed under the control of a hepatocyte promoter to limit expression in non-hepatic tissues. Targeting the ENHO virus to the liver is beneficial for several reasons. For example, as the site of the majority of endogenous adropin production, the liver contains the necessary packaging and secretion mechanisms to deliver adropin to the circulation. In addition, the liver contains 10-15% of the body blood volume, making it an ideal site of circulating protein production. Finally, the half-life of differentiated adult hepatocytes is 200-300 days, meaning that individual treatments are likely to maintain adropin protein production for 3-6+ months.

[0108] In certain embodiments, the virus disclosed herein includes a nucleic acid molecule encoding ENHO or a functional fragment thereof. In certain embodiments, the nucleic acid molecule encodes human ENHO or a functional fragment thereof. In certain embodiments, the human ENHO has a nucleic acid sequence that is at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the nucleic acid sequence set forth in GenBank / NCBI database accession no. NM 198573.3. In certain embodiments, the nucleic acid molecule encoding human ENHO can contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the nucleic acid sequence set forth in GenBank / NCBI database accession no. NM 198573.3, that do not significantly alter the function or activity of the human adropin.

[0109] Alternatively, the nucleic acid molecule encoding human ENHO comprises or consists of a nucleic acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or 100% homologous or identical to the nucleic acid sequence set forth in SEQ ID NO: 3, which is provided below. AGACCGCCCGCGGCAGAGGCCGCCCCGGTCGCGCCGCGGCGGGAGCGGCCGGT GGAGGCTGCGCGGCCGAGGGGGAGGGCCGGGGGAGCGGACGTCGCCTCTGCT GGTCTCCCACCTCCCGCCGCCCCCCGCCCGCAGGCTCCCAAGCCTTAGTCGGCG CCGAGCATCCCGCTGCCCCGGACCCTCCCGCGGGCGCGCACCAGGCTCAACTC AGGCTCAGGACTGCAGGTAGACATCTCCACTGCCCAGGAATCACTGAGCGTGC AGACAGCACAGCCTCCTCTGAAGGCCGGCCATACCAGAGTCCTGCCTCGGCAT GGGCCTCACCATTGAGGCAGCTCCACTGTCTGTGCTGGTCTGAGGGTGCTGCCT GTCATGGGGGCAGCCATCTCCCAGGGGGCCCTCATCGCCATCGTCTGCAACGGT CTCGTGGGCTTCTTGCTGCTGCTGCTCTGGGTCATCCTCTGCTGGGCCTGCCATT CTCGCTCTGCCGACGTTGACTCTCTCTCTGAATCCAGTCCCAACTCCAGCCCTG GCCCCTGTCCTGAGAAGGCCCCACCACCCCAGAAGCCCAGCCATGAAGGCAGC TACCTGCTGCAGCCCTGAAGGCCCCTGGCCTAGCCTGGAGCCCAGGACCTAAG TCCACCTCACCTAGAGCCTGGAATTAGGATCCCAGAGTTCAGCCAGCCTGGGGT CCAGAACTCAAGAGTCCGCCTGCTTGGAGCTGGACCCAGCGGCCCAGAGTCTA GCCAGCTTGGCTCCAATAGGAGCTCAGTGGCCCTAAGGAGATGGGCCTGGGGT GGGGGCTTATGAGTTGGTGCTAGAGCCAGGGCCATCTGGACTATGCTCCATCCC AAGGGCCAAGGGTCAGGGGCCGGGTCCACTCTTTCCCTAGGCTGAGCACCTCT AGGCCCTCTAGGCTGGGGAAGCAAACTGGAACCCATGGCAATAATAGGAGGGT GTCCAGGCTGGGCCCCTCCCCTGGTCCTCCCAGTGTTTGCTGGATAATAAATGG AACTATGGCTCTA (SEQ ID NO. : 3)

[0110] In certain embodiments, the nucleic acid molecule encoding ENHO is integrated into the genome of the virus, where the expression of the nucleic acid molecule is operably linked to a promoter that is active or activatable in the virus infected cell. As used herein, “operably linked” means that a promoter is in a correct functional location and / or orientation in relation to a nucleic acid locus to control transcriptional initiation and / or expression of that locus. In certain embodiments, the nucleic acid encoding ENHO is operably linked to a promoter which induces expression in hepatocytes. Non-limiting examples of promoters include thyroxine binding globulin (TBG), albumin (ALB), hepatitis virus (HBV), alpha- 1 antitrypsin (AAT), and human cytomegalovirus (CMV).

[0111] Alternatively or in addition, the adropin-based therapy is a composition comprising a virus, wherein the vims comprises a heterologous nucleic acid encoding an Energy Homeostasis-associated (ENHO) gene. In certain embodiments, the virus is administered into the liver of the subject. In certain embodiments, the vims is administered to hepatocytes of the subject. In certain embodiments, the virus is an adenoviral associated virus (AAV). In certain embodiments, the AAV isotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV / DJ, and AAV / DJ8. In certain embodiments, the virus is administered intravenously, intraperitoneally, or subcutaneously.

[0112] In certain embodiments, the virus is administered in an amount between about IO5to about IO20genome copies per kg, between about 105to about IO10genome copies per kg, between about IO10to about IO13genome copies per kg, between about 1013to about 102ugenome copies per kg, about 103genome copies per kg, about 106genome copies per kg, about 107genome copies per kg, about 108genome copies per kg, about 109genome copies per kg, about IO10genome copies per kg, about 1011genome copies per kg, about 1012genome copies per kg, about 1013genome copies per kg, about 1014genome copies per kg, about 1015genome copies per kg, about 10lbgenome copies per kg, about IO17genome copies per kg, about 1018genome copies per kg, about 1019genome copies per kg, or about IO20genome copies per kg. In certain embodiments, the virus is administered in an amount that is at least about 105genome copies per kg, at least about 106genome copies per kg, at least about 107genome copies per kg, at least about 108genome copies per kg, at least about IO9genome copies per kg, at least about IO10genome copies per kg, at least about 1011genome copies per kg, at least about 1012genome copies per kg, at least about 1013genome copies per kg, at least about 1014genome copies per kg, at least about IO15genome copies per kg, at least about 1016genome copies per kg, at least about 1017genome copies per kg, at least about 1018genome copies per kg, at least about IO19genome copies per kg, or at least about 102ugenome copies per kg.

[0113] 4. Pharmaceutical Compositions

[0114] The present disclosure further provides pharmaceutical compositions that include a virus that expresses adropin or a functional fragment thereof. For example, but not by way of limitation, the methods can include administering to the subject a virus that comprises a nucleic acid that encodes adropin, e.g., human adropin, or a functional fragment thereof. In certain embodiments, the pharmaceutical composition includes an effective amount of the virus. In certain embodiments, the pharmaceutical composition can be prepared as solutions, dispersions in glycerol, liquid polyethylene glycols, and any combinations thereof in oils, in solid dosage forms, as inhalable dosage forms, as intranasal dosage forms, as liposomal formulations, dosage forms comprising nanoparticles, dosage forms comprising microparticles, polymeric dosage forms, or any combinations thereof.

[0115] Alternatively or in addition, the pharmaceutical composition described herein further includes a pharmaceutically acceptable carrier, e.g., an excipient. In certain embodiments, the pharmaceutically acceptable carrier includes any carrier which does not interfere with the effectiveness of the biological activity of the active ingredients and / or that is not toxic to the patient to whom it is administered. Non-limiting examples of suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents and sterile solutions. Additional non-limiting examples of pharmaceutically acceptable carriers include gels, bioadsorbable matrix materials, implantation elements containing the virus, and any other suitable vehicle, delivery, or dispensing means or material.

[0116] In certain embodiments, the pharmaceutically acceptable carrier can be a buffering agent. Non-limiting examples of suitable buffering agents can include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate. As a buffering agent, sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium glucomate, aluminium hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, di sodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide other calcium salts, and combinations thereof.

[0117] In certain embodiments, the virus disclosed herein can be propagated in suitable host cells, isolated from host cells, and stored in conditions that promotes stability and integrity of the virus, such that loss of infectivity over time is minimized. In certain embodiments, the virus disclosed herein can be stored by freezing or drying, such as by lyophilization. In certain embodiments, prior to administration, the stored virus can be reconstituted (if dried for storage) and diluted in a pharmaceutically acceptable carrier for administration.

[0118] In certain embodiments, the pharmaceutical compositions disclosed herein can be provided systemically. In certain embodiments, the presently disclosed viruses or pharmaceutical compositions are directly injected into an organ of interest (e.g., liver). Alternatively, the presently disclosed viruses or pharmaceutical compositions are provided indirectly to the organ of interest, for example, by administration into the circulatory system. The viruses or pharmaceutical compositions can be provided intravenously, intraperitoneally, or subcutaneously. When administering a therapeutic composition of the presently disclosed subject matter (e.g., a pharmaceutical composition comprising a presently disclosed virus), it can be formulated in a unit dosage injectable form (solution, suspension, emulsion).

[0119] Alternatively or in addition, the pharmaceutical composition comprises a therapeutically effective amount of an adropin-based therapy. In certain embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In certain embodiments, the adropin-based therapy is an AAV, wherein the AAV comprises a heterologous nucleic acid encoding an ENHO gene. In certain embodiments, the ENHO gene comprises SEQ ID NO.: 3. In certain embodiments, the adropin-based therapy is a recombinant adropin peptide. In certain embodiments, the recombinant adropin peptide comprises SEQ ID NO.: 1. In certain embodiments, the recombinant adropin peptide comprises SEQ ID NO.: 2. In certain embodiments, the pharmaceutical composition is administered intravenously, intraperitoneally, or subcutaneously.

[0120] EXAMPLES

[0121] The present disclosure will be better understood by reference to the following Examples, which are provided as exemplary of the presently disclosed subject matter, and not by way of limitation.

[0122] EXAMPLE 1: Adropin Level as a Biomarker for HFpEF

[0123] Adropin is a circulating, liver-derived peptide that can be measured through proteomic (e.g. mass spectrometry) or biochemical (e.g. ELISA) techniques in human blood. Adropin levels were evaluated in human patients with cardiometabolic HFpEF.

[0124] Age- and gender-matched blood samples were obtained from healthy control and HFpEF patients. Adropin concentrations in blood plasma were significantly decreased in affected patients (FIGS. 1A and 2A). Thus, circulating (blood) adropin levels were significantly decreased in HFpEF patients. ROC analysis (FIG. IB) demonstrated that circulating adropin levels display sensitivity and specificity levels that would be useful as a HFpEF biomarker.

[0125] EXAMPLE 2: Recombinant Adropin Therapy

[0126] Therapeutic interventions that result in reduced body weight and lower blood glucose (e.g. treatment with the GLP-1 receptor agonist semaglutide [1]) improve patient symptoms in cardiometabolic HFpEF. This finding demonstrates that correcting systemic metabolic dysfunction is central to tackling the disease. In the heart itself, HFpEF patients display evidence of reduced fatty acid utilization [2] and altered glycolytic function [3], Whether these intrinsic metabolic changes drive cardiac dysfunction and HFpEF disease progression remains to be fully determined, and represents a logical next step in understanding HFpEF pathophysiology. Therefore, recombinant adropin, an antihyperglycemic hepatokine that regulates cardiac fuel substrate flexibility [4], was investigated for restricting maladaptive metabolic remodeling and cardiac dysfunction in HFpEF.

[0127] Twelve-week-old C57BL / 6J wildtype male mice were exposed to normal chow, or a combination of 60% high fat diet plus L-NAME (0.5 g / L in drinking water) to induce HFpEF. After eight weeks, HFpEF mice were randomly assigned to either a control HFpEF group, or to a HFpEF group receiving daily intraperitoneal injections of recombinant adropin (450 nmol / kg) for the remaining four weeks of the study (HFpEF+Adr.) (FIG. 2B).

[0128] Daily adropin treatment significantly increased whole-body glucose tolerance in HFpEF mice (FIGS. 2C and 2D). In contrast to semaglutide treatment in HFpEF patients [1], there was no significant change in body weight between HFpEF and HFpEF+Adr. mice (43.8 g vs. 40.1 g; P = 0.27). Echocardiographic and histological analyses (FIG. 2E) revealed that HFpEF mice developed cardiac diastolic dysfunction, increased interstitial fibrosis, and cardiomyocyte hypertrophy relative to control mice (FIGS. 2F-2J). In each case, adropin treatment significantly attenuated these dysfunctional parameters (FIGS. 2F-2J).

[0129] Bulk RNA-seq analysis of HFpEF and HFpEF+Adr. hearts revealed that adropin significantly reduced the expression of extracellular matrix remodeling and fibrosis genes (FIGS. 3A, 3B, and 9A-9L), matching the reduced interstitial fibrosis observed (FIG. 21). Untargeted metabolomic analysis showed a significant change in metabolites from the hexosamine biosynthesis pathway (HBP) (FIG. 3C), which generates the substrates for protein O-GlcNAc modification. These changes corresponded with a significant increase in total cardiac protein O-GlcNAcylation in HFpEF hearts relative to control mice, which was largely reversed (P < 0.06) in adropin-treated HFpEF animals (FIG. 3D). Interrogation of the HBP / O-GlcNAc pathway in HFpEF and HFpEF+Adr. mice showed no significant changes in the abundance of rate-limiting (GF AT) or regulatory (OGT and OGA) enzymes controlling protein O-GlcNAcylation (FIG. 4). Instead, a significant increase in metabolites related to HBP / O-GlcNAc activity (e.g. GlcNAc-6-P, CoA, Uridine) in HFpEF+Adr. mice was found relative to control HFpEF animals (FIG. 4), demonstrating reduced entry of HBP metabolites into the O-GlcNAcylation pathway after adropin treatment. Energy metabolism studies in patients show that HFpEF hearts use less free fatty acids, which correlates with reduced cardiac function [2], Long-chain acyl-CoA dehydrogenase (LCAD) displayed elevated O-GlcNAcylation in HFpEF hearts, which correlated with reduced enzymatic activity (FIGS. 5A-5D). Adropin treatment reversed the increase in LCAD O-GlcNAcylation, and led to a corresponding restoral of LCAD enzymatic activity (FIGS. 5B-5D). To confirm its regulatory effect on LCAD activity, in vitro O-GlcNAcylation assays were performed. LCAD activity was significantly decreased in the presence of the O-GlcNAcylation substrate (UDP-GlcNAc) and transferase enzyme (OGT), but not the substrate alone (FIG. 5E). Additionally, PLIN5 abundance (a marker of lipid droplet accumulation) was elevated in HFpEF hearts, and this was significantly reversed by adropin treatment (FIGS. 5F-5G). Overall, these data demonstrate that adropin treatment alleviates the inhibition of fatty acid utilization in HFpEF hearts, and reduces abnormal lipid accumulation.

[0130] Finally, the dependency of in vivo adropin function on GPR19, an orphan GPCR and putative receptor for this hepatokine in vitro [5], was investigated. Female wildtype or GPR19 knockout (GPR19 KO) mice on a C57BL / 6N background were placed on the same regimen as above (FIGS. 2B and 6 A), and subjected to echocardiography after two weeks of treatment. Wildtype mice developed diastolic dysfunction on a HFpEF diet, which was reversed by adropin treatment (FIG. 6B). In contrast, adropin failed to improve diastolic dysfunction in GPR19 KO mice, showing that this receptor is essential for adropin function in vivo (FIGS. 6B and 6C).

[0131] In summary, long-term recombinant adropin treatment restricts metabolic remodeling in a preclinical HFpEF model. Mechanistically, HFpEF induces O-GlcNAc-mediated inhibition of cardiac fatty acid oxidation, which is alleviated by adropin treatment. Combined, these data demonstrate that targeted measures to restore normal cardiac fuel substrate metabolism can represent a direct route in tackling cardiac dysfunction in HFpEF.

[0132] EXAMPLE 3: Recombinant Adropin Therapy

[0133] Cardiometabolic heart failure with preserved ejection fraction (HFpEF) is a heterogenous metabolic disease, which in the heart presents as left ventricle diastolic dysfunction, ventricular stiffness, and myocardial structural remodeling. Deleterious changes in cardiac metabolism are central to HFpEF pathophysiology, and proposed treatments for the disease have focused on repairing these defects. In this study, a preclinical mouse model that recapitulates cardiometabolic HFpEF was used to elucidate the molecular mechanisms driving cardiac dysfunction, and tested whether recombinant adropin (a liver- and brain-derived peptide hormone) could reverse observed defects. Long-term treatment with adropin reversed multiple markers of HFpEF-related cardiac dysfunction (including fibrosis, diastolic dysfunction, and cardiomyocyte hypertrophy). Using untargeted metabolomics, adropin treatment restricted deleterious metabolite entry into the hexosamine biosynthesis pathway, leading to a reduction in the inhibitory O-GlcNAcylation of the cardiac fatty acid oxidation enzyme long chain acyl-CoA dehydrogenase. Adropin can restore cardiac metabolic function in HFpEF, and targeting this pathway is a novel therapeutic avenue for this disease.

[0134] Heart failure is a complex clinical syndrome that results from any structural or functional impairments of ventricular filling and / or ejection of blood, and remains the leading cause of morbidity and mortality worldwide (Heidenreich et al., 2023). Heart failure with preserved ejection fraction (HFpEF), a subtype of heart failure, is primarily characterized by cardiac diastolic dysfunction. This presentation of the disease currently accounts for >50% of all heart failure cases, with a global prevalence of approximately 32 million (Shah et al., 2020, Redfield and Borlaug 2023). HFpEF is recognized as a heterogenous syndrome, whose underlying pathophysiological mechanisms include several extracardiac abnormalities such as metabolic derangements, arterial hypertension, microvascular endothelial dysfunction, inflammation, and renal insufficiency (Shah et al., 2016, Shah et al., 2020).

[0135] Partly due to a lack of specifically-designed drugs for the disease, treatment for HFpEF currently focuses on the management of comorbidities (e.g. obesity, type 2 diabetes, hypertension), with new guidelines recommending the use of glycemia-reducing therapeutics (e.g. SGLT2 inhibitors) that improve cardiovascular outcomes in heart failure (Kittleson et al., 2023). The recent STEP -HFpEF trial in obesity-related HFpEF demonstrated that therapy-mediated reductions in body weight can also be a key tool in disease regression (Kosiborod et al., 2023). These developments suggest that approaches targeting metabolic defects can offer substantial benefits in this disease. On this basis, whether treatment with recombinant adropin, a brain- and liver-derived endogenous peptide hormone (Kumar et al., 2008), can have beneficial effects in a preclinical model of cardiometabolic HFpEF was examined.

[0136] Previous studies have shown that adropin improves physical activity (Wong et al, 2014, Ganesh-Kumar et al., 2012), attenuates hepatic steatosis and injury (Kumar et al., 2008, Chen et al., 2019), and reduces vascular stiffness in diet- induced obesity (Jurrissen et al., 2022). In the heart, it was demonstrated that short-term adropin treatment can improve cardiac fuel substrate flexibility by restoring glucose oxidation in the diabetic heart (Thapa et al., 2019b). Combined, these beneficial features raised the question of whether this peptide can be used as a therapeutic intervention in HFpEF. Here, it is shown that long-term treatment with recombinant adropin restored cardiac diastolic function, reduced cardiac hypertrophy and fibrosis, and improved whole-body glucose tolerance. These changes were linked to a reversal of metabolic remodeling and improved fatty acid oxidation enzyme activity, supporting that adropin treatment mitigates the impact of HFpEF stimuli via improved cardiometabolic function.

[0137] Methods

[0138] Animal Care and Use. Male C57BL / 6J 8-week-old mice were obtained from the Jackson Laboratory and maintained on chow for 4 weeks to acclimate to their new environment. Animals were housed under standard conditions, with ad libitum access to water and food, and maintained on a constant 12-hour light / dark cycle. At 12 weeks of age, mice were exposed to normal chow (Chow; 60% carbohydrate, 26% protein, 14% fat; ProLab IsoPro RMH 3000), or a combination of high-fat diet (HFD; 20% carbohydrate, 20% protein, 60% fat; Research Diets D12492) plus N-co-nitro-L-arginine methyl ester (L-NAME: 0.5 g / L, pH 7.4; ThermoFisher) supplemented in drinking water to induce HFpEF (Schiattarella et al., 2019). After 8 weeks of diet, mice were randomly assigned to a control or treatment group receiving daily intraperitoneal injections of either vehicle or recombinant adropin (Adr.) at 450 nmol / kg animal weight, prepared in PBS with 0.1% BSA, for the remaining 4 weeks of the study. L-NAME supplemented drinking water was changed weekly, and adropin was made fresh and sterile filtered before each use. Animals were euthanized by isofluorane anesthesia followed by cervical dislocation.

[0139] Echocardiography. Mice were anesthetized using isofluorane (1.5%-2.0% v / v by inhalation) and monitored for cardiac functional parameters in the supine position using a Visual Sonics Vevo 3100. Core temperature was maintained at 37 °C by imaging mice while on a heating pad, and heart rates were kept consistent between experimental groups (~400-500 beats per min). Short axis M-mode was used to assess LV dimensions and motion patterns, pulsed-wave (PW) doppler mode to evaluate blood flow velocities across the mitral valve, and tissue doppler mode to measure mitral annular plane velocity. Doppler profiles were acquired in the parasternal long axis (PLAX) apical 4 chamber view. The left atrial area was quantified in the apical 4-chamber view by tracing the border of the left atrium. Markers of systolic and diastolic function were calculated using standard echocardiography equations. At the end of the procedure, all mice recovered from anesthesia without difficulties. Image analysis was performed independently by a blinded sonographer, and all parameters were measured at least 3 times with averages presented.

[0140] Intraperitoneal Glucose Tolerance Test (IPGTT), Glucose tolerance tests were performed as previously described with minor modifications (Jurczak et al., 2011). After an overnight fast (~16 h), mice were set-up in restrainers with tails snipped <5mm to prime for blood collection and left to acclimate under a heated bulb for 2 h prior to GTT start time at 9 a.m. Basal plasma samples (t = 0) were collected, and D-glucose was diluted in water (20% final concentration) and sterile filtered for intraperitoneal administration at 1.5 g / kg body weight. Blood glucose was measured by tail bleed at set time points using a Bayer Contour Next EZ handheld glucometer (t = 15, 30, 45, 60, and 120 min).

[0141] Histology. Preparation and staining of all histological samples were conducted by the Pitt Biospecimen Core at the University of Pittsburgh. All mice were sacrificed, and tissue harvested for analysis within one hour of completion of the IPGTT study. The apex of the heart was excised from each mouse following euthanasia, fixed in 10% buffered formalin phosphate overnight at shaking incubation, washed 3 times for 5 min with IX phosphate-buffered saline (PBS), and then transferred to 70% ethanol. Samples were then embedded in paraffin, sectioned into 4 pm slides and stained with hematoxylin and eosin (H&E), wheat germ agglutinin (WGA) or Masson’s Trichrome for analysis. Histology sections were visualized with the Evos FL Auto 2 Microscope, observed changes in structure were quantified using ImageJ software, and representative images are shown.

[0142] Transcriptomics. Total RNA was isolated from the left ventricle of the heart using the RNeasy Plus Mini Kit (Qiagen REF 74134). Approximately 2 pg RNA was used for bulk RNA sequencing performed by Azenta / GENEWIZ, based on company recommendations to identify differential gene expression patterns between the experimental groups.

[0143] Quantitative Metabolomics. Metabolic quenching and polar metabolite pool extraction was performed by adding ice cold 80% methanol (aqueous) at a ratio of 1:15 wt input tissuewol. (13Cl)-creatinine, (D3)-taurine, (D3)-lactate and (D3)-alanine (Sigma-Aldrich) were added to the sample lysates as an internal standard for a final concentration of 10 pM. Samples are homogenized using an MP Bio FastPrep system using Matrix D (ceramic sphere) for 60 s at 60 hz. The supernatant was then cleared of protein by centrifugation at 16,000 g. Cleared supernatant (2 pL) was subjected to online LC-MS analysis. Analyses were performed by untargeted liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Briefly, samples were injected via a Thermo Vanquish UHPLC and separated over a reversed phase. Thermo HyperCarb porous graphite column (2.1x100 mm, 3 pm particle size) maintained at 55 °C. For the 20 min LC gradient, the mobile phase consisted of the following: solvent A (water / 0.1% FA) and solvent B (ACN / 0.1% FA). The gradient used was: 0-1 min 1% B, increase to 15% B over 5 min, continue increasing to 98% B over 5 min, hold at 98% B for 5 min, re-equilibrate at 1% B for 5 min. The Thermo IDX tribrid mass spectrometer was operated in both positive and negative ion mode, scanning in ddMS2 mode (2 pscans) from 70 to 800 m / z at 120,000 resolution with an AGC target of 2e5 for full scan, 2e4 for ms2 scans using HCD fragmentation at stepped 15, 35, 50 collision energies. Source ionization setting was 3.0 and 2.4 kV spray voltage, respectively, for positive and negative mode. Source gas parameters were 35 sheath gas, 12 auxiliary gas at 320 °C, and 8 sweep gas. Calibration was performed prior to analysis. Integrated peak areas were then extracted manually using Quan Browser (Thermo Fisher Xcalibur ver. 2.7). Untargeted differential comparisons were performed using Compound Discoverer 3.0 (Thermo Fisher) to generate a ranked list of significant compounds with tentative identifications from BioCyc, KEGG, and internal compound databases. Purified standards were then purchased and compared in retention time, m / z, along with ms2 fragmentation patterns to validate the identity of significant hits.

[0144] Protein Isolation and Immunoblotting. Heart tissues were rapidly harvested following euthanasia, weighed, RV discarded, and LV flash-frozen in liquid nitrogen. For protein isolation, tissues were minced and lysed in CHAPS buffer (1% CHAPS, 150 mM NaCl, 10 mM HEPES, pH 7.4) using a VWR 4-Place Mini Bead Mill, then incubated on ice for ~ 2.5 h. Homogenates were spun at 10,000 g at 4 °C for 10 min, and the supernatants collected for immunoblotting. For immunoblotting, protein lysates were quantitated using a BioDrop pLITE Analyzer, prepared in LDS sample buffer, separated using Bolt SDS-PAGE 4-12% or 12% Bis-Tris Plus gels, and transferred to nitrocellulose membranes (all Invitrogen). Membranes were blocked using SuperBlock (PBS) Blocking Buffer and incubated overnight in the following primary antibodies: mouse O-GlcNAc (9875), rabbit GF AT (D12F4), rabbit OGT (D1D8Q), rabbit OGA (E9C5U), rabbit GAPDH (2118S) from Cell Signaling Technologies; rabbit CD36 (18836-1-AP), rabbit LCAD (17526-1-AP) from Protein Tech, and GCN5L1 as previously reported (Scott et al., 2012). Protein loading was confirmed using GAPDH as a loading control. Fluorescent anti -goat or anti -rabbit secondary antibodies (red, 700 nm; green, 800 nm) from LiCor were used to detect expression levels. Images were obtained using Licor Odyssey CLx System and protein densitometry was measured using the LiCor Image Studio Lite Ver. 5.2 Software. Co-Immunoprecipitation, For co-immunoprecipitation experiments, protein lysates were harvested in CHAPS lysis buffer (1% CHAPS, 150 mMNaCl, 10 mMHEPES, pH 7.4), and 500 pg of protein was incubated overnight at 4o C with 5 pL mouse O-GlcNAc antibody (Cell Signaling). Immunocaptured proteins were isolated using Protein-G and Protein-A agarose beads (Cell Signaling Technology, catalog number 9007), washed 5 times with 500 pL 1% CHAPS buffer, and then eluted in LDS sample buffer (Life Technologies) at 95 °C. Samples were separated on 4-12% Bis-Tris Bolt gels, transferred to nitrocellulose membranes, and probed with appropriate antibodies. Images were obtained using LiCor Odyssey CLx System and protein densitometry was measured using the LiCor Image Studio Lite Ver. 5.2 Software.

[0145] Biochemical Assays. To assess the activity of long chain acyl-CoA dehydrogenase enzymes (LCAD), homogenized protein samples were incubated with palmitoyl-CoA as described previously (Thapa et al., 2017). Briefly, ~10 pg of protein was incubated with 0.1 M potassium phosphate, 50 pM 2,6- dichlorophenolindophenol, 2 mM phenazine ethosulfate, 0.2 mM N-ethylmaleimide, 0.4 mM potassium cyanide, and 0.1% Triton X-100 at 37 °C for 4 min. The reaction was initiated with 60 pM palmitoyl-CoA, and the rate of absorbance change was measured / ob served at 600 nm over 45 min. Activities were converted to moles of substrate oxidized / min / mass of protein. In vitro LCAD activity was measured after incubating a reaction mixture of 4 pg of recombinant LCAD, 0.8 mM UDP-GlcNAc (Sigma, Catalog # U4375), 40 pg / mL of recombinant human O-GlcNAc Transferase (rhOGT) (Catalog # 8446-GT) in assay buffer (25 mM Tris, 10 mM CaC12, pH 7.5 and 10 mM MgC12) at 37 °C for 30 min.

[0146] Statistics. Means ± SEM were calculated for all data sets. Data were analyzed using one-way ANOVA with Tukey’s post-hoc testing to determine differences between genotypes and feeding / treatment groups. Time course data was analyzed using one-way ANOVA with Sidak’s post- hoc testing. P < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 9.5 Software.

[0147] Results

[0148] Circulating adropin levels are diminished in HFpEF patients. Circulating adropin levels are known to decrease in patients with type 2 diabetes (Soltani et al, 2023), aging (Yang et al, 2018), and hypertension (Gulen et al, 2016). While these are all comorbidities for HFpEF, there have been no published studies examining circulating adropin levels in patients with this disease. Therefore, plasma levels of adropin were measured in deidentified age- and gender-matched control and HFpEF patient samples (n = 19-20) obtained from a University of Pittsburgh biobank (Table 1). Consistent with the studies in comorbid disease states, there was a significant decrease in plasma adropin levels in HFpEF patients relative to non-affected controls (FIG. 1 A). These data suggest that a reduction in circulating adropin levels can be linked to HFpEF disease development or progression.

[0149] Table 1. Control and HFpEF patient characteristics. M = male, F = female; W = white, NH / NL = non-Hispanic / non-Latino. N = 19-20.

[0150] Control HFpEF

[0151] ID Age Gender Race Ethnicity ID Age Gender Race Ethnicity 758 78 M W NH / NL 166 77 M W NH / NL 843 61 F W NH / NL 434 58 F W NH / NL 854 77 M w NH / NL 442 76 M w NH / NL 872 75 F w NH / NL 445 72 F w NH / NL 884 73 F w NH / NL 459 72 F w NH / NL 909 75 F w NH / NL 462 74 F w NH / NL 933 56 F w NH / NL 466 58 F w NH / NL 935 72 M w NH / NL 470 74 M w NH / NL 896 62 F w NH / NL 476 59 F w NH / NL 958 65 F w NH / NL 482 65 F w NH / NL 930 63 F w NH / NL 492 68 F w NH / NL 946 45 M w NH / NL 499 46 M w NH / NL 947 72 F w NH / NL 514 74 F w NH / NL 974 66 M w NH / NL 562 69 M w NH / NL 1137 59 F w NH / NL 576 70 F w NH / NL 992 74 M w NH / NL 583 63 F w NH / NL 1014 73 F w NH / NL 633 71 M w NH / NL 1122 50 M w NH / NL 636 74 F w NH / NL 1052 72 M w NH / NL 663 48 M w NH / NL

[0152]

[0153] 675 72 M w NH / NL

[0154] Recombinant adropin treatment prevents cardiac dysfunction in a mouse model of HFpEF, The potential link between reduced circulating adropin and HFpEF in patients supported the premise that therapeutic restoration of adropin levels can be protective against the disease. Therefore, a recently developed two-hit mouse model of HFpEF (Schiattarella et al, 2019) was used to test this hypothesis. Male C57BL / 6J mice were placed on either a chow (control) or HFpEF (60% high fat diet plus L-NAME-supplemented drinking water) diet for 8 weeks. After 8 weeks, HFpEF mice were randomly given either vehicle (PBS) or adropin (450 nmol / kg / day; Thapa et al, 2019b) by intraperitoneal (IP.) injection once daily for 4 weeks (FIG. 2B). Daily adropin treatment significantly increased circulating adropin levels in HFpEF mice (15.9 vs. 2.2 ng / mL, P = 0.0067), demonstrating that administration of recombinant adropin could restore plasma concentrations to those observed in healthy humans (FIG. 1A).

[0155] The recent STEP-HFpEF Phase 3 clinical trial showed that the GLP-1 receptor agonist semaglutide improved patient-reported outcomes in HFpEF, which was at least partially related to weight loss (Kosiborod et al, 2023). Therefore, whether adropin treatment promoted weight loss in mice was examined; there was no significant difference in body weight between vehicle- and adropin-treated HFpEF animals (FIGS. 9A-9B). It has been previously shown that short-term adropin treatment (< 3 days) leads to improved whole-body glucose tolerance in diet-induced obese mice (Gao et al, 2015; Thapa et al, 2019a). Whether long-term adropin treatment (4 weeks) in HFpEF mice had the same effect was tested; there was a significant decrease in insulin resistance in HFpEF mice treated with adropin relative to non-treated HFpEF controls (FIG. 2D). Combined, these data support that adropin treatment can have a positive effect on whole-body physiology by reducing glucose intolerance. However, unlike semaglutide in humans, this improvement is unrelated to the beneficial effects of weight loss.

[0156] Next, the effect of adropin treatment on cardiac functional parameters and tissue structure was examined (FIG. 2E). Mice with HFpEF displayed a significant increase in cardiac weight and a reduction in cardiac output, both of which were absent in adropin-treated HFpEF mice (FIGS. 8A-8B). As expected from a HFpEF model, all mouse groups displayed normal systolic function as measured by left ventricular ejection fraction and isovolumetric contraction time (FIG. 2K, Table 2). While both HFpEF groups displayed a -25% increase in E / e’ ratio, adropin-treated mice displayed a significant decrease in isovolumetric relaxation time, and a trend towards a decreased E / A ratio relative to untreated HFpEF mice (FIG. 2G, Table 2). These data indicated that adropin can increase diastolic function in HFpEF by improving ventricular relaxation, whilst having a more limited impact on left ventricle filling pressures.

[0157] Table 2. Echocardiograph analysis of Chow, HFpEF, and HFpEF+Adr. mice. LV = left ventricle; IVCT = isovolumetric contraction time; IVRT = isovolumetric relaxation time; AW = anterior wall; PW = posterior wall; MPI = myocardial performance (Tau) index; LAA = left atrial area; sys = systolic; dia = diastolic; * = P < 0.05 vs. Chow; # = P < 0.05 vs. HFpEF. Chow HFpEF HFpEF+Adr

[0158] LF Mass 128.6 ± 18.05 157.8 ± 25.15* 149.8 ± 24.26 Heart Rate 491.9 ±28.93 448.1 ± 37.75 488.3 ± 42.43 Ejection Fraction

[0159] 64.70 ± 3.326 62.16 ± 3.941 63.73 ± 6.176

[0160] (%)

[0161] E / e' 21.66 ± 3.249 26.09 ± 5.871 27.01 ± 5.694

[0162] E / A 1.601 ± 0.2996 1.699 ± 0.3553 1.562 ± 0.3157

[0163] IVCT 13.58 ±2.293 14.28 ± 1.511 12.60 ± 1.528

[0164] IVRT 12.67 ± 1.259 16.44 ± 2.118* 14.52 ± 1.801* Stroke Volume 47.79 ± 7.432 41.89 ± 8.713 45.01 ± 9.634 Cardiac Output 23.38 ±2.799 18.58 ± 3.167* 21.82 ± 4.132

[0165] LV AW; sys 1.401 ± 0.06618 1.548 ± 0.1178* 1.490 ± 0.1205 LV AW; dia 0.8807 ± 0.06180 1.121 ± 0.1100* 1.035 ± 0.1213* LV PW; sys 1.211 ± 0.1044 1.255 ± 0.1376 1.312 ± 1.653 LV PW; dia 0.7841 ± 0.1113 0.8945 ± 0.07463 0.8639 ± 0.1161

[0166] 0.6154 ± 0.5330 ±

[0167] LV MPI (Tau) 0.5290 ± 0.03861

[0168] 0.08687* 0.06274#

[0169]

[0170] LAA 4.909 ± 0.4121 4.788 ± 0.7369 5.002 ± 0.6408

[0171] Histological analysis of cardiac tissue sections demonstrated that HFpEF mice displayed a significant increase in myocardial interstitial fibrosis and cardiomyocyte cross-sectional area, which was completely reversed following long-term adropin treatment (FIGS.

[0172] 2E, 21, and 2J). The positive impact of adropin on reducing myocardial fibrosis was evident in bulk RNA-seq analysis or untreated vs. adropin-treated HFpEF hearts, with extracellular matrix processes being identified as the top hit in pathway analyses (FIG. 3A). Closer examination demonstrated that multiple genes related to extracellular matrix formation and fibrosis (e.g. Postn, Fnl, Collal) were significantly downregulated in HFpEF hearts after adropin treatment (FIGS. 3B, 9A-9L). Combined, these data demonstrate that long-term adopin treatment in HFpEF promoted whole-body metabolic homeostasis, improved myocardial relaxation, prevented maladaptive cardiac tissue remodeling, and limited cardiomyocyte hypertrophy.

[0173] Adropin treatment limits cardiac protein O-GlcNAcylation in HFpEF mice. The decrease in cardiac fibrosis in adropin-treated HFpEF mice is likely to account for at least some of the functional improvements observed, particularly in terms of myocardial relaxation. However, it is unlikely to explain some of the other improvements that were found to result from adropin treatment. To better understand the underlying processes involved, targeted and untargeted metabolomic analyses was performed of hearts from untreated and adropin-treated HFpEF mice. Surprisingly, there were few significant changes between the hearts of the two HFpEF groups in our targeted analysis of amino acids, energy metabolites, and TCA cycle intermediates. In contrast, the untargeted analysis demonstrated that metabolites related to the hexosamine biosynthesis pathway (HBP) were specifically enriched in untreated vs. adropin-treated HFpEF hearts (FIG. 3C). As the HBP provides substrates for the generation of UDP-GlcNAc, the co-factor for protein O-GlcNAcylation, the abundance of this posttranslational modification in control and HFpEF hearts was examined.

[0174] Immunoblotting using an O-GlcNAc-specific antibody demonstrated that there was a significant increase in cardiac protein O-GlcNAcylation in untreated HFpEF hearts, which was largely reversed (P = 0.06 vs. HFpEF) following long-term adropin treatment (FIG. 3C). O-GlcNAcylation is controlled at the protein level by the rate-limiting enzyme GF AT, and by the opposing activities of the O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) which, respectively, add and remove this modification to / from other proteins (e.g., Umapathy et al., 2021, Tran et al., 2020, Prakoso et al., 2022). Unexpectedly, despite the difference in O-GlcNAcylation levels observed in untreated and adropin-treated HFpEF hearts, there were no significant changes in the abundance of any of these regulatory enzymes (FIG. 4).

[0175] To determine if changes in HBP substrate abundance, rather that enzyme activity, was controlling the levels of O-GlcNAcylation observed, the metabolomic data was examined for HBP metabolites. Hexose levels in untreated HFpEF mice were significantly increased relative control mice, suggesting that there can be a restriction in glucose moving through the glycolysis pathway (FIG. 4). Downstream, an accumulation of GlcNAc-6-P, Uridine, and CoA was observed in adropin-treated HFpEF hearts relative to control and untreated HFpEF mice. As these metabolites feed into the HBP at different points, their accumulation supports a reduction of metabolite flux into and through the pathway to generate UDP-GlcNAc, the co-factor for protein O-GlcNAcylation. Combined, these data suggest that part of the function of adropin in protecting from HFpEF is through its modulatory effect on cardiac protein O-GlcNAcylation.

[0176] Adropin-mediated reductions in LCAD O-GlcNAcylation improve its enzymatic activity. Studies in humans and mice have demonstrated that pathways related to cardiac energy metabolism, particularly those in fatty acid utilization, are negatively impacted by HFpEF progression (Tong et al, 2021; Hahn et al, 2023; O’Sullivan et al, 2024). Therefore, whether specific fatty acid oxidation enzymes were impacted by the increased protein O-GlcNAcylation observed above was examined. Using O-GlcNAc-specific immunoprecipitation, it was observed that the beta oxidation enzyme long-chain acyl-CoA dehydrogenase (LCAD) displayed elevated O-GlcNAcylation in HFpEF hearts, which was reversed by adropin treatment (FIGS. 5A-5B). The effect of O-GlcNAcylation on LCAD enzymatic activity in vivo was examined; it was found that its ability to oxidize C16 palmitoyl-CoA was reduced in vehicle-treated HFpEF hearts, but not those treated with adropin (FIG. 5C). Combined, there was a significant negative correlation between LCAD O-GlcNAcylation abundance and its enzymatic activity across all cardiac tissues (FIG. 5D), which was unrelated to LCAD protein abundance (FIG. 10). To confirm the regulatory effect of increased O-GlcNAcylation on LCAD activity, a series of in vitro assays was performed. LCAD activity was significantly decreased in the presence of the O-GlcNAcylation substrate (UDP-GlcNAc) and transferase enzyme (OGT), but not the substrate alone (FIG. 5E). Finally, whether changes in lipid handling are related to the observed decrease in LCAD enzymatic activity was examined, and the abundance of the key cardiac lipid droplet membrane protein perilipin-5 (PLIN5) was measured. In untreated HFpEF mice there was a significant increase in PLIN5, (suggestive of increased lipid droplet size) compared to control mice, which was absent following adropin treatment (FIGS. 5F-5G). Combined, these data suggest that increased O-GlcNAcylation of fatty acid enzymes like LCAD in HFpEF can decrease their activity and lead to myocardial lipid accumulation, which can be reversed by adropin treatment.

[0177] Discussion

[0178] Studies on adropin signaling in cardiometabolic disease have focused on its effects on fuel energy metabolism, with limited exploration of its role on myocardial structure and function (reviewed in Mushala and Scott, 2021). Short-term adropin treatment improved cardiac work and efficiency ex vivo, which was accompanied by enhanced insulin signaling in lean mice (Altamimi et al., 2019). In contrast, exposure to a high-fat diet inhibited acute adropin-driven improvements in contractile function, and insulin stimulation failed to compensate for this impaired cardiac contractility in diet-induced obese mice ex vivo (Thapa et al., 2022). This study demonstrated that chronic adropin treatment in vivo restores cardiac function, specifically left ventricular diastolic function, in the failing diabetic myocardium of mice.

[0179] Diastolic dysfunction is a hallmark of not only HFpEF, but also diabetic cardiomyopathy (DCM), where relaxation and filling pressure of the left ventricle is impaired (reviewed in Jia et al., 2018). Abnormalities of LV passive elasticity are often caused by structural impediments, such as increased cardiomyocyte diameter and extracellular matrix deposition, which contribute to diastolic dysfunction Aurigemma et al., 2006). These data show that adropin treatment attenuates LV hypertrophy and fibrosis, indicative of improved structural remodeling. In addition, transcriptomic analysis demonstrated similar increases in pro-fibrotic pathways as seen in HFpEF patients (Ye et al., 2023) which was attenuated upon adropin treatment, providing further evidence of restored myocardial structure and function. Therefore, these findings collectively highlight the therapeutic potential of prolonged adropin treatment in preventing cardiac remodeling and functional decline in HFpEF and / or DCM.

[0180] Data presented in this study add to the growing understanding of the transcriptomic and metabolomic signature of the HFD + L-NAME HFpEF model, providing preclinical evidence of known mechanisms underlying the pathogenesis of cardiometabolic HFpEF in patients, such as fuel substrate inflexibility (Hahn et al., 2023). Unlike previous acute studies, the ability of adropin treatment to regulate cardiac substrate utilization was independent of the inhibitory effects of PDK4 or GCN5L1 on pyruvate dehydrogenase activity (Thapa et al., 2019b, Altamimi et al., 2019). Instead, it was found that adropin treatment can reduce flux through the HBP, which inhibits cardiac protein O-GlcNAcylation to promote fatty acid oxidation. The HBP is a non-oxidative branch of glycolysis where the rate-limiting enzyme, GF AT, uses fructose-6-phosphate and glutamine to catalyze its conversion into glucosamine-6-phosphate, which is subsequently metabolized through a series of reactions utilizing substrates from major metabolic pathways to generate the end-product UDP-GlcNAc (Umapathi et al., 2021). UDP-GlcNAc serves as the co-factor for the attachment of an O-linked P-N-acetyl glucosamine moiety (O-GlcNAc) to serine and threonine residues of nuclear, cytoplasmic, and mitochondrial proteins, using 2-3% of the total cellular glucose found in the heart (Tran et al., 2020).

[0181] The dynamic on- and off-cycling of the O-GlcNAc modification is regulated by the activity of two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively (Prakoso et al., 2022). Several studies have implicated aberrant regulation of the HBP, and consequent maladaptive protein O-GlcNAcylation, in heart failure (e.g., Umapathy et al., 2021, Tran et al., 2020, Prakoso et al., 2022). These studies have identified changes in OGT and OGA enzyme expression as the predominant mechanism that triggers excessive protein O-GlcNAcylation in diabetic hearts. However, here it was found that changes in substrate entry from amino acid metabolism (glutamine), fatty acid metabolism (coenzyme A), and nucleotide metabolism (uridine) increased UDP-GlcNAc availability to promote increased cardiac protein O-GlcNAcylation levels. Notably, previous comparative proteomics studies revealed dysregulated mitochondrial O-GlcNAcylation in diabetic hearts (Ma et al., 2016), with key targets in fatty acid oxidation that have subsequently been linked to increased enzyme activity in pressure-overload hypertrophy (Zhu et al., 2022).Laczy et al. (2011) showed that increased cardiac O-GlcNAcylation was associated with a decrease in glucose oxidation and a stimulation of fatty acid oxidation ex vivo, through increased CD36 expression and O-GlcNAcylation. While a significant increase in CD36 protein abundance was observed, which was significantly attenuated by adropin treatment (FIGS. 11A-11B), direct evidence of O-GlcNAc modification on CD36 was not detected (blots not shown). Instead, the FAO enzyme LOAD was identified as a target of O-GlcNAcylation in HFpEF mice, and adropin treatment showed moderate inhibition of the modification of these targets. Interestingly, it was found that LOAD O-GlcNAcylation was associated with decreased cardiac enzyme activity in HFpEF mouse hearts, which was restored upon adropin treatment. The inhibitory effect of O-GlcNAcylation on LOAD enzyme activity was confirmed in vitro, further suggesting that adropin treatment can augment FAO activity via this mechanism. Further studies need to be conducted to determine whether adropin-mediated changes of cardiac FAO enzyme O-GlcNAcylation regulates fatty acid utilization in the heart in vivo.

[0182] In summary, HFpEF-driven disruptions to normal cardiac energy metabolism led to an apparent shunt of glycolytic intermediates into the HBP, which promoted an increase in cardiac protein O-GlcNAcylation and decreased fatty acid oxidation enzyme activity. Remarkably, long-term adropin treatment reversed this metabolic remodeling in the heart, leading to restored cardiac structure and diastolic function. These findings are the first to report the long-term effects of adropin signaling on cardiac function and substrate flexibility in HFpEF, highlighting this pathway as a potential target for future therapeutic interventions.

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[0223] * * *

[0224] Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the present disclosure. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, and compositions of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the present disclosure of the presently disclosed subject matter, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized according to the presently disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps. Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, this present disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A method for diagnosing a cardiometabolic disease, comprising:a) determining the level of adropin in one or more samples from a subject;b) comparing the level of adropin to a reference level; andc) diagnosing the subject as having a high risk of cardiometabolic disease if the level of the adropin in the one or more samples is decreased with respect to the reference level.

2. The method of claim 1, further comprising administering a therapeutically effective amount of one or more therapeutic agent.

3. The method of claim 2, wherein the one or more therapeutic agent comprises an antiinflammatory agent, an anti-fibrotic agent, a diuretic, an angiotensin receptor-neprilysin inhibitor (ARNI), an angiotensin receptor blockers (ARB), a mineralocorticoid antagonist (MRA), a sodium-glucose cotransporter-2 (SGLT2) inhibitor, metformin, a glucagon-like peptide-1 (GLP-1) agonist, a gastric inhibitory polypeptide (GIP) antagonist, an angiotensinconverting enzyme (ACE) inhibitor, a beta-blocker, a nondihydropyridine calcium channel blocker, or a combination thereof.

4. The method of claim 2, wherein the one or more therapeutic agent comprises an adropin-based therapy.

5. The method of claim 1, further comprising providing surgical intervention or a pacemaker to the subject.

6. The method of claim 1, wherein the one or more samples are collected from at least one selected from the group consisting of whole blood, blood plasma, and combinations thereof.

7. The method of claim 1, wherein the reference level is the amount of adropin in one or more reference samples collected from a healthy individual.

8. The method of claim 1, wherein the reference level is the level of adropin in the same subject at an earlier timepoint.

9. The method of claim 1, wherein the level of adropin in the one or more samples is determined using ELISA or mass spectrometry.

10. The method of claim 1, wherein the cardiometabolic disease is selected from the group consisting of heart failure with preserved ejection fraction (HFpEF), hypertension, or non-alcoholic fatty liver disease (NAFLD).

11. The method of claim 10, wherein the cardiometabolic disease is heart failure with preserved ejection fraction (HFpEF).

12. The method of claim 4, wherein the adropin-based therapy is administered intravenously, intraperitoneally, or subcutaneously.

13. The method of claim 4, wherein the adropin-based therapy is a recombinant adropin peptide.

14. The method of claim 13, wherein the recombinant adropin peptide comprises SEQ ID NO.: 1 or SEQ ID NO.: 2.

15. The method of claim 13, wherein the recombinant adropin peptide is administered in an amount between about 1 ng / kg to about 200 mg / kg.

16. The method of claim 4, wherein the adropin-based therapy is a composition comprising a virus, wherein the virus comprises a heterologous nucleic acid encoding an Energy Homeostasis-associated (ENHO) gene.

17. The method of claim 16, wherein the virus is administered into the liver of the subj ect.

18. The method of claim 16, wherein the virus is administered to hepatocytes of the subject.

19. The method of claim 16, wherein the heterologous nucleic acid is operably linked to a promoter selected from the group consisting of thyroxine binding globulin (TBG), albumin (ALB), hepatitis virus (HBV), alpha- 1 antitrypsin (AAT), and human cytomegalovirus (CMV).

20. The method of claim 16, wherein the virus is an adenoviral associated virus (AAV).

21. The method of claim 20, wherein the AAV isotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV / DJ, and AAV / DJ8.

22. The method of claim 16, wherein the ENHO gene comprises SEQ ID NO.: 3.

23. The method of claim 1, wherein step (c) comprises diagnosing the subject as having a high risk of cardiometabolic disease if the level of adropin in the one or more samples is decreased by at least about 20% with respect to the reference level.

24. A method for treating a cardiometabolic disease in a subject in need thereof, comprising:a) determining the level of adropin in one or more samples from a subject;b) comparing the level of adropin to a reference level; andc) determining that the level of adropin in the one or more samples is decreased with respect to the reference level.

25. The method of claim 24, further comprising administering a therapeutically effective amount of one or more therapeutic agent.

26. The method of claim 25, wherein the one or more therapeutic agent comprises an anti-inflammatory agent, an anti-fibrotic agent, a diuretic, an angiotensin receptor-neprilysin inhibitor (ARNI), an angiotensin receptor blockers (ARB), a mineralocorticoid antagonist (MRA), a sodium-glucose cotransporter-2 (SGLT2) inhibitor, metformin, a glucagon-like peptide-1 (GLP-1) agonist, a gastric inhibitory polypeptide (GIP) antagonist, an angiotensinconverting enzyme (ACE) inhibitor, a beta-blocker, a nondihydropyridine calcium channel blocker, or a combination thereof.

27. The method of claim 25, wherein the one or more therapeutic agent comprises an adropin-based therapy.

28. The method of claim 24, further comprising providing surgical intervention or a pacemaker to the subject.

29. The method of claim 24, wherein the one or more samples are collected from at least one selected from the group consisting of whole blood, blood plasma, and combinations thereof.

30. The method of claim 24, wherein the reference level is the amount of adropin in one or more reference samples collected from a healthy individual.

31. The method of claim 24, wherein the reference level is the level of adropin in the same subject at an earlier timepoint.

32. The method of claim 24, wherein the level of adropin in the one or more samples is determined using ELISA or mass spectrometry.

33. The method of claim 24, wherein the cardiometabolic disease is selected from the group consisting of heart failure with preserved ejection fraction (HFpEF), hypertension, or non-alcoholic fatty liver disease (NAFLD).

34. The method of claim 33, wherein the cardiometabolic disease is heart failure with preserved ejection fraction (HFpEF).

35. The method of claim 27, wherein the adropin-based therapy is administered intravenously, intraperitoneally, or subcutaneously.

36. The method of claim 27, wherein the adropin-based therapy is a recombinant adropin peptide.

37. The method of claim 36, wherein the recombinant adropin peptide comprises SEQ ID NO.: 1 or SEQ ID NO.: 2.

38. The method of claim 36, wherein the recombinant adropin peptide is administered in an amount between about 1 ng / kg to about 200 mg / kg.

39. The method of claim 24, wherein the adropin-based therapy is a composition comprising a virus, wherein the virus comprises a heterologous nucleic acid encoding an Energy Homeostasis-associated (ENHO) gene.

40. The method of claim 39, wherein the virus is administered into the liver of the subject.

41. The method of claim 39, wherein the virus is administered to hepatocytes of the subject.

42. The method of claim 39, wherein the heterologous nucleic acid is operably linked to a promoter selected from the group consisting of thyroxine binding globulin (TBG), albumin(ALB), hepatitis virus (HBV), alpha- 1 antitrypsin (AAT), and human cytomegalovirus (CMV).

43. The method of claim 39, wherein the virus is an adenoviral associated virus (AAV).

44. The method of claim 43, wherein the AAV isotype is selected from the group consisting of AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV / DJ, and AAV / DJ8.

45. The method of claim 39, wherein the ENHO gene comprises SEQ ID NO.: 3.

46. The method of claim 24, wherein step (c) comprises determining that the level of adropin in the one or more samples is decreased by at least about 20% with respect to the reference level.

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