Immune checkpoint inhibitors for use in cardiac repair

Abstract

Methods of using inhibitors of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) for treating heart disease are provided. In addition, methods are provided for screening candidate agents for inhibition of CTLA-4 and effectiveness of candidate inhibitors of CTLA-4 in improving cardiac function, decreasing or preventing damage to the heart, including reducing chamber dilatation, ventricular remodeling, and / or amount of fibrotic scar tissue, and increasing survival.

Description

IMMUNE CHECKPOINT INHIBITORS FOR USE IN CARDIAC REPAIRCROSS-REFERENCE TO RELATED APPLICATION

[0001] Pursuant to 35 U.S.C. § 119(e), this application claims priority to the filing dates of United States Provisional Patent Application Serial No. 63 / 640,119 filed April 29, 2024, the disclosure of which application is herein incorporated by reference in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML file, “UCSF- 784WO” created on April 9, 2025, and having a size of 22,850 bytes. The contents of the Sequence Listing XML file are incorporated by reference herein in their entireties.INTRODUCTION

[0003] Myocardial infarction (MI) is a leading cause of death worldwide, contributing significantly to the global burden of cardiovascular diseases (CVDs). According to the World Health Organization, an estimated 17.9 million people died from CVDs in 2019, representing 32% of all global deaths. Of these deaths, 85% were due to heart attack and stroke (World Health Organization. June 11, 2021. “Cardiovascular Diseases (CVDs).” who.int / newsroom / fact- sheets / detail / cardiovascular-diseases-(cvds)). The average annual direct and indirect cost of CVDs in the United States was an estimated $407.3 billion from 2018 to 2019 (Tsao et al. (2023) Circulation 147:e93-e621).

[0004] The therapeutic arena for MI encompasses a multitude of methodologies, but all face critical challenges: Conventional therapies like aspirin, thrombolytics, and betablockers, as well as stenting or bypass surgery, aim to improve blood flow and reduce heart damage. Their effectiveness is time-sensitive and comes with risks like bleeding, artery stenosis, surgical issues, and medication side effects. In 2019, the U.S. saw over 1 million angioplasties and 500,000 bypass surgeries, with about 40% of patients facing complications (McNichols et al. (2021) Cardiol. Ther. 10:89-109). While stem cell therapies and tissue engineering are promising avenues, major challenges remain for cell engraftment, immune rejection, and scalability (Abubakar et al. (2023) Cureusl5:e41533). Precision Medicine for MI is constrained by high costs, few predictive genetic markers, individual variability understanding gaps, extensive clinical validation needs, and limited patient benefits (Abubakar et al., supra).

[0005] Importantly, most of the methodology addressing MI involve acute revascularizing of the blocked vessel that would normally lead to myocardial death. However, as medical community has improved acute revascularization though both medical (e.g., aspirin, anti-platelettherapies, anticoagulation) and interventional approaches (catheterization / angioplasty), there is a growing number of patients who survive the initial MI but later develop heart failure. This transition from MI to heart failure occurs due to cardiac remodeling over time. There have been few approved therapies for this cardiac remodeling including renin angiotensin inhibitors but few recent therapies address the critical issue of cardiac remodeling.

[0006] Thus, there remains a need for better methods of not only treating the acute Ml but also treating and ideally preventing the subsequent cardiac remodeling that occurs over time.SUMMARY

[0007] Methods of using inhibitors of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) for treating heart disease are provided. In addition, methods are provided for screening candidate agents for inhibition of CTLA-4 and effectiveness of candidate inhibitors of CTLA-4 in improving cardiac function, decreasing or preventing damage to the heart, including reducing chamber dilatation, ventricular remodeling, and / or amount of fibrotic scar tissue, and increasing survival.

[0008] In one aspect, a method of treating heart disease in a subject is provided, the method comprising administering a therapeutically effective amount of an inhibitor of CTLA-4 to the subject.

[0009] In certain embodiments, the inhibitor of CTLA-4 is an antibody that specifically binds to CTLA-4. In some embodiments, the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a Fvfragment, and a nanobody. In some embodiments, the antibody is ipilimumab or tremelimumab.

[0010] In certain embodiments, the heart disease is myocardial infarction, myocardial ischemia, myocardial injury, heart failure, atherosclerosis, coronary artery disease, or a cardiomyopathy.

[0011] In certain embodiments, the inhibitor of CTLA-4 is administered intravenously or intra-arterially.

[0012] In certain embodiments, the inhibitor of CTLA-4 is administered locally to damaged heart tissue or a site of cardiac ischemia or infarction.

[0013] In certain embodiments, multiple therapeutically effective doses of the inhibitor of CTLA-4 are administered to the subject. In some embodiments, the inhibitor of CTLA-4 is administered according to a daily dosing regimen or intermittently.

[0014] In certain embodiments, the inhibitor of CTLA-4 is administered after a myocardial infarction.

[0015] In certain embodiments, the inhibitor of CTLA-4 is administered prophylactically to protect against or delay or prevent myocardial infarction.

[0016] In certain embodiments, the subject has cardiovascular disease.

[0017] In certain embodiments, the treatment increases left ventricular ejection fraction, reduces chamber dilatation, reduces ventricular remodeling, reduces area of fibrotic scar tissue, reduces the level of plasma troponin, and / or increases survival compared to in the absence of the treatment.

[0018] In certain embodiments, the treatment with the inhibitor of CTLA-4 increases proliferation of Foxp3+ regulatory T cells compared to in the absence of the treatment.

[0019] In certain embodiments, the inhibitor of CTLA-4 inhibits binding of CTLA-4 to B7.1 and B7.2 antigen-presenting cell ligands.

[0020] In certain embodiments, the inhibitor of CTLA-4 stimulates proliferation of a regulatory T cell (Treg). In some embodiments, the Treg is a Foxp3+Treg.

[0021] In certain embodiments, the subject is human.

[0022] In another aspect, a method of treating ventricular remodeling in a subject is provided, the method comprising administering a therapeutically effective amount of an inhibitor of CTLA-4 to the subject.

[0023] hi certain embodiments, the inhibitor of CTLA-4 is administered prophylactically or therapeutically after myocardial infarction, myocardial ischemia, myocardial injury, atherosclerosis, or heart failure to prevent, decrease, or delay ventricular remodeling.

[0024] In certain embodiments, the ventricular remodeling comprises changes in left ventricular (LV) geometry, mass, and / or volume.

[0025] In certain embodiments, the inhibitor of CTLA-4 is an antibody that specifically binds to CTLA-4. In some embodiments, the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a Fvfragment, and a nanobody. In some embodiments, the antibody is ipilimumab or tremelimumab.

[0026] In certain embodiments, the inhibitor of CTLA-4 is administered after a myocardial infarction.

[0027] In another aspect, a method of screening for an inhibitor of cytotoxic T- lymphocyte-associated protein 4 (CTLA-4) for treating heart disease is provided, the method comprising: (a) contacting CTLA-4 with a candidate agent; (b) measuring inhibition of CTLA-4 by the candidate agent to determine if the candidate agent is an inhibitor of CTLA-4; (c) administering the candidate agent to a mammalian non-human animal before, during, or after performing left anterior descending artery (LAD) ligation on the mammalian non-human animal,if the candidate agent is determined to be an inhibitor of CTLA-4; and (d) measuring left ventricular ejection fraction, chamber size, chamber dilatation, ventricular remodeling, area of fibrotic scar tissue, survival, level of plasma troponin, or any combination thereof in the mammalian non-human animal after said performing the LAD, wherein increased left ventricular ejection fraction, reduced chamber dilatation, reduced ventricular remodeling, reduced area of fibrotic scar tissue, increased survival, reduced level of plasma troponin, or any combination thereof, compared to in absence of said administering the candidate agent indicates that the candidate agent is useful for treating heart disease.

[0028] In certain embodiments, the candidate agent is a small molecule, a peptide, a protein, a peptoid, an aptamer, an antibody that specifically binds to CTLA-4, an antibody mimetic, an inhibitory nucleic acid, or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system. In some embodiments, the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a Fvfragment, and a nanobody. In some embodiments, the inhibitory nucleic acid is selected from the group consisting of a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a Piwi- interacting RNA (piRNA), a small nuclear RNA (snRNA), an antisense oligonucleotide, and a peptide nucleic acid. In some embodiments, the inhibitory nucleic acid inhibits CTLA-4 transcription or protein translation. In some embodiments, the CRISPR system targets a CTLA-4 gene or a CTLA-4 RNA transcript, or makes epigenetic changes that reduce CTLA-4 expression. In some embodiments, the CRISPR system comprises Cas9, Casl2a, Casl2d, Cast 3a, Casl3b, Casl3d, or a dead Cas9 (dCas9).

[0029] In certain embodiments, the non-human animal model is a rodent or primate. In some embodiments, the rodent is a mouse.

[0030] In certain embodiments, measuring the inhibition of CTLA-4 comprises measuring inhibition of binding of CTLA-4 to B7.1 and B7.2 antigen-presenting cell ligands.

[0031] In certain embodiments, measuring inhibition of CTLA-4 comprises measuring proliferation of a Foxp3+ regulatory T cell (Treg).

[0032] In certain embodiments, the level of plasma troponin is measured using an immunoassay (e.g., ELISA) or liquid chromatography-tandem mass spectrometry.

[0033] In certain embodiments, the chamber size, the chamber dilatation, the ventricular remodeling, or the area of fibrotic scar tissue is measured by echocardiography, ultrasound, or histology.

[0034] In certain embodiments, the left ventricular ejection fraction is measured by echocardiography, magnetic resonance imaging (MRI), computerized tomography (CT),radionuclide angiography, gated myocardial perfusion single-photon emission computed tomography (SPECT), or gated myocardial perfusion positron emission tomography (PET).

[0035] In certain embodiments, the method further comprises contacting a cardiomyocyte with the candidate agent; and measuring contractility, calcium (Ca2+)dynamics, extracellular field potential (EFP), pro-arrhythmic properties, or any combination thereof. In some embodiments, the cardiomyocyte is a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM).

[0036] In another aspect, a composition comprising an inhibitor of CTLA-4 for use in a method of treating heart disease or ventricular remodeling is provided.

[0037] In certain embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0038] In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier selected from the group consisting of a cream, emulsion, gel, liposome, nanoparticle, or ointment.

[0039] In certain embodiments, the inhibitor of CTLA-4 in the composition is an antibody that specifically binds to CTLA-4. In some embodiments, the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a Fvfragment, and a nanobody. In some embodiments, the antibody is ipilimumab or tremelimumab.

[0040] In certain embodiments, the inhibitor of CTLA-4 in the composition inhibits binding of CTLA-4 to B7.1 and B7.2 antigen-presenting cell ligands.

[0041] In certain embodiments, the inhibitor of CTLA-4 increases proliferation of a Foxp3+ Treg.

[0042] In certain embodiments, the heart disease is myocardial infarction, myocardial ischemia, myocardial injury, heart failure, atherosclerosis, coronary artery disease, or a cardiomyopathy.

[0043] In another aspect, use of an inhibitor of CTLA-4 in the manufacture of a medicament or pharmaceutical composition for treating heart disease in a subject in need thereof is provided.BRIEF DESCRIPTION OF THE DRAWINGS

[0044] FIGS. 1A-1B. (FIG. 1A) Immune checkpoints inhibit T-cell activation. (FIG. IB) ICIs permit T cell activation to kill cancer.

[0045] FIGS. 2A-2D. Higher expression of CD80 / 86 in Ml and significantly correlated with poor left ventricular ejection fraction. (FIG. 2A) Normalized expression levels (RPKM) for selected Immune checkpoints ligand genes. Average expression levels across time points areshown in black symbols and dashed lines. Expression levels for individual replicates are shown in colored symbols (n=4) (mean ± SEM., *p<0.05). (FIG. 2B) Expression of ICI ligands genes are determined by qPCR in the isolated cardiomyocytes 2 days post-MI (mean ± SEM., *p<0.05, n=5). (FIG. 2C) Correlation analysis was conducted with the expression level of these immune checkpoints and the cardiac function in each animal (n=14). (FIG. 2D) RNA-seq diverse set of immune checkpoint ligands expression in the injured heart post-MI.

[0046] FIGS. 3A-3F. Cardiac function and remodeling post-MI in ICI-treated mice. (FIG. 3A). Schematic overview of experimental design. (FIG. 3B). Kaplan-Meier overall survival analysis of anti-CTLA4 and anti-PD-1 compared to isotype control IgG isotype treated mice after MI. (n=16 / Control, n=14 / anti-CTLA4, n=8 / anti-PD-l and n=22 / sham). (FIGS. 3C-3E). Echocardiography to examine the cardiac contractile performance Ejection fraction (EF), and chamber dilatation LV dimensions in end-diastole (LVID; d) and end-systole (LVID; s) at D7 post-MI in all the groups following ischemic injury (n=15 / Control, n=10 / anti-PD-l, n=15 / anti- CTLA4, and n=22 / sham). (FIG. 3F). Infarct size at D28 post-MI in all the groups following ischemic injury. (n=10 / Control, n=10 / anti-PD-l, and n=7 / anti-CTLA4).

[0047] FIGS. 4A-4D. CTLA4 inhibition increases Treg number in peripheral blood and injured heart. (FIG. 4A) Periphery blood cell population and (FIG. 4B) FoxP3+CD4+ Treg cells was evaluated by flow-cytometry in mice treated with n=4 / anti-CTLA-4, n=4 / IgG control and n=3 / Abatacept and n=3 / anti-CD28 *p<0.05 versus IgG control animals. FoxP3+CD4+ Treg cells in (FIG. 4C) peripheral blood and (FIG. 4D) cardiac ischemic / borderzone area evaluated by flowcytometry 2 days Post-MI in mice treated with n=3 / anti-CTLA-4 or n=4 / IgG control.

[0048] FIG. 5. Research strategies to study infiltrated immune cells in cardiac remodeling via scRNA-seq.DETAILED DESCRIPTION

[0049] Methods of using inhibitors of CTLA-4 for treating heart disease are provided. In addition, methods are provided for screening candidate agents for inhibition of CTLA-4 and effectiveness of candidate inhibitors of CTLA-4 in improving cardiac function, decreasing or preventing damage to the heart, including reducing chamber dilatation, ventricular remodeling, and / or amount of fibrotic scar tissue, and increasing survival.

[0050] Before exemplary embodiments of the present invention are described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0051] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0052] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and exemplary methods and materials may now be described. Any and all publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0053] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the inhibitor" includes reference to one or more inhibitors and equivalents thereof, e.g., antagonists, known to those skilled in the art, and so forth.

[0054] It is further noted that the claims may be drafted to exclude any element which may be optional. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only” and the like in connection with the recitation of claim elements, or the use of a “negative” limitation.

[0055] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. To the extent such publications may set out definitions of a term that conflicts with the explicit or implicit definition of the present disclosure, the definition of the present disclosure controls.

[0056] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.DEFINITIONS

[0057] The term "about," particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.

[0058] The term “heart disease” is used herein to refer to any disease or condition resulting in reduced blood flow to the cardiac muscle, ischemia, and heart damage. Heart diseases include, but are not limited to, myocardial infarction, myocardial ischemia, myocardial injury, heart failure, atherosclerosis, coronary artery disease, and cardiomyopathies.

[0059] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease. The term “treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and / or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and / or symptom(s), i.e., arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and / or symptom(s). Those in need of treatment include those already inflicted (e.g., those with a heart disease or ventricular remodeling) as well as those in which prevention is desired (e.g., those with increased susceptibility or a genetic predisposition to developing heart disease or ventricular remodeling).

[0060] A therapeutic treatment is one in which the subject is inflicted prior to administration and a prophylactic treatment is one in which the subject is not inflicted prior to administration. In some embodiments, the subject has an increased likelihood of becoming inflicted or is suspected of being inflicted prior to treatment. In some embodiments, the subject is suspected of having an increased likelihood of becoming inflicted.

[0061] The term "survival" as used herein means the time from the start of treatment to the time of death.

[0062] The terms “individual”, “subject”, and “patient”, are used interchangeably herein and refer to mammals, including human and non-human mammals such as, but not limited to, non-human primates, including chimpanzees and other apes and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domestic animals such as dogs and cats; and farm animals such as sheep, goats, pigs, horses and cows. In some cases, the subject methods find use in experimental animals, in veterinary applications, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, primates, and transgenic animals.

[0063] The term "inhibitor of CTLA-4" or “CTLA-4 inhibitor” as used herein refers to any molecule (e.g., small molecule inhibitor, protein, polypeptide, peptide, fusion protein, inhibitory nucleic acid (e.g., siRNA, miRNA, shRNA, antisense nucleic acid), peptide nucleic acid, peptoid, antibody, antibody mimetic, aptamer) or CRISPR system targeting the CTLA-4 gene (e.g., Cas9, Casl2a), CTLA-4 RNA transcripts (e.g., Casl3), or epigenome (e.g., dCas9 fusion protein) that inhibits CTLA-4 biological activity (e.g., binding of CTLA-4 to B7.1 and B7.2 antigen-presenting cell ligands), and / or CTLA-4 expression (e.g., transcription or translation). The CTLA-4 inhibitor may inhibit one or more CTLA-4 isoforms, including soluble and transmembrane isoforms. In some embodiments, the CTLA-4 inhibitor selectively inhibits one CTLA-4 isoform. Inhibition may be complete or partial (i.e., all activity, some activity, or most activity is blocked by an inhibitor). For example, an inhibitor may reduce the activity of CTLA- 4 by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between as compared to native or control levels.

[0064] An "effective amount" of a CTLA-4 inhibitor (e.g., small molecule inhibitor, protein, polypeptide, peptide, fusion protein, inhibitory nucleic acid (e.g., siRNA, miRNA, shRNA, antisense nucleic acid, or peptide nucleic acid), peptoid, antibody, antibody mimetic, or aptamer) or CRISPR system targeting the CTLA-4 gene (e.g., Cas9, Casl2a), RNA (Casl3), or epigenome (dCas9 fusion protein) is an amount sufficient to inhibit the biological activity of CTLA-4, for example, by inhibiting binding of CTLA-4 to B7.1 and B7.2 antigen-presenting cell ligands and / or interfering with CTLA-4 gene expression (e.g., transcription or translation). An effective amount can be administered in one or more administrations, applications, or dosages.

[0065] By "therapeutically effective dose or amount" of an inhibitor of CTLA-4 is intended an amount that, when administered as described herein, brings about a positive therapeutic response in treatment of heart disease, such as an amount that increases left ventricular ejection fraction, reduces chamber dilatation, reduces ventricular remodeling, reduces area of fibrotic scar tissue, reduces level of plasma troponin, and / or increases survival. The exact amount required will vary from subject to subject, depending on the species, age, and general conditionof the subject, the severity of the condition being treated, the particular drug or drugs employed, mode of administration, and the like. An appropriate "effective" amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.

[0066] The term "administering" is intended to include routes of administration which allow the agent to perform its intended function of inhibiting biological activity or expression of CTLA-4 and / or increasing left ventricular ejection fraction, reducing chamber dilatation, reducing ventricular remodeling, and / or reducing amount of fibrotic scar tissue. Examples of routes of administration which can be used include injection (subcutaneous, intravenous, parenterally, intraarterially, endotracheal, intraosseous, intracardiac, intraperitoneally, intrathecal, etc.), oral, pulmonary, and transdermal, and local administration into the heart at a site of damaged heart tissue or a site of cardiac ischemia or infarction. Injections can be administered as bolus injections or by continuous infusion. Depending on the route of administration, the agent can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally affect its ability to perform its intended function. The agent may be administered alone, or in conjunction with a pharmaceutically acceptable carrier. Further, the agent may be coadministered with a pharmaceutically acceptable carrier. The agent also may be administered as a prodrug, which is converted to its active form in vivo.

[0067] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

[0068] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts. Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0069] "Substantially purified" generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, peptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample, a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the artand include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0070] “Isolated” refers to an entity of interest that is in an environment different from that in which it may naturally occur. “Isolated” is meant to include entities that are within samples that are substantially enriched for the entity of interest and / or in which the entity of interest is partially or substantially purified.

[0071] The term “derived from” is used herein to identify the original source of a molecule but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant means.

[0072] As used herein, the term “determining” refers to both quantitative and qualitative determinations and as such, the term “determining” is used interchangeably herein with “assaying,” “measuring,” and the like.

[0073] The terms "quantity", "amount", and "level" are used interchangeably herein and may refer to an absolute quantification of a molecule or an analyte in a sample, or to a relative quantification of a molecule or analyte in a sample, i.e., relative to another value such as relative to a reference value as taught herein, or to a range of values for the molecule or analyte. These values or ranges can be obtained from a single patient or from a group of patients.

[0074] The term "antibody" encompasses polyclonal antibodies, monoclonal antibodies as well as hybrid antibodies, altered antibodies, chimeric antibodies, and humanized antibodies. The term antibody includes: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab')2 and F(ab) fragments; Fvmolecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules (scFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); nanobodies or single-domain antibodies (sdAb) (see, e.g., Wang et al. (2016) Int J Nanomedicine 11:3287-3303, Vincke et al. (2012) Methods Mol Biol 911:15-26; dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31: 1579-1584; Cumber et al. (1992) J Immunology 149B: 120-126); humanized antibody molecules (see, e.g., Riechmann et al. (1988) Nature 332:323-327 ; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and, any functional fragments obtained from such molecules, wherein such fragments retain specific -binding properties of the parent antibody molecule.

[0075] The phrase "specifically (or selectively) binds" with reference to binding of an antibody to an antigen (e.g., CTLA-4) refers to a binding reaction that is determinative of the presence of the antigen in a heterogeneous population of proteins and other biologies. Thus, underdesignated immunoassay conditions, the specified antibodies bind to a particular antigen at least two times the background and do not substantially bind in a significant amount to other antigens present in the sample. Specific binding to an antigen under such conditions may require an antibody that is selected for its specificity for a particular antigen. For example, antibodies raised to an antigen from specific species such as rat, mouse, or human can be selected to obtain only those antibodies that are specifically immunoreactive with the antigen and not with other proteins, except for polymorphic variants and alleles. This selection may be achieved by subtracting out antibodies that cross-react with molecules from other species. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular antigen. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane. Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 100 times background.

[0076] The terms "microRNA," "miRNA," and MiR" are interchangeable and refer to endogenous or artificial non-coding RNAs that are capable of regulating gene expression. It is believed that miRNAs function via RNA interference. When used herein in the context of inactivation, the use of the term microRNAs is intended to include also long non-coding RNAs, piRNAs, siRNAs, and the like. Endogenous (e.g., naturally occurring) miRNAs are typically expressed from RNA polymerase II promoters and are generated from a larger transcript.

[0077] The terms "siRNA" and "short interfering RNA" are interchangeable and refer to single-stranded or double-stranded RNA molecules that are capable of inducing RNA interference. SiRNA molecules typically have a duplex region that is between 18 and 30 base pairs in length.

[0078] The terms "shRNA" and "small hairpin RNA" are interchangeable and refer to RNA molecules, typically about 80 base pairs in length, that form a hairpin structure. shRNA molecules are processed within a cell to form siRNA which knock down gene expression through RNA interference.

[0079] The terms "piRNA" and "Piwi-interacting RNA" are interchangeable and refer to a class of small RNAs involved in gene silencing. PiRNA molecules typically are between 26 and 31 nucleotides in length.

[0080] The terms "snRNA" and "small nuclear RNA" are interchangeable and refer to a class of small RNAs involved in a variety of processes including RNA splicing and regulation of transcription factors. The subclass of small nucleolar RNAs (snoRNAs) is also included. The termis also intended to include artificial snRNAs, such as antisense derivatives of snRNAs comprising antisense sequences directed against the CTLA-4 gene.

[0081] The term "antisense", as used herein, refers to any composition containing nucleotide sequences which are complementary to a specific DNA or RNA sequence. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to the "sense" strand. Antisense molecules include peptide nucleic acids and may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and block either transcription or translation. The designation "negative" is sometimes used in reference to the antisense strand, and "positive" is sometimes used in reference to the sense strand.

[0082] The terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule" are used herein to include a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded DNA, as well as triple-, double- and single-stranded RNA. It also includes modifications, such as by methylation and / or by capping, and unmodified forms of the polynucleotide. More particularly, the terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule" include polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D- ribose), any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, Oreg., as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. There is no intended distinction in length between the terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule," and these terms will be used interchangeably. Thus, these terms include, for example, 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3’ P5’ phosphoramidates, 2'-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and single-stranded RNA, microRNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA, and also include known types of modifications, for example, labels which are known in the art, methylation, "caps," substitution of one or more of the naturally occurring nucleotides with an analog (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine, C5- propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7- deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), internucleotide modifications such as, for example, those with uncharged linkages(e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide. The term also includes locked nucleic acids (e.g., comprising a ribonucleotide that has a methylene bridge between the 2'-oxygen atom and the 4’-carbon atom). See, for example, Kurreck et al. (2002) Nucleic Acids Res. 30: 1911-1918; Elayadi et al. (2001) Curr. Opinion Invest. Drugs 2: 558-561 ; Orum et al. (2001) Curr. Opinion Mol. Ther. 3: 239-243; Koshkin et al. (1998) Tetrahedron 54: 3607-3630; Obika et al. (1998) Tetrahedron Lett. 39: 5401-5404.

[0083] The term "homologous region" refers to a region of a nucleic acid with homology to another nucleic acid region. Thus, whether a "homologous region" is present in a nucleic acid molecule is determined with reference to another nucleic acid region in the same or a different molecule. Further, since a nucleic acid is often double-stranded, the term "homologous, region," as used herein, refers to the ability of nucleic acid molecules to hybridize to each other. For example, a single-stranded nucleic acid molecule can have two homologous regions which are capable of hybridizing to each other. Thus, the term "homologous region" includes nucleic acid segments with complementary sequence. Homologous regions may vary in length, but will typically be between 4 and 40 nucleotides (e.g., from about 4 to about 40, from about 5 to about 40, from about 5 to about 35, from about 5 to about 30, from about 5 to about 20, from about 6 to about 30, from about 6 to about 25, from about 6 to about 15, from about 7 to about 18, from about 8 to about 20, from about 8 to about 15, etc.).

[0084] The term "complementary" and "complementarity" are interchangeable and refer to the ability of polynucleotides to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in antiparallel polynucleotide strands or regions. Complementary polynucleotide strands or regions can base pair in the Watson-Crick manner (e.g., A to T, A to U, C to G). 100% complementary refers to the situation in which each nucleotide unit of one polynucleotide strand or region can hydrogen bond with each nucleotide unit of a second polynucleotide strand or region. Less than perfect complementarity refers to the situation in which some, but not all, nucleotide units of two strands or two regions can hydrogen bond with each other and can be expressed as a percentage.

[0085] A "target site" or "target sequence" for an inhibitory nucleic acid is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by an antisense oligonucleotide or inhibitory RNA molecule.

[0086] The term "transfection" is used to refer to the uptake of foreign DNA or RNA by a cell. A cell has been "transfected" when exogenous DNA or RNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA or RNA moieties into suitable host cells. The term refers to both stable and transient uptake of the genetic material, and includes uptake, for example, of microRNA, siRNA, piRNA, IncRNA, or antisense nucleic acids.

[0087] A “CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.

[0088] The term "Cas9" as used herein encompasses type II clustered regularly interspaced short palindromic repeats (CRISPR) system Cas9 endonucleases from any species, and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks).

[0089] A Cas9 endonuclease binds to and cleaves DNA at a site comprising a sequence complementary to its bound guide RNA (gRNA). For purposes of Cas9 targeting, a gRNA may comprise a sequence "complementary" to a target sequence (e.g., major or minor allele), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.

[0090] By "selectively binds" with reference to a guide RNA is meant that the guide RNA binds preferentially to a target sequence of interest or binds with greater affinity to the target sequence than to other genomic sequences. For example, a gRNA will bind to a substantially complementary sequence and not to unrelated sequences. A gRNA that "selectively binds" to a particular allele, such as a particular mutant allele (e.g., allele comprising a substitution, insertion,or deletion), denotes a gRNA that binds preferentially to the particular target allele, but to a lesser extent to a wild-type allele or other sequences. A gRNA that selectively binds to a particular target DNA sequence will selectively direct binding of Cas9 to a substantially complementary sequence at the target site and not to unrelated sequences.

[0091] The term "donor polynucleotide" refers to a polynucleotide that provides a sequence of an intended edit to be integrated into the genome at a target locus by homology directed repair (HDR).

[0092] A "target site" or "target sequence" is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by a guide RNA (gRNA) or a homology arm of a donor polynucleotide. The target site may be allele-specific (e.g., a major or minor allele).

[0093] By "homology arm" is meant a portion of a donor polynucleotide that is responsible for targeting the donor polynucleotide to the genomic sequence to be edited in a cell. The donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3’ genomic target sequence flanking a nucleotide sequence comprising the intended edit to the genomic DNA. The homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide. The 5’ and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively. The nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR or recombineering at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3’ homology arms.

[0094] "Administering" a nucleic acid, such as an inhibitory or regulatory nucleic acid (e.g., microRNA, siRNA, piRNA, snRNA, antisense nucleic acid, or IncRNA), or a CRISPR system (expressing, e.g., a donor polynucleotide, guide RNA, Cas protein (e.g., Cas9, Casl2a, Casl2d, Casl3, or dCas9)) to a cell comprises transducing, transfecting, electroporating, translocating, fusing, phagocytosing, shooting or ballistic methods, etc., i.e., any means by which a nucleic acid can be transported across a cell membrane.Inhibitors of CTLA-4

[0095] The inventors have discovered that inhibitors of CTLA-4 are useful for promoting cardiac repair after myocardial infarction (see Examples). Immune cells migrate to the injured heart after myocardial infarction and regulate the immune response during the healing process. Inhibition of CTLA-4 leads to improved early post-infarct cardiac repair, reduced cardiac infarctsize, increased survival, and better preservation of cardiac function, including increased left ventricular ejection fraction, reduced chamber dilatation, and reduced ventricular remodeling. Therefore, inhibitors of CTLA-4 may be useful in treating myocardial infarction as well as other heart diseases where cardiac repair and regeneration are crucial such as, but not limited to, myocardial ischemia, myocardial injury, heart failure, atherosclerosis, coronary artery disease, and cardiomyopathies.

[0096] An "inhibitor of CTLA-4" can be any molecule (e.g., small molecule inhibitor, protein, polypeptide, peptide, fusion protein, inhibitory nucleic acid (e.g., siRNA, miRNA, antisense nucleic acid), peptide nucleic acid, peptoid, antibody, antibody mimetic, aptamer) or CRISPR system targeting the CTLA-4 gene (e.g., Cas9, Casl2a), CTLA-4 RNA transcripts (e.g., Casl3), or epigenome (e.g., dCas9 fusion protein) that inhibits CTLA-4 biological activity (e.g., binding of CTLA-4 to B7.1 and B7.2 antigen-presenting cell ligands), and / or CTLA-4 expression (e.g., transcription or translation). The CTLA-4 inhibitor may inhibit one or more CTLA-4 isoforms, including soluble and transmembrane isoforms. In some embodiments, the CTLA-4 inhibitor selectively inhibits a single CTLA-4 isoform. Inhibition may be complete or partial (i.e., all activity, some activity, or most activity is blocked by an inhibitor). For example, an inhibitor may reduce the activity of CTLA-4 by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between as compared to native or control levels.

[0097] An exemplary inhibitor of CTLA-4 is ipilimumab, a monoclonal antibody that binds to CTLA-4 and inhibits CTLA-4 activity. Formulations of ipilimumab (Yervoy®) are commercially available, for example, from Bristol Myers Squibb (San Francisco, CA) and have been approved by the U.S. Food and Drug Administration (FDA) previously for the treatment of certain cancers, including melanoma, non-small cell lung cancer (NSCLC), pleural mesothelioma, esophageal cancer, kidney cancer (RCC), liver cancer, and colorectal cancer.

[0098] Another exemplary inhibitor of CTLA-4 is tremelimumab, a different monoclonal antibody that also binds to CTLA-4 and inhibits CTLA-4 activity. Formulations of tremelimumab (Imjudo®) are commercially available, for example, from AstraZeneca (Cambridge, United Kingdom) and have been approved by the U.S. Food and Drug Administration (FDA) previously for the treatment of certain cancers, including hepatocellular carcinoma and metastatic non-small cell lung cancer.Pharmaceutical Compositions

[0099] Inhibitors of CTLA-4 such as ipilimumab and tremelimumab or other inhibitors identified by the screening methods described herein, can be formulated into pharmaceutical compositions, optionally comprising one or more pharmaceutically acceptable excipients.Exemplary excipients include, without limitation, carbohydrates, inorganic salts, antimicrobial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for injectable compositions include water, alcohols, polyols, glycerine, vegetable oils, phospholipids, and surfactants. A carbohydrate such as a sugar, a derivatized sugar such as an alditol, aldonic acid, an esterified sugar, and / or a sugar polymer may be present as an excipient. Specific carbohydrate excipients include, for example: monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. The excipient can also include an inorganic salt or buffer such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

[0100] A composition can also include an antimicrobial agent for preventing or deterring microbial growth. Nonlimiting examples of antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.

[0101] An antioxidant can be present in the composition as well. Antioxidants are used to prevent oxidation, thereby preventing the deterioration of the CTLA-4 inhibitor, or other components of the preparation. Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, mono thioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0102] A surfactant can be present as an excipient. Exemplary surfactants include: polysorbates, such as "Tween 20" and "Tween 80," and pluronics such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids, such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (although preferably not in liposomal form), fatty acids and fatty esters; steroids, such as cholesterol; chelating agents, such as EDTA; and zinc and other such suitable cations.

[0103] Acids or bases can be present as an excipient in the composition. Nonlimiting examples of acids that can be used include those acids selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof. Examples of suitable bases include, without limitation, bases selected fromthe group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumerate, and combinations thereof.

[0104] The amount of the CTLA-4 inhibitor (e.g., when contained in a drug delivery system) in the composition will vary depending on a number of factors but will optimally be a therapeutically effective dose when the composition is in a unit dosage form or container (e.g., a vial). A therapeutically effective dose can be determined experimentally by repeated administration of increasing amounts of the composition in order to determine which amount produces a clinically desired endpoint.

[0105] The amount of any individual excipient in the composition will vary depending on the nature and function of the excipient and particular needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing varying amounts of the excipient (ranging from low to high), examining the stability and other parameters, and then determining the range at which optimal performance is attained with no significant adverse effects. Generally, however, the excipient(s) will be present in the composition in an amount of about 1 % to about 99% by weight, preferably from about 5% to about 98% by weight, more preferably from about 15 to about 95% by weight of the excipient, with concentrations less than 30% by weight most preferred. These foregoing pharmaceutical excipients along with other excipients are described in "Remington: The Science & Practice of Pharmacy", 19th ed., Williams & Williams, (1995), the "Physician’s Desk Reference", 52nd ed., Medical Economics, Montvale, NJ (1998), and Kibbe, A.H., Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, D.C., 2000.

[0106] The compositions encompass all types of formulations and in particular those that are suited for injection, e.g., powders or lyophilates that can be reconstituted with a solvent prior to use, as well as ready for injection solutions or suspensions, dry insoluble compositions for combination with a vehicle prior to use, and emulsions and liquid concentrates for dilution prior to administration. Examples of suitable diluents for reconstituting solid compositions prior to injection include bacteriostatic water for injection, dextrose 5% in water, phosphate buffered saline, Ringer’s solution, saline, sterile water, deionized water, and combinations thereof. With respect to liquid pharmaceutical compositions, solutions and suspensions are envisioned. Additional preferred compositions include those for oral, ocular, or localized delivery.

[0107] The pharmaceutical preparations herein can also be housed in a syringe, an implantation device, or the like, depending upon the intended mode of delivery and use.Preferably, the composition comprising a CTLA-4 inhibitor is in unit dosage form, meaning an amount of the CTLA-4 inhibitor or a conjugate or composition of the invention appropriate for a single dose in a premeasured or pre-packaged form.

[0108] The compositions herein may optionally include one or more additional agents, such as one or more other drugs for treating heart disease or other medications. For example, compounded preparations may include at least one CTLA-4 inhibitor and one or more other drugs for treating heart disease such as, but not limited to, cholesterol lowering medications such as statins, fibrates, ezetimibe, lomitapide, and proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors; beta-blockers such as propranolol, bucindolol, carteolol, carvedilol, labetalol, nadolol, oxprenolol, penbutolol, pindolol, sotalol, timolol, acebutolol, atenolol, betaxolol, bisoprolol, celiprolol, metoprolol, esmolol, bisoprolol, butaxamine, ICI- 118,551 , SR 59230A, and nebivolol; nitroglycerin; and calcium channel blockers such as amlodipine (Norvasc), aranidipine (Sapresta), azelnidipine (Calblock), bamidipine (HypoCa), benidipine (Coniel), cilnidipine (Atelec, Cinalong, Siscard), clevidipine (Cleviprex), efonidipine (Landel), felodipine (Plendil), isradipine (DynaCirc, Preseal), lacidipine (Motens, Lacipil), lercanidipine (Zanidip), manidipine (Calslot, Madipine), nicardipine (Cardene, Carden SR), nifedipine (Procardia, Adalat), nilvadipine (Nivadil), nimodipine (Nimotop), nisoldipine (Baymycard, Sular, Syscor), nitrendipine (Cardif, Nitrepin, Baylotensin), pranidipine (Acalas), fendiline, gallopamil, verapamil (Calan, Isoptin), mibefradil, bepridil, flunarizine, fluspirilene, and fendiline. gabapentin and pregabalin; and other drugs for treating heart disease, or other medications used to treat a subject for a condition or disease. Alternatively, such agents can be contained in a separate composition from the composition comprising the CTLA-4 inhibitor and co- administered concurrently, before, or after the composition comprising the CTLA-4 inhibitor.Administration

[0109] At least one therapeutically effective cycle of treatment with an inhibitor of CTLA- 4 will be administered to a subject for treatment of heart disease. Heart diseases include any disease or condition resulting in reduced blood flow to the cardiac muscle, ischemia, and / or heart damage such as but are not limited to, myocardial infarction, myocardial ischemia, myocardial injury, heart failure, atherosclerosis, coronary artery disease, and cardiomyopathies.

[0110] By "therapeutically effective dose or amount" of an inhibitor of CTLA-4 is intended an amount that, when administered as described herein, brings about a positive therapeutic response in treatment of heart disease, such as an amount that increases left ventricular ejection fraction, reduces chamber dilatation, reduces ventricular remodeling, reduces area of fibrotic scar tissue, and / or increases survival. Additionally, a therapeutically effective dose oramount may inhibit binding of CTLA-4 to B7.1 and B7.2 antigen-presenting cell ligands and / or increase proliferation of Foxp3+Tregs.

[0111] In certain embodiments, multiple therapeutically effective doses of compositions comprising an inhibitor of CTLA-4, and / or one or more other therapeutic agents, such as other drugs for treating a heart disease, or other medications will be administered. The compositions of the present invention are typically, although not necessarily, administered orally, via injection (subcutaneously, intravenously, intramuscularly, or intraperitoneally), by infusion, or locally. Additional modes of administration are also contemplated, such as intra-arterial, intracardiac, pulmonary, endotracheal, intraosseous, rectal, transdermal, transmucosal, intrathecal, pericardial, and so forth.

[0112] The preparations are also suitable for local treatment. In a particular embodiment, a composition is used for localized delivery of an inhibitor of CTLA-4 for the treatment of heart disease. For example, compositions may be administered locally to damaged heart tissue or a site of cardiac ischemia or infarction. The particular preparation and appropriate method of administration are chosen to target the inhibitor of CTLA-4 to the site where repair of heart damage is needed.

[0113] The pharmaceutical preparation can be in the form of a liquid solution or suspension immediately prior to administration, but may also take another form such as a syrup, cream, ointment, tablet, capsule, powder, gel, matrix, suppository, or the like. The pharmaceutical compositions comprising an inhibitor of CTLA-4 and / or other agents may be administered using the same or different routes of administration in accordance with any medically acceptable method known in the art.

[0114] In another embodiment, the pharmaceutical compositions comprising an inhibitor of CTLA-4 and / or other agents are administered prophylactically, e.g., to prevent myocardial infarction and / or ventricular remodeling. Such prophylactic uses will be of particular value for subjects who have a genetic predisposition or a condition such as obesity, high levels of cholesterol, high blood pressure, lack of exercise, smoking, or diabetes that increases the risk of developing coronary artery disease.

[0115] In another embodiment, the pharmaceutical compositions comprising an inhibitor of CTLA-4 and / or other agents are in a sustained-release formulation, or a formulation that is administered using a sustained-release device. Such devices are well known in the art, and include, for example, transdermal patches, and miniature implantable pumps that can provide for drug delivery over time in a continuous, steady-state fashion at a variety of doses to achieve a sustained- release effect with a non-sustained-release pharmaceutical composition.

[0116] The disclosure also provides a method for administering a conjugate comprising an inhibitor of CTLA-4 to a patient suffering from heart disease or a condition that is responsive to treatment with an inhibitor of CTLA-4 contained in the conjugate or composition. The method comprises administering, via any of the herein described modes, a therapeutically effective amount of the conjugate or drug delivery system, preferably provided as part of a pharmaceutical composition. The method of administering may be used to treat any condition that is responsive to treatment with an inhibitor of CTLA-4.

[0117] Those of ordinary skill in the art will appreciate which conditions a specific inhibitor of CTLA-4 can effectively treat. The actual dose to be administered will vary depending upon the age, weight, and general condition of the subject as well as the particular disorder associated with protein misfolding being treated, the severity of the condition being treated, the judgment of the health care professional, and the particular CTLA-4 inhibitor or conjugate being administered. Therapeutically effective amounts can be determined by those skilled in the art, and will be adjusted to the particular requirements of each particular case.

[0118] In certain embodiments, multiple therapeutically effective doses of an inhibitor of CTLA-4 will be administered according to a daily dosing regimen or intermittently. For example, a therapeutically effective dose can be administered, one day a week, two days a week, three days a week, four days a week, or five days a week, and so forth. By “intermittent” administration is intended the therapeutically effective dose can be administered, for example, every other day, every two days, every three days, once a week, every other week, and so forth. For example, in some embodiments, a composition comprising an inhibitor of CTLA-4 will be administered once- weekly, twice-weekly or thrice-weekly for an extended period of time, such as for 1, 2, 3, 4, 5, 6, 7, 8...10...15...24 weeks, and so forth. By “twice-weekly” or “two times per week” is intended that two therapeutically effective doses of the agent in question is administered to the subject within a 7 day period, beginning on day 1 of the first week of administration, with a minimum of 72 hours, between doses and a maximum of 96 hours between doses. By “thrice weekly” or “three times per week” is intended that three therapeutically effective doses are administered to the subject within a 7 day period, allowing for a minimum of 48 hours between doses and a maximum of 72 hours between doses. For purposes of the present disclosure, this type of dosing is referred to as “intermittent” therapy. In accordance with the methods described herein, a subject can receive intermittent therapy (i.e., once- weekly, twice-weekly or thrice-weekly administration of a therapeutically effective dose) for one or more weekly cycles until the desired therapeutic response is achieved. The agents can be administered by any acceptable route of administration as noted herein below. The amount administered will depend on the potency of the specific CTLA-4inhibitor, the particular disorder associated with protein misfolding that is treated, the magnitude of the effect desired, and the route of administration.

[0119] A purified CTLA-4 inhibitor (again, preferably provided as part of a pharmaceutical preparation) can be administered alone or in combination with one or more other therapeutic agents for treating heart disease such as, but not limited to, cholesterol lowering medications such as statins, fibrates, ezetimibe, lomitapide, and proprotein convertase subtilisin / kexin type 9 (PCSK9) inhibitors; beta-blockers such as propranolol, bucindolol, carteolol, carvedilol, labetalol, nadolol, oxprenolol, penbutolol, pindolol, sotalol, timolol, acebutolol, atenolol, betaxolol, bisoprolol, celiprolol, metoprolol, esmolol, bisoprolol, butaxamine, ICI-118,551, SR 59230A, and nebivolol; nitroglycerin; and calcium channel blockers such as amlodipine (Norvasc), aranidipine (Sapresta), azelnidipine (Calblock), bamidipine (HypoCa), benidipine (Coniel), cilnidipine (Atelec, Cinalong, Siscard), clevidipine (Cleviprex), efonidipine (Landel), felodipine (Plendil), isradipine (DynaCirc, Preseal), lacidipine (Motens, Lacipil), lercanidipine (Zanidip), manidipine (Calslot, Madipine), nicardipine (Cardene, Carden SR), nifedipine (Procardia, Adalat), nilvadipine (Nivadil), nimodipine (Nimotop), nisoldipine (Baymycard, Sular, Syscor), nitrendipine (Cardif, Nitrepin, Baylotensin), pranidipine (Acalas), fendiline, gallopamil, verapamil (Calan, Isoptin), mibefradil, bepridil, flunarizine, fluspirilene, and fendiline. gabapentin and pregabalin; or other medications used to treat a particular condition or disease according to a variety of dosing schedules depending on the judgment of the clinician, needs of the patient, and so forth. The specific dosing schedule will be known by those of ordinary skill in the art or can be determined experimentally using routine methods. Exemplary dosing schedules include, without limitation, administration five times a day, four times a day, three times a day, twice daily, once daily, three times weekly, twice weekly, once weekly, twice monthly, once monthly, and any combination thereof. Preferred compositions are those requiring dosing no more than once a day.

[0120] An inhibitor of CTLA-4 can be administered prior to, concurrent with, or subsequent to other agents. If provided at the same time as other agents, the inhibitor of CTLA-4 can be provided in the same or in a different composition. Thus, CTLA-4 inhibitor and / or other agents can be presented to the individual by way of concurrent therapy. By “concurrent therapy” is intended administration to a subject such that the therapeutic effect of the combination of the substances is caused in the subject undergoing therapy. For example, concurrent therapy may be achieved by administering a dose of a pharmaceutical composition comprising an inhibitor of CTLA-4 and a dose of a pharmaceutical composition comprising at least one other agent, such as another drug for treating a heart disease, which in combination comprise a therapeutically effective dose, according to a particular dosing regimen. Similarly, an inhibitor of CTLA-4 andone or more other therapeutic agents can be administered in at least one therapeutic dose. Administration of the separate pharmaceutical compositions can be performed simultaneously or at different times (i.e., sequentially, in either order, on the same day, or on different days), as long as the therapeutic effect of the combination of these substances is caused in the subject undergoing therapy.

[0121] The methods described herein can be used for treating a human subject for heart disease. The methods described herein will also find use in veterinary applications for treatment of such heart diseases or conditions, for example, in domestic animals including, without limitation, pets, such as dogs and cats, and farm animals, such as sheep, goats, pigs, horses and cattle.Screening for Inhibitors of CTLA-4

[0122] Inhibitors of CTLA-4 may be useful in treating heart disease. Accordingly, screening methods for identifying candidate agents that inhibit CTLA-4 activity and can be potentially used for treating heart disease are provided. A variety of assays may be used for this purpose, and in many embodiments, a candidate agent will be tested in different assays to confirm inhibitory capability as well as efficacy in treating heart disease. For example, biochemical assays may determine the ability of an agent to inhibit biological activity of CTLA-4 (e.g., inhibition of binding of CTLA-4 to B7.1 and B7.2 antigen-presenting cell ligands, proliferation of Foxp3+ Tregs). In addition, an animal model of myocardial infarction having left anterior descending artery (LAD) ligation may be tested in the absence or presence of a candidate agent to evaluate effects, for example, on heart function (e.g., left ventricular ejection fraction), chamber dilatation, ventricular remodeling, amount of fibrotic scar tissue, plasma troponin levels, and survival.

[0123] An inhibitor of CTLA-4 can be any molecule including, without limitation, a small molecule inhibitor, protein, polypeptide, peptide, fusion protein, nucleic acid, oligonucleotide, peptide nucleic acid, peptoid, antibody or fragment thereof, antibody mimetic, aptamer, or a CRISPR system targeting the CTLA-4 gene (e.g., Cas9, Casl2a), RNA transcripts (e.g., Casl3), or epigenome (e.g., dCas9 fusion protein) that inhibits CTLA-4 activity and / or CTLA-4 expression (e.g., transcription or translation). Inhibition may be complete or partial (i.e., all activity, some activity, or most activity is blocked by an inhibitor). For example, an inhibitor may reduce the activity of CTLA-4 or reduce CTLA-4 mRNA or protein levels by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between as compared to native or control levels. The CTLA-4 inhibitor may inhibit one or more isoforms of CTLA-4, including soluble and transmembrane isoforms. In some embodiments, the CTLA-4 inhibitor selectively inhibits one CTLA-4 isoform.

[0124] In some embodiments, a CTLA-4 inhibitor reduces biological activity of CTLA-4 by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between as compared to native or control levels. In some embodiments, a CTLA-4 inhibitor inhibits binding of CTLA-4 to B7.1 and B7.2 antigen-presenting cell ligands or increases proliferation of Foxp3+ Tregs.

[0125] For purposes of the assay methods, CTLA-4 may be provided as an isolated protein. Alternatively, the CTLA-4 protein can be present in the context of a cell, tissue, heart organ or organoid, or animal. Any convenient format may be used for the assay, e.g. wells, plates, flasks, etc., preferably a high throughput format, such as multi-well plates. A test agent of interest is added to the reaction mixture with the CTLA-4 protein and the effect of the agent on CTLA-4 activity is determined.

[0126] Inhibitors can be identified by contacting CTLA-4 with a candidate agent; and measuring inhibition of CTLA-4 biological activity (e.g., inhibition of binding of CTLA-4 to B7.1 and B7.2 antigen-presenting cell ligands or proliferation of Foxp3+ Tregs) by the candidate agent. Assays can be performed in the presence of the candidate agent and in the absence of the candidate agent to allow results to be compared.

[0127] Binding of CTLA-4 by either of its ligands, CD80 (B7-1) or CD86 (B7-2) on an antigen presenting cell (APC) inhibits CD28 co-stimulation of T cell activation, cell proliferation and cytokine production. Binding of CTLA-4 to CD80 (B7-1) and CD86 (B7-2) may be measured in the presence and absence of a candidate agent by surface plasmon resonance (SPR) to determine if a candidate agent inhibits binding. SPR can be performed, for example, as described by van der Merwe et al. (1997) J. Exp. Med. 185(3):393-403, Greene et al. (1996) J. Biol. Chem. 271(43):26762-26771; herein incorporated by reference.

[0128] Alternatively, a CTLA-4 blockade assay using T cells expressing CTLA-4 and antigen presenting cells presenting CD80 (B7-1) and CD86 (B7-2) can be used to detect T cell activation in the presence and absence of a candidate agent. Such an assay is commercially available from Promega Corporation (Madison, WI), which uses lurkat T cells expressing human CTLA-4 and a luciferase reporter driven by a native promoter that responds to activation of the T cell receptor (TCR) and CD28. The lurkat T cells are used in combination with artificial antigen presenting cells. In this assay, the artificial antigen presenting cells are Raji cells expressing an engineered cell surface protein designed to activate cognate TCRs in an antigen-independent manner and the CTLA-4 ligands, CD80 and CD86. The Jurkat T cells and artificial antigen presenting cells are co-cultured, resulting in competition of CTLA-4 with CD28 for their shared ligands, CD80 and CD86, which inhibits activation of the CD28 pathway and promoter-mediatedluminescence. Addition of an inhibitor of CTLA-4 blocks the interaction of CTLA-4 with CD80 and CD86 and results in detectable promoter-mediated luminescence.

[0129] In some embodiments, a test agent that is shown to inhibit CTLA-4 activity is further tested for its ability to induce proliferation of Foxp3+ Tregs in a cell-based assay. In these embodiments, a test agent of interest is contacted with Foxp3+ Tregs and the effect, if any, of the test agent on the Foxp3+ Tregs is determined. For example, activation and proliferation of Foxp3+ Tregs can be measured in the presence and absence of a candidate agent by flow cytometry. Cell proliferation of Foxp3+ Tregs can also be detected and quantified, for example, using a cell counter or staining of Foxp3+Tregs with a fluorescent tracking dye, such as carboxyfluorescein succinimidyl ester (CFSE).

[0130] In some embodiments, a test agent is tested in a cell-based model of cardiotoxicity. Such a cell-based model can be created using human induced pluripotent stem cells, which are differentiated into cardiomyocytes (hiPSC-CMs) using known protocols (see, e.g., Example 8 and Lian, X., et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt / beta-catenin signaling under fully defined conditions. Nat Protoc 8, 162-175 (2013); Kleinsorge, M. & Cyganek, L. Subtype-Directed Differentiation of Human iPSCs into Atrial and Ventricular Cardiomyocytes. STAR Protoc 1, 100026 (2020); herein incorporated by reference in their entireties), AC 16 transformed human ventricular cardiomyocytes (Davidson, M.M., et al. Novel cell lines derived from adult human ventricular cardiomyocytes. J Mol Cell Cardiol 39, 133-147 (2005), herein incorporated by reference in its entirety), or HL-1 immortalized mouse atrial cardiomyocytes (Claycomb, W.C., et al. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci USA. 95(6), 2979-2984 (1998); White, S.M., Constantin, P.E. & Claycomb, W.C. Cardiac physiology at the cellular level: use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cell structure and function. Am J Physiol Heart Circ Physiol 286, H823-829 (2004)). Cell viability can be evaluated using annexin V (AxV) with propidium iodide (PI) staining for early apoptosis and cell stress (Vermes, I., Haanen, C., Steffens-Nakken, H. & Reutelingsperger, C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. I Immunol Methods 184, 39-51 (1995), herein incorporated by reference in its entirety). Monolayers of cells plated on multi-electrode arrays can be monitored using extracellular field potential (EFP) recordings. Suitable multi-electrode arrays are commercially available including, without limitation, the Nanion CardioExcyte96 system from Nanion Technologies (Miinchen, Germany), a high- throughput screening platform which allows for the simultaneous recording of cell impendence (as a marker of cellular contractility) and extracellular field potentials (EFP, a measure ofelectrophysiologic activity). Multi-electrode arrays can be used to detect the effects of test agents on pro- arrhythmic properties (see, e.g., Doerr, L., et al. New easy-to-use hybrid system for extracellular potential and impedance recordings. J Lab Autom 20, 175-188 (2015); Ziegler, R., Hausermann, F., Kirchner, S. & Polonchuk, L. Cardiac Safety of Kinase Inhibitors - Improving Understanding and Prediction of Liabilities in Drug Discovery Using Human Stem Cell-Derived Models. Front Cardiovasc Med 8, 639824 (2021); herein incorporated by reference in their entireties).

[0131] The effects of test agents on contractility and calcium (Ca2+)dynamics of cardiomyocytes can be tested using a custom optimized multicell Calcium and Contractility System (lonOptix, Westwood, MA). See, e.g., Wust, R.C., et al. Rapid frequency-dependent changes in free mitochondrial calcium concentration in rat cardiac myocytes. J Physiol 595, 2001- 2019 (2017); herein incorporated by reference in its entirety. Cells are cultured at low confluency to enable the detection of Ca2+signals at the single-cell level. This system allows simultaneous data acquisition from multiple cells ensuring high throughput. Fluorescent signals of Ca2+are measured using a ratiometric Ca2+indicator, such as Fura-2-acetoxymethyl ester (Furo-2AM, Ex: 360 / 380 nm, Em: 510 nm; 1 pM; abeam), with 1 Hz field electrical stimulation (Martinez, M., Martinez, N.A. & Silva, W.I. Measurement of the Intracellular Calcium Concentration with Fura- 2 AM Using a Fluorescence Plate Reader. Bio Protoc 7, e2411 (2017); herein incorporated by reference in its entirety). Properties of Ca2+dynamics, including Ca2+amplitude, time-to-peak, Ca2+reuptake decay constant (r) are compared in the presence and absence of a test agent. Contractility of cardiomyocytes is evaluated by measuring shortening and re-lengthening of sarcomeres or measuring cell length. Test agents can be applied to cells through a perfusion system.

[0132] Assays may further include suitable controls (e.g., a sample comprising the CTLA- 4 protein in the absence of the test agent). Generally, a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.

[0133] A variety of other reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc., including agents that are used to facilitate optimal binding activity and / or reduce non-specific or background activity. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used. The components of the assay mixture are added in any order that provides for the requisite activity. Incubations are performed at any suitable temperature, typically between 4DC and 40 DC. Incubation periods are selected for optimum activity but mayT1also be optimized to facilitate rapid high-throughput screening. In some embodiments, between 0.1 hour and 1 hour, between 1 hour and 2 hours, or between 2 hours and 4 hours, will be sufficient.

[0134] A variety of different test agents may be screened. Candidate agents encompass numerous chemical classes, e.g., small organic compounds having a molecular weight of more than 50 daltons and less than about 10,000 daltons, less than about 5,000 daltons, or less than about 2,500 daltons. Test agents can comprise functional groups necessary for structural interaction with proteins, e.g., hydrogen bonding, and can include at least an amine, carbonyl, hydroxyl or carboxyl group, or at least two of the functional chemical groups. The test agents can comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Test agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[0135] Test agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Moreover, screening may be directed to known pharmacologically active compounds and chemical analogs thereof, or to new agents with unknown properties such as those created through rational drug design.

[0136] In some embodiments, test agents are synthetic compounds. A number of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. See for example WO 94 / 24314, hereby expressly incorporated by reference, which discusses methods for generating new compounds, including random chemistry methods as well as enzymatic methods.

[0137] In another embodiment, the test agents are provided as libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts that are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, including enzymatic modifications, to produce structural analogs.

[0138] In some embodiments, the test agents are organic moieties. In this embodiment, test agents are synthesized from a series of substrates that can be chemically modified. “Chemically modified” herein includes traditional chemical reactions as well as enzymatic reactions. These substrates generally include, but are not limited to, alkyl groups (including alkanes, alkenes, alkynes and heteroalkyl), aryl groups (including arenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepins, beta-lactams, tetracylines, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecodysone, etc.), alkaloids (including ergots, vinca, curare, pyrollizdine, and mitomycines), organometallic compounds, hetero-atom bearing compounds, amino acids, and nucleosides. Chemical (including enzymatic) reactions may be done on the moieties to form new substrates or candidate agents which can then be tested using the present invention.

[0139] In some embodiments test agents are assessed for any cytotoxic activity it may exhibit toward a living eukaryotic cell, using well-known assays, such as trypan blue dye exclusion, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide) assay, and the like. Agents that do not exhibit significant cytotoxic activity are considered candidate agents.

[0140] In some embodiments, the test agent is an antibody that specifically binds to and inhibits biological activity of CTLA-4. Any type of antibody may be screened for the ability to inhibit CTLA-4 by the methods described herein, including polyclonal antibodies, monoclonal antibodies, hybrid antibodies, altered antibodies, chimeric antibodies and, humanized antibodies, as well as: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab')2 and F(ab) fragments; Fvmolecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659- 2662; and Ehrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules (sFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); nanobodies or single-domain antibodies (sdAb) (see, e.g., Wang et al. (2016) hit J Nanomedicine 11:3287-3303, Vincke et al. (2012) Methods Mol Biol 911: 15-26; dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumber et al. (1992) J Immunology 149B:120-126); humanized antibody molecules (see, e.g., Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and, any functional fragments obtained from such molecules, wherein such fragments retain specific-binding properties of the parent antibody molecule.

[0141] In other embodiments, the test agent is an aptamer that specifically binds to and inhibits biological activity of CTLA-4. Aptamers may be isolated from a combinatorial library and improved by directed mutation or repeated rounds of mutagenesis and selection. For a description of methods of producing aptamers, see, e.g., Aptamers: Tools for Nanotherapy and Molecular Imaging (R.N. Veedu ed., Pan Stanford, 2016), Nucleic Acid and Peptide Aptamers: Methods and Protocols (Methods in Molecular Biology, G. Mayer ed., Humana Press, 2009), Aptamers Selected by Cell-SELEX for Theranostics (W. Tan, X. Fang eds., Springer, 2015), Cox et al. (2001) Bioorg. Med. Chem. 9(10):2525-2531; Cox et al. (2002) Nucleic Acids Res. 30(20): el08, Kenan et al. (1999) Methods Mol. Biol. 118:217-231; Platelia et al. (2016) Biochim. Biophys. Acta Nov 16 pii: S0304-4165(16)30447-0, and Lyu et al. (2016) Theranostics 6(9): 1440- 1452; herein incorporated by reference in their entireties.

[0142] In yet other embodiments, the test agent is an antibody mimetic that specifically binds to and inhibits biological activity of CTLA-4. Any type of antibody mimetic may be used as an inhibitor, including, but not limited to, affibody molecules (Nygren (2008) FEBS J. 275 (11):2668-2676), affilins (Ebersbach et al. (2007) J. Mol. Biol. 372 (1): 172- 185), affimers (lohnson et al. (2012) Anal. Chem. 84 (15):6553-6560), affitins (Krehenbrink et al. (2008) I. Mol. Biol. 383 (5): 1058-1068), alphabodies (Desmet et al. (2014) Nature Communications 5:5237), anticalins (Skerra (2008) FEBS J. 275 (ll):2677-2683), avimers (Silverman et al. (2005) Nat. Biotechnol. 23 (12): 1556-1561), darpins (Stumpp et al. (2008) Drug Discov. Today 13 (15- 16):695-701), fynomers (Grabulovski et al. (2007) J. Biol. Chem. 282 (5):3196-3204), and monobodies (Koide et al. (2007) Methods Mol. Biol. 352:95-109).The candidate agents may be further tested for efficacy in treating heart disease in vivo, e.g., in an animal model. For example, candidate agents can be tested in a mammalian animal model of myocardial infarction having left anterior descending artery ligation. An exemplary murine model of myocardial infarction having left anterior descending artery ligation is described in Example 2. Left ventricular ejection fraction, chamber dilatation, ventricular remodeling, amount of fibrotic scar tissue, plasma troponin levels, or survival, or any combination thereof can be measured in the presence and absence of candidate agents to evaluate efficacy. For example, a Kaplan-Meier survival analysis (see, e.g., Hua et al. (2021) Heart Lung Circ. 30(7):978-985; Zhao et al. (2023) Heart Surg. Forum 26(5):E478-E484.; Goel et al. (2010) Int. J. Ayurveda Res. l(4):274-278; herein incorporated by reference) can be used in tracking survival rates and wall rupture-induced mortality. Echocardiography can be used to evaluate cardiac function, chamber size, ventricular remodeling, and infarct size after left anterior descending artery ligation. Echocardiographic imaging in a parasternal long-axis view can be used to estimate the extent of the fibrotic scar formed after left anterior descending artery ligation. Ultrasound can also be usedto assess infarct size and left ventricular function. Alternatively or additionally, hearts can be removed from animals and evaluated for end-point measurements. For example, the size of the fibrotic scar, chamber size and dilatation, and / or ventricular remodeling can be determined by histological analysis. See, e.g., Baudouy et al. (2017) J. Vis. Exp. 128:1-9, Rutledge et al. (2020) Cardiovasc Ultrasound 18:9, Yuan et al. (2011) J. Am. Soc. Echocardiogr 24 219-226, Dawson et al. (2004) Circulation 110: 1632-1637, Kanno et al. (2002) J. Am. Soc. Echocardiogr. 15: 601- 609, 2002. Dann et al. (2022) Am. J. Physiol. Heart Circ. Physiol. 322(3):H359-H372; herein incorporated by reference in their entireties. The left ventricular ejection fraction can be measured by echocardiography, magnetic resonance imaging (MRI), computerized tomography (CT), radionuclide angiography, gated myocardial perfusion single-photon emission computed tomography (SPECT), or gated myocardial perfusion positron emission tomography (PET). See, e.g., Foley et al. (2012) European Cardiology 8(2): 108-114, Picard et al. (2008) J. Am. Soc. Echocardiogr. 21(1): 14-21, Jenkins et al. (2007) Am. J. Cardiol. 99(3):300-306; herein incorporated by reference in their entireties. Additionally, cardiac damage can be evaluated by quantifying plasma troponin levels, for example, using an enzyme-linked immunosorbent assay (ELISA) or other immunoassay technique or liquid chromatography-tandem mass spectrometry. See, e.g., Bodor et al. (1992) Clin. Chem. 38(11):2203-2214, Penttila et al. (1997) Eur. J. Clin. Chem. Clin. Biochem. 35(10):767-774, Wu et al. (2013) Clin. Biochem. 46(12):969-978, Schneck et al. (2018) Anal. Bioanal. Chem. 410(11): 2805-2813; herein incorporated by reference in their entireties. ELISA kits for measuring troponin are commercially available, for example, from Thermo Fisher Scientific (Waltham, MA), Abeam (Cambridge, United Kingdom), and Sigma- Aldrich (St. Louis, MO).

[0143] An animal model can be used not only to determine efficacy but also the toxicity or side effects of treatment with a candidate agent. Alternatively, an agent identified, as described herein, can be used in an animal model to determine the mechanism of action of such an agent. Monitoring the efficacy of agents (e.g., drugs) on heart disease can be applied not only in basic drug screening, but also in clinical trials. Furthermore, this disclosure pertains to uses of inhibitors of CTLA-4, identified by the above-described screening assays for treatment of heart diseases such as, but not limited to, myocardial infarction, myocardial ischemia, myocardial injury, heart failure, atherosclerosis, coronary artery disease, and cardiomyopathies.Screening for Inhibitors of CTLA-4 Gene Expression

[0144] Alternatively, a CTLA-4 inhibitor may reduce CTLA-4 gene expression (e.g., transcription or translation). Candidate agents are identified by contacting a cell with a candidate compound and measuring the expression of CTLA-4, as determined by e.g., mRNA or polypeptidelevels. The level of expression of CTLA-4 mRNA or protein in the presence of the candidate compound is compared to the level of expression of CTLA-4 mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified based on this comparison. For example, when expression of CTLA-4 mRNA or protein in cells is decreased (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an agent that inhibits CTLA-4 expression. Alternatively, when expression of CTLA-4 mRNA or protein is the same or increased (statistically significantly more) in the presence of the candidate compound than in its absence, the candidate compound is likely not an agent that inhibits CTLA-4 expression or activity.

[0145] In some embodiments, an inhibitor of CTLA-4 gene expression reduces CTLA-4 mRNA or protein levels by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between as compared to native or control levels. Inhibitors of CTLA-4 gene expression can include, but are not limited to, antisense oligonucleotides, inhibitory RNA molecules, such as miRNAs, siRNAs, shRNAs, piRNAs, and snRNAs, peptide nucleic acids, small molecule inhibitors, and CRISPR systems designed for genome, RNA transcript, or epigenome editing. Various types of inhibitors for inhibiting nucleic acid function are well known in the art. See e.g., International patent application WO / 2012 / 018881; U.S. patent application 2011 / 0251261; U.S. patent no. 6,713,457; Kole et al. (2012) Nat. Rev. Drug Discov. ll(2): 125-40; Sanghvi (2011) Curr. Protoc. Nucleic Acid Chem. Chapter 4:Unit 4.1.1-22; herein incorporated by reference in their entireties.

[0146] Inhibitors can be single stranded or double stranded polynucleotides and may contain one or more chemical modifications, such as, but not limited to, locked nucleic acids, peptide nucleic acids, sugar modifications, such as 2'-O-alkyl (e.g., 2'-O-methyl, 2'-O- methoxy ethyl), 2'-fluoro, and 4'-thio modifications, and backbone modifications, such as one or more phosphorothioate, morpholino, or phosphonocarboxylate linkages. In addition, inhibitory RNA molecules may have a "tail" covalently attached to their 3'- and / or 5'-end, which may be used to stabilize the RNA inhibitory molecule or enhance cellular uptake. Such tails include, but are not limited to, intercalating groups, various kinds of reporter groups, and lipophilic groups attached to the 3' or 5' ends of the RNA molecules. In certain embodiments, the RNA inhibitory molecule is conjugated to cholesterol or acridine. See, for example, the following for descriptions of syntheses of 3'-cholesterol or 3'-acridine modified oligonucleotides: Gamper, H. B., Reed, M. W., Cox, T., Virosco, J. S., Adams, A. D., Gall, A., Scholler, J. K., and Meyer, R. B. (1993) Facile Preparation and Exonuclease Stability of 3'-Modified Oligodeoxynucleotides. Nucleic Acids Res. 21 145-150; and Reed, M. W., Adams, A. D., Nelson, J. S., and Meyer, R. B., Jr. (1991) Acridine and Cholesterol-Derivatized Solid Supports for Improved Synthesis of 3'-ModifiedOligonucleotides. Bioconjugate Chem. 2217-225 (1993); herein incorporated by reference in their entireties. Additional lipophilic moieties that can be used, include, but are not limited to, oleyl, retinyl, and cholesteryl residues, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3- (oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. Additional compounds, and methods of use, are set out in US Patent Publication Nos. 2010 / 0076056, 2009 / 0247608 and 2009 / 0131360; herein incorporated by reference in their entireties.

[0147] In one embodiment, inhibition of CTLA-4 function may be achieved by administering antisense oligonucleotides targeting the CTLA-4 gene. The antisense oligonucleotides may be ribonucleotides or deoxyribonucleotides. Preferably, the antisense oligonucleotides have at least one chemical modification. Antisense oligonucleotides may be comprised of one or more "locked nucleic acids". "Locked nucleic acids" (LNAs) are modified ribonucleotides that contain an extra bridge between the 2’ and 4' carbons of the ribose sugar moiety resulting in a "locked" conformation that confers enhanced thermal stability to oligonucleotides containing the LNAs. Alternatively, the antisense oligonucleotides may comprise peptide nucleic acids (PNAs), which contain a peptide-based backbone rather than a sugar-phosphate backbone. The antisense oligonucleotides may contain one or more chemical modifications, including, but are not limited to, sugar modifications, such as 2'-O-alkyl (e.g. 2'-O- methyl, 2'-O-methoxyethyl), 2'-fluoro, and 4' thio modifications, and backbone modifications, such as one or more phosphorothioate, morpholino, or phosphonocarboxylate linkages (see, for example, U.S. Pat. Nos. 6,693,187 and 7,067,641, which are herein incorporated by reference in their entireties). In some embodiments, suitable antisense oligonucleotides are 2'-O-methoxyethyl "gapmers" which contain 2'-O-methoxyethyl-modified ribonucleotides on both 5' and 3' ends with at least ten deoxyribonucleotides in the center. These "gapmers" are capable of triggering RNase H-dependent degradation mechanisms of RNA targets. Other modifications of antisense oligonucleotides to enhance stability and improve efficacy, such as those described in U.S. Pat. No. 6,838,283, which is herein incorporated by reference in its entirety, are known in the art and are suitable for use in the methods of the invention. Antisense oligonucleotides may comprise a sequence that is at least partially complementary to a CTLA-4 target sequence, e.g., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to the CTLA-4 target sequence. In some embodiments, the antisense oligonucleotide may be substantially complementary to the CTLA-4 target sequence, that is at least about 95%, 96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence. In one embodiment, theantisense oligonucleotide comprises a sequence that is 100% complementary to the CTLA-4 target sequence.

[0148] In another embodiment, the inhibitor of CTLA-4 is an inhibitory RNA molecule (e.g., a miRNA, a siRNA, shRNA, a piRNA, or a snRNA) having a single-stranded or doublestranded region that is at least partially complementary to the target sequence of CTLA-4, e.g., about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence of CTLA-4. In some embodiments, the inhibitory RNA comprises a sequence that is substantially complementary to the target sequence of CTLA-4, e.g., about 95%, 96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence. In other embodiments, the inhibitory RNA molecule may contain a region that has 100% complementarity to the target sequence. In some embodiments, the inhibitory molecules may target the CTLA-4 sequence of SEQ ID NO:1 (human CTLA-4 gene), SEQ ID NO:2 (human CTLA-4 mRNA transcript encoding transmembrane isoform), or SEQ ID NO: 3 (human CTLA-4 mRNA transcript encoding soluble isoform). In certain embodiments, the inhibitory RNA molecule may be a double-stranded, small interfering RNA or a short hairpin RNA molecule (shRNA) comprising a stem-loop structure.

[0149] Inhibitors can be detectably labeled by well-known techniques. Detectable labels include, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels and enzyme labels. Such labeled inhibitors can be used to determine cellular uptake efficiency, quantitate binding of inhibitors at target sites, or visualize inhibitor localization.CRISPR System for Targeting CTLA-4

[0150] In some embodiments, a CRISPR / Cas system is used to inactivate or reduce expression of an endogenous CTLA-4 gene in heart tissue. For example, a CRISPR / Cas system can be used to delete, inactivate, or mutate an endogenous CTLA-4 gene to eliminate or reduce CTLA-4 gene expression or protein activity. CTLA-4 gene knockout may be achieved through either the non-homologous end joining (NHEJ) or the microhomology-mediated end-joining (MMEJ) DNA repair pathways, which generate small nucleotide insertions or deletions (indels) at a site of a double-strand break (DSB) in the DNA. Gene knockout results if an indel shifts the reading frame or introduces a premature stop codon in a CTLA-4 coding sequence. In some embodiments, a CRISPR system is used with a guide RNA (gRNA) to direct a Cas9 RNA-guided nuclease to create a DSB at a target site in an exon of a CTLA-4 gene. For example, CTLA-4 can be inactivated by using a CRISPR system with a gRNA designed to guide a Cas9 nuclease to a target site to create a site-specific DNA break in an exon, wherein introduction of an indel in the exon results in knockout of the CTLA gene. Alternatively, CRISPR can be performed with a donorDNA template using homologous recombination (HR) to replace a portion of the genomic sequence with a modified sequence. Genome modification can be performed, for example, using homology directed repair (HDR) with a donor polynucleotide comprising a sequence comprising an intended genome edit flanked by a pair of homology arms responsible for targeting the donor polynucleotide to the target locus (e.g., CTLA-4 gene) to be edited in a heart tissue. The donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence. The homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide. The 5’ and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively.

[0151] The homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the donor polynucleotide and genomic DNA at the target locus. For example, a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3’ homology arms.

[0152] In certain embodiments, the corresponding homologous nucleotide sequences in the genomic target sequence (i.e., the "5’ target sequence" and "3' target sequence") flank a specific site for cleavage and / or a specific site for introducing the intended edit. The distance between the specific cleavage site and the homologous nucleotide sequences (e.g., each homology arm) can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides). In most cases, a smaller distance may give rise to a higher gene targeting rate. In a preferred embodiment, the donor polynucleotide is substantially identical to the target genomic sequence, across its entire length except for the sequence changes to be introduced to a portion of the genome that encompasses both the specific cleavage site and the portions of the genomic target sequence to be altered.

[0153] A homology arm can be of any length, e.g. 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more,1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc. In some instances, the 5' and 3’ homology arms are substantially equal in length to one another, e.g. one may be 30% shorter or less than the other homology arm, 20% shorter or less than the other homology arm, 10% shorter or less than the other homology arm, 5% shorter or less than the other homology arm, 2% shorter or less than the other homology arm, or only a few nucleotides less than the other homology arm. In other instances, the 5' and 3' homology arms are substantially different in length from one another, e.g. one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm.

[0154] The donor polynucleotide is used in combination with an RNA-guided nuclease, which is targeted to a particular genomic sequence (i.e., genomic target sequence to be modified) by a guide RNA (gRNA). A target- specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-gRNA complex by hybridization at the target site. For example, the gRNA can be designed with a sequence complementary to a target sequence in the CTLA-4 gene. In some embodiments, the gRNA is designed with a sequence complementary to a specific CTLA-4 mutation to target the nuclease-gRNA complex to the site of a mutation in heart tissue. The mutation may comprise an insertion, a deletion, or a substitution. For example, the mutation may include a single nucleotide variation, gene fusion, translocation, inversion, duplication, frameshift, missense, nonsense, or other mutation. The targeted minor allele may be a common genetic variant or a rare genetic variant. In certain embodiments, the gRNA is designed to selectively bind to a minor allele with single base-pair discrimination, for example, to allow binding of the nuclease-gRNA complex to a single nucleotide polymorphism (SNP). In particular, the gRNA may be designed to target disease-relevant mutations of interest for the purpose of genome editing to delete or deactivate the CTLA-4 gene in heart tissue.

[0155] In certain embodiments, the RNA-guided nuclease used for genome modification is a CRISPR system Cas nuclease. Any RNA-guided Cas nuclease capable of catalyzing site- directed cleavage of DNA to allow integration of donor polynucleotides by the HDR mechanism can be used in genome editing, including CRISPR system type I, type II, or type III Cas nucleases. Examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), CasF, CasG, CasH, Csyl , Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2,Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions thereof.

[0156] In certain embodiments, a type II CRISPR system Cas9 endonuclease is used. Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks) may be used to perform genome modification as described herein. The Cas9 need not be physically derived from an organism, but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP_002989955, WP_038434062, WP_011528583); Campylobacter jejuni (WP_022552435, YP_002344900), Campylobacter coli (WP_060786116); Campylobacter fetus (WP_059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP_033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC_021314); Belliella baltica (NC_018010); Psychroflexus torquisl (NC_018721); Streptococcus thermophilus (YP_820832), Streptococcus mutans (WP_061046374, WP_024786433); Listeria innocua (NP_472073); Listeria monocytogenes (WP_061665472); Legionella pneumophila (WP_062726656); Staphylococcus aureus (WP_001573634); Francisella tularensis (WP_032729892, WP_014548420), Enterococcus faecalis (WP_033919308); Lactobacillus rhamnosus (WP_048482595, WP_032965177); and Neisseria meningitidis (WP_061704949, YP_002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol. 198(5):797-807, Shmakov et al. (2015) Mol. Cell. 60(3):385-397, and Chylinski et al. (2014) Nucleic Acids Res. 42(10):6091-6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.

[0157] The CRISPR-Cas system naturally occurs in bacteria and archaea where it plays a role in RNA-mediated adaptive immunity against foreign DNA. The bacterial type II CRISPR system uses the endonuclease, Cas9, which forms a complex with a guide RNA (gRNA) that specifically hybridizes to a complementary genomic target sequence, where the Cas9 endonuclease catalyzes cleavage to produce a double-stranded break. Targeting of Cas9 typicallyfurther relies on the presence of a 5' protospacer-adjacent motif (PAM) in the DNA at or near the gRNA-binding site.

[0158] The genomic target site will typically comprise a nucleotide sequence that is complementary to the gRNA, and may further comprise a protospacer adjacent motif (PAM). In certain embodiments, the target site comprises 20-30 base pairs in addition to a 3 base pair PAM. Typically, the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen. Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide. In certain embodiments, the allele targeted by a gRNA comprises a mutation that creates a PAM within the allele, wherein the PAM promotes binding of the Cas9- gRNA complex to the allele.

[0159] In certain embodiments, the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length. The guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules.

[0160] In another embodiment, the CRISPR nuclease from Prevotella and Francisella 1 (Cpfl, also known as Casl2a) is used. Cpfl is another class II CRISPR / Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Cpfl does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cpfl for targeting than Cas9. Cpfl is capable of cleaving either DNA or RNA. The PAM sites recognized by Cpfl have the sequences 5’-YTN-3' (where "Y" is a pyrimidine and "N" is any nucleobase) or 5'-TTN-3', in contrast to the G-rich PAM site recognized by Cas9. Cpfl cleavage of DNA produces double-stranded breaks with a sticky-ends having a 4 or 5 nucleotide overhang. For a discussion of Cpfl, see, e.g., Ledford et al. (2015) Nature. 526 (7571): 17-17 , Zetsche et al. (2015) Cell. 163 (3):759-771, Murovec et al. (2017) Plant Biotechnol. J. 15(8):917-926, Zhang et al. (2017) Front. Plant Sci. 8:177, Fernandes et al. (2016) Postepy Biochem. 62(3):315-326; herein incorporated by reference.

[0161] Casl2b (C2cl) is another class II CRISPR / Cas system RNA-guided nuclease that may be used. C2cl, similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites. For a description of Cas l2b, see, e.g., Shmakov et al. (2015) Mol Cell. 60(3):385- 397, Zhang et al. (2017) Front Plant Sci. 8:177; herein incorporated by reference.

[0162] In yet another embodiment, an engineered RNA-guided FokI nuclease may be used. RNA-guided FokI nucleases comprise fusions of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting on FokI. For a description of engineered RNA-guided FokI nucleases, see, e.g., Havlicek et al. (2017) Mol. Ther. 25(2):342-355, Pan et al. (2016) Sci Rep. 6:35794, Tsai et al. (2014) Nat Biotechnol. 32(6):569-576; herein incorporated by reference.

[0163] An RNA-guided nuclease can be provided in the form of a protein, such as the nuclease complexed with a gRNA, or provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector). In some embodiments, the RNA-guided nuclease and the gRNA are both provided by vectors. Both can be expressed by a single vector or separately on different vectors. The vector(s) encoding the RNA- guided nuclease an gRNA may be included in a CRISPR expression system to target the CTLA- 4 gene in heart tissue.

[0164] Codon usage may be optimized to improve production of an RNA-guided nuclease in a particular cell or organism. For example, a nucleic acid encoding an RNA-guided nuclease or reverse transcriptase can be modified to substitute codons having a higher frequency of usage in a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the RNA-guided nuclease is introduced into cells (e.g., Foxp3+ Tregs), the protein can be transiently, conditionally, or constitutively expressed in the cell.

[0165] In another embodiment, CRISPR interference (CRISPRi) is used to repress CTLA- 4 gene expression. CRISPRi is performed with a complex of a catalytically inactive Cas9 (dCas9) with a guide RNA that targets the CTLA-4 gene. An engineered nuclease-deactivated Cas9 (dCas9) is used to allow sequence-specific targeting without cleavage. Nuclease-deactivated forms of Cas9 may be engineered by mutating catalytic residues at the active site of Cas9 to destroy nuclease activity. Any such nuclease deficient Cas9 protein from any species may be used as long as the engineered dCas9 retains gRNA-mediated sequence- specific targeting. In particular, the nuclease activity of Cas9 from Streptococcus pyogenes can be deactivated by introducing two mutations (D10A and H841 A) in the RuvCl and HNH nuclease domains. Other engineered dCas9 proteins may be produced by similarly mutating the corresponding residues in other bacterial Cas9 isoforms. For a description of engineered nuclease-deactivated forms of Cas9, see, e.g., Qi et al. (2013) Cell 152: 1173-1183, Dominguez et al. (2016) Nat. Rev. Mol. Cell. Biol. 17(1):5-15; herein incorporated by reference in their entireties.

[0166] The dCas9 protein can be designed to target the CTLA-4 gene by altering its guide RNA sequence. A target- specific single guide RNA (sgRNA) comprises a nucleotide sequencethat is complementary to a target site, and thereby mediates binding of the dCas9-sgRNA complex by hybridization at the target site. CRISPRi can be used to sterically repress transcription by blocking either transcriptional initiation or elongation by designing a sgRNA with a sequence complementary to a CTLA-4 promoter or exonic sequence. The sgRNA may be complementary to the non-template strand or the template strand, but preferably is complementary to the nontemplate strand to more strongly repress transcription.

[0167] The target site will typically comprise a nucleotide sequence that is complementary to the sgRNA, and may further comprise a protospacer adjacent motif (PAM). In certain embodiments, the target site comprises 20-30 base pairs in addition to a 3 base pair PAM. Typically, the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen. Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide.

[0168] In certain embodiments, the sgRNA comprises 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, 19-21 nucleotides, and any length between the stated ranges, including, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0169] The sgRNAs are readily synthesized by standard techniques, e.g., solid phase synthesis via phosphoramidite chemistry, as disclosed in U.S. Patent Nos. 4,458,066 and 4,415,732, incorporated herein by reference; Beaucage et al., Tetrahedron (1992) 48:2223-2311; and Applied Biosystems User Bulletin No. 13 (1 April 1987). Other chemical synthesis methods include, for example, the phosphotriester method described by Narang et al., Meth. Enzymol. (1979) 68:90 and the phosphodiester method disclosed by Brown et al., Meth. Enzymol. (1979) 68:109.

[0170] In some embodiments, the dCas9 is fused to a transcriptional repressor domain capable of further repressing transcription of the CTLA-4 gene, e.g., by inducing heterochromatinization. For example, a Kruppel associated box (KRAB) can be fused to dCas9 to repress transcription of a target gene in human cells (see, e.g., Gilbert et al. (2013) Cell. 154 (2): 442-45, O'Geen et al. (2017) Nucleic Acids Res. 45(17):9901-9916; herein incorporated by reference).

[0171] Alternatively, dCas9 can be used to introduce epigenetic changes that reduceCTLA-4 gene expression by fusion of dCas9 to an epigenetic modifier such as a chromatinmodifying epigenetic enzyme. The promoter for CTLA-4 can be silenced, for example, by methylation or acetylation (e.g. histone H3 lysine 9 [H3K9] methylation, histone H3 lysine 27 [H3K27] methylation, and / or DNA methylation). For example, fusion of dCas9 to a DNAmethyltransferase such as DNA methyltransferase 3 alpha (DNMT3A) or a chimeric Dnmt3a / Dnmt3L methyltransferase (DNMT3A3L) allows targeted DNA methylation. Fusion of dCas9 to histone demethylase LSD1 allows targeted histone demethylation (see, e.g., Liu et al. (2016) Cell 167(l):233-247, Lo et al. (2017) FlOOORes. 6. pii: F1000 Faculty Rev-747, and Stepper et al. (2017) Nucleic Acids Res. 45(4):1703-1713; herein incorporated by reference).

[0172] In yet other embodiments, an RNA-targeting CR1SPR-Casl3 system is used to perform RNA interference to reduce CTLA-4 expression. Members of the Casl3 family are RNA- guided RNases containing two HEPN domains having RNase activity. In particular, Casl3a (C2c2), Casl3b (C2c6), and Casl3d can be used for RNA knockdown. Casl3 proteins can be made to target and cleave CTLA-4 transcribed RNA using a gRNA with complementarity to the target transcript sequence. The gRNA is typically about 64 nucleotides in length with a short hairpin crRNA and a 28-30 nucleotide spacer that is complementary to the target site on the CTLA-4 RNA transcript. Casl3 recognition and cleavage of a target transcript results in degradation of the transcript as well as nonspecific degradation of any nearby transcripts. See, e.g., Abudayyeh et al. (2017) Nature 550:280-284, Hameed et al. (2019) Microb. Pathog. 133:103551, Wang et al. (2019) Biotechnol Adv. 37(5):708-729, Aman et al. (2018) Viruses 10(12). pii: E732, and Zhang et al. (2018) Cell 175(l):212-223; herein incorporated by reference.Determining Levels of Expression of CTLA-4

[0173] An "effective amount" of a CTLA-4 inhibitory nucleic acid (e.g., microRNA, siRNA, shRNA, piRNA, snRNA, antisense oligonucleotide) or a CRISPR system targeting the CTLA-4 gene (e.g., Cas9, Casl2a), RNA (e.g., Casl3), or epigenome (e.g., dCas9 fusion protein) is an amount sufficient to effect beneficial or desired results, such as an amount that reduces CTLA-4 activity, for example, by interfering with transcription of CTLA-4 or interfering with translation of CTLA-4. In some embodiments, a CTLA-4 inhibitory nucleic acid or CRISPR system reduces the CTLA-4 mRNA levels or protein levels by at least about 10% to about 100%, 20% to about 100%, 30% to about 100%, 40% to about 100%, 50% to about 100%, 60% to about 100%, 70% to about 100%, 10% to about 90%, 20% to about 85%, 40% to about 84%, 60% to about 90%, including any percent within these ranges, such as but not limited to 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%.

[0174] Any convenient protocol may be used for evaluating CTLA-4 expression by detecting CTLA-4 protein or mRNA levels in the presence or absence of a candidate CTLA-4 inhibitor. For measuring protein levels in a sample, various antibody-based methods, including without limitation immunoassays, e.g., enzyme-linked immunosorbent assays (ELISAs),immunohistochemistry, and flow cytometry (FACS) may be used. Any convenient antibody can be used that specifically binds to the CTLA-4 protein. The terms "specifically binds" or "specific binding" as used herein refer to preferential binding to a molecule relative to other molecules or moieties in a solution or reaction mixture (e.g., an antibody specifically binds to a particular polypeptide or epitope relative to other available polypeptides or epitopes). In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is characterized by a Kd (dissociation constant) of 10'5M or less (e.g., 10’6M or less, 10’7M or less, 10’8M or less, 10'9M or less, IO-10M or less, 10"11M or less, 1012M or less, 1013M or less, 10"14M or less, 10"15M or less, or 10’16M or less). By "Affinity" it is meant the strength of binding, increased binding affinity being correlated with a lower Kd.

[0175] While a variety of different manners of assaying for protein levels are known in the art, one representative and convenient type of protocol for assaying protein levels is the enzyme-linked immunosorbent assay (ELISA). In ELISA and ELISA-based assays, one or more antibodies specific for the proteins of interest may be immobilized onto a selected solid surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate. After washing to remove incompletely adsorbed material, the assay plate wells are coated with a non-specific "blocking" protein that is known to be antigenically neutral with regard to the test sample such as bovine serum albumin (BSA), casein or solutions of powdered milk. This allows for blocking of non-specific adsorption sites on the immobilizing surface, thereby reducing the background caused by non-specific binding of antigen onto the surface. After washing to remove unbound blocking protein, the immobilizing surface is contacted with the sample to be tested under conditions that are conducive to immune complex (antigen / antibody) formation. Such conditions include diluting the sample with diluents such as BSA or bovine gamma globulin (BGG) in phosphate buffered saline (PBS)ZTween or PBS / Triton-X 100, which also tend to assist in the reduction of nonspecific background, and allowing the sample to incubate for about 2-4 hours at temperatures on the order of about 25°-27° C. (although other temperatures may be used). Following incubation, the antisera-contacted surface is washed so as to remove nonimmunocomplexed material. An exemplary washing procedure includes washing with a solution such as PBS / Tween, PBS / Triton-X 100, or borate buffer. The occurrence and amount of immunocomplex formation may then be determined by subjecting the bound immunocomplexes to a second antibody having specificity for the target that differs from the first antibody and detecting binding of the second antibody. In certain embodiments, the second antibody will have an associated enzyme, e.g. urease, peroxidase, or alkaline phosphatase, which will generate a color precipitate upon incubating with an appropriate chromogenic substrate. For example, a urease or peroxidase-conjugated anti-human IgG may be employed, for a period of time and underconditions which favor the development of immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS / Tween). After such incubation with the second antibody and washing to remove unbound material, the amount of label is quantified, for example by incubation with a chromogenic substrate such as urea and bromocresol purple in the case of a urease label or 2,2'-azino-di-(3-ethyl-benzthiazoline)-6- sulfonic acid (ABTS) and H2O2, in the case of a peroxidase label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer. The preceding format may be altered by first binding the sample to the assay plate. Then, primary antibody is incubated with the assay plate, followed by detecting of bound primary antibody using a labeled second antibody with specificity for the primary antibody.

[0176] The solid substrate upon which the antibody or antibodies are immobilized can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin particle, etc. The substrate may be chosen to maximize signal to noise ratios, to minimize background binding, as well as for ease of separation and cost. Washes may be effected in a manner most appropriate for the substrate being used, for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, or rinsing a bead, particle, chromatographic column or filter with a wash solution or solvent.

[0177] Alternatively, non-ELISA based-methods for measuring the levels of the CTLA-4 protein in a sample may be employed, and any convenient method may be used. Representative examples known to one of ordinary skill in the art include but are not limited to other immunoassay techniques such as radioimmunoassays (RIA), sandwich immunoassays, fluorescent immunoassays, enzyme multiplied immunoassay technique (EMIT), capillary electrophoresis immunoassays (CEIA), and immunoprecipitation assays; mass spectrometry, or tandem mass spectrometry, proteomic arrays, xMAP microsphere technology, western blotting, immunohistochemistry, flow cytometry, cytometry by time-of-flight (CyTOF), multiplexed ion beam imaging (MIBI), and detection in body fluid by electrochemical sensor. In, for example, flow cytometry methods, the quantitative level of gene products of the one or more genes of interest are detected on cells in a cell suspension by lasers. As with ELIS As and immunohistochemistry, antibodies (e.g., monoclonal antibodies) that specifically bind the polypeptides encoded by the genes of interest are used in such methods.

[0178] As another example, electrochemical sensors may be employed. In such methods, a capture aptamer or an antibody that is specific for a target protein (the "analyte") is immobilized on an electrode. A second aptamer or antibody, also specific for the target protein, is labeled with, for example, pyrroquinoline quinone glucose dehydrogenase ((PQQ)GDH). The sample of body fluid is introduced to the sensor either by submerging the electrodes in body fluid or by addingthe sample fluid to a sample chamber, and the analyte allowed to interact with the labeled aptamer / antibody and the immobilized capture aptamer / antibody. Glucose is then provided to the sample, and the electric current generated by (PQQ)GDH is observed, where the amount of electric current passing through the electrochemical cell is directly related to the amount of analyte captured at the electrode.

[0179] Flow cytometry can be used to distinguish subpopulations of cells expressing different cellular markers and to determine their frequency in a population of cells. Typically, whole cells are incubated with antibodies that specifically bind to the cellular markers. The antibodies can be labeled, for example, with a fluorophore, isotope, or quantum dot to facilitate detection of the cellular markers. The cells are then suspended in a stream of fluid and passed through an electronic detection apparatus. In addition, fluorescence-activated cell sorting (FACS) can be used to sort a heterogeneous mixture of cells into separate containers. (See, e.g., Shapiro Practical Flow Cytometry, Wiley-Liss, 4thedition, 2003; Loken Immunofluorescence Techniques in Flow Cytometry and Sorting, Wiley, 2ndedition, 1990; Flow Cytometry: Principles and Applications, (ed. Macey), Humana Press 1stedition, 2007; herein incorporated by reference in their entireties.)

[0180] In other embodiments, the amount or level in the sample of mRNA encoded by the CTLA-4 gene is determined. Any convenient method for measuring mRNA levels in a sample may be used, e.g. hybridization-based methods, e.g. northern blotting and in situ hybridization (Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999)), RNase protection assays (Hod, Biotechniques 13:852-854 (1992)), and PCR-based methods (e.g. reverse transcription PCR (RT-PCR) (Weis et al., Trends in Genetics 8:263-264 (1992)).

[0181] For measuring mRNA levels, the starting material may be total RNA, i.e. unfractionated RNA, or poly A+ RNA isolated from a suspension of cells (e.g. heart tissue). General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). RNA isolation can also be performed using a purification kit, buffer set and protease from commercial manufacturers, according to the manufacturer's instructions. For example, RNA from cell suspensions can be isolated using Qiagen RNeasy mini-columns, and RNA from cell suspensions or homogenized tissue samples can be isolated using the TRIzol reagent-based kits (Invitrogen), MasterPure Complete DNA and RNA Purification Kit (EPICENTRE, Madison, Wis.), Paraffin Block RNA Isolation Kit (Ambion, Inc.) or RNA Stat- 60 kit (Tel-Test).

[0182] The mRNA levels may be measured by any convenient method. Examples of methods for measuring mRNA levels may be found in, e.g., the field of differential geneexpression analysis. One representative and convenient type of protocol for measuring mRNA levels is array-based gene expression profiling. Such protocols are hybridization assays in which a nucleic acid that displays "probe" nucleic acids for each of the genes to be assayed / profiled in the profile to be generated is employed. In these assays, a sample of target nucleic acids is first prepared from the initial nucleic acid sample being assayed, where preparation may include labeling of the target nucleic acids with a label, e.g., a member of signal producing system. Following target nucleic acid sample preparation, the sample is contacted with the array under hybridization conditions, whereby complexes are formed between target nucleic acids that are complementary to probe sequences attached to the array surface. The presence of hybridized complexes is then detected, either qualitatively or quantitatively.

[0183] Specific hybridization technology which may be employed in the subject methods includes that described in U.S. Pat. Nos. 5,143,854; 5,288,644; 5,324,633; 5,432,049; 5,470,710; 5,492,806; 5,503,980; 5,510,270; 5,525,464; 5,547,839; 5,580,732; 5,661,028; 5,800,992; the disclosures of which are herein incorporated by reference; as well as WO 95 / 21265; WO 96 / 31622; WO 97 / 10365; WO 97 / 27317; EP 373 203; and EP 785 280. In these methods, an array of "probe" nucleic acids that includes a probe for each of the phenotype determinative genes whose expression is being assayed is contacted with target nucleic acids as described above. Contact is carried out under hybridization conditions, e.g., stringent hybridization conditions, and unbound nucleic acid is then removed. The term "stringent assay conditions" as used herein refers to conditions that are compatible to produce binding pairs of nucleic acids, e.g., surface bound and solution phase nucleic acids, of sufficient complementarity to provide for the desired level of specificity in the assay while being less compatible to the formation of binding pairs between binding members of insufficient complementarity to provide for the desired specificity. Stringent assay conditions are the summation or combination (totality) of both hybridization and wash conditions.

[0184] The resultant pattern of hybridized nucleic acid provides information regarding expression for each of the genes that have been probed, where the expression information is in terms of whether or not the gene is expressed and, typically, at what level, where the expression data, i.e., expression profile (e.g., in the form of a transcriptosome), may be both qualitative and quantitative.

[0185] Additionally or alternatively, non-array based methods for quantitating the level of one or more nucleic acids in a sample may be employed. These include those based on amplification protocols, e.g., Polymerase Chain Reaction (PCR)-based assays, including quantitative PCR, reverse-transcription PCR (RT-PCR), real-time PCR, and the like, e.g. TaqMan, RT-PCR, MassARRAY System, BeadArray technology, and Luminex technology; and those thatrely upon hybridization of probes to filters, e.g. Northern blotting and in situ hybridization. Serial Analysis Gene Expression (SAGE) can also be used to determine RNA abundances in a cell sample. See, e.g., Velculescu et al., 1995, Science 270:484-7; Carulli, et al., 1998, Journal of Cellular Biochemistry Supplements 30 / 31:286-96; herein incorporated by reference in their entireties. SAGE analysis does not require a special device for detection, can be used for simultaneously detecting the expression of large numbers of transcription products.UTILITY

[0186] Inhibitors of CTLA-4 are useful for cardiac repair after myocardial infarction. Immune cells migrate to the injured heart after myocardial infarction and regulate the immune response during the healing process. Inhibition of CTLA-4 leads to improved early post-infarct cardiac repair, reduced cardiac infarct size, increased survival, and better preservation of cardiac function, including increased left ventricular ejection fraction, reduced chamber dilatation, and reduced ventricular remodeling (see Examples). Inhibitors of CTLA-may also be useful in treating other heart diseases where cardiac repair and regeneration are crucial such as, but not limited to, myocardial ischemia, myocardial injury, heart failure, atherosclerosis, coronary artery disease, and cardiomyopathies.Examples of Non-Limiting Aspects of the Disclosure

[0187] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1-61 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:1. A method of treating heart disease in a subject, the method comprising administering a therapeutically effective amount of an inhibitor of cytotoxic T-lymphocyte- associated protein 4 (CTLA-4) to the subject.2. The method of aspect 1 , wherein the inhibitor of CTLA-4 is an antibody that specifically binds to CTLA-4.3. The method of aspect 2, wherein the antibody is ipilimumab or tremelimumab.4. The method of aspect 2, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a Fvfragment, and a nanobody.5. The method of any one of aspects 1-4, wherein the heart disease is myocardial infarction, myocardial ischemia, myocardial injury, heart failure, atherosclerosis, coronary artery disease, and cardiomyopathies.6. The method of any one of aspects 1-5, wherein the inhibitor of CTLA-4 is administered intravenously or intra-arterially.7. The method of any one of aspects 1-5, wherein the inhibitor of CTLA-4 is administered locally to damaged heart tissue or a site of cardiac ischemia or infarction.8. The method of any one of aspects 1-7, wherein multiple therapeutically effective doses of the inhibitor of CTLA-4 are administered to the subject.9. The method of aspect 8, wherein the inhibitor of CTLA-4 is administered according to a daily dosing regimen or intermittently.10. The method of any one of aspects 1-9, wherein the inhibitor of CTLA-4 is administered after a myocardial infarction.11. The method of any one of aspects 1-9, wherein the inhibitor of CTLA-4 is administered prophylactically to protect against or delay or prevent myocardial infarction.12. The method of any one of aspects 1-11, wherein the subject has cardiovascular disease.13. The method of any one of aspects 1-12, wherein treatment with the inhibitor of CTLA-4 increases left ventricular ejection fraction, reduces chamber dilatation, reduces ventricular remodeling, reduces area of fibrotic scar tissue, reduces plasma troponin levels, and / or increases survival compared to in absence of the treatment.14. The method of any one of aspects 1-13, wherein treatment with the inhibitor of CTLA-4 increases proliferation of Foxp3+ regulatory T cells compared to in absence of the treatment.15. The method of any one of aspects 1-14, wherein the subject is human.16. A method of treating ventricular remodeling in a subject, the method comprising administering a therapeutically effective amount of an inhibitor of cytotoxic T-lymphocyte- associated protein 4 (CTLA-4) to the subject.17. The method of aspect 16, wherein the inhibitor of CTLA-4 is administered prophylactically or therapeutically after myocardial infarction, myocardial ischemia, myocardial injury, atherosclerosis, or heart failure to prevent, decrease, or delay ventricular remodeling.18. The method of aspect 16 or 17, wherein the ventricular remodeling comprises changes in left ventricular (LV) geometry, mass, and / or volume.19. The method of any one of aspects 16-18, wherein the inhibitor of CTLA-4 is an antibody that specifically binds to CTLA-4.20. The method of aspect 19, wherein the antibody is ipilimumab or tremelimumab.21. The method of aspect 19, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a Fvfragment, and a nanobody.22. The method of any one of aspects 16-21, wherein the inhibitor of CTLA-4 inhibits binding of CTLA-4 to B7.1 and B7.2 antigen-presenting cell ligands.23. The method of any one of aspects 16-22, wherein the inhibitor of CTLA-4 stimulates proliferation of a regulatory T cell (Treg).24. The method of aspect 23, wherein the Treg is a Foxp3+Treg.25. The method of any one of aspects 16-24, wherein the subject has a myocardial infarction, myocardial ischemia, myocardial injury, heart failure, atherosclerosis, coronary artery disease, or a cardiomyopathy.26. The method of any one of aspects 16-25, wherein the inhibitor of CTLA-4 is administered intravenously or intra-arterially.27. The method of any one of aspects 16-25, wherein the inhibitor of CTLA-4 is administered locally to damaged heart tissue or a site of cardiac ischemia or infarction.28. The method of any one of aspects 16-27, wherein multiple therapeutically effective doses of the inhibitor of CTLA-4 are administered to the subject.29. The method of aspect 28, wherein the inhibitor of CTLA-4 is administered according to a daily dosing regimen or intermittently.30. A method of screening for an inhibitor of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) for treating heart disease, the method comprising:(a) contacting CTLA-4 with a candidate agent;(b) measuring inhibition of CTLA-4 by the candidate agent to determine if the candidate agent is an inhibitor of CTLA-4;(c) administering the candidate agent to a mammalian non-human animal before, during, or after performing left anterior descending artery (LAD) ligation on the mammalian non-human animal, if the candidate agent is determined to be an inhibitor of CTLA-4; and(d) measuring left ventricular ejection fraction, chamber size, chamber dilatation, ventricular remodeling, area of fibrotic scar tissue, survival, level of plasma troponin, or any combination thereof in the mammalian non-human animal after said performing the LAD, wherein increased left ventricular ejection fraction, reduced chamber dilatation, reduced ventricular remodeling, reduced area of fibrotic scar tissue, increased survival, reduced level of plasma troponin, or any combination thereof, compared to in absence of said administering the candidate agent indicates that the candidate agent is useful for treating heart disease.31 . The method of aspect 30, wherein the candidate agent is a small molecule, a peptide, a protein, a peptoid, an aptamer, an antibody that specifically binds to CTLA-4, anantibody mimetic, an inhibitory nucleic acid, or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system.32. The method of aspect 31, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a Fvfragment, and a nanobody.33. The method of aspect 31, wherein the inhibitory nucleic acid is selected from the group consisting of a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), an antisense oligonucleotide, and a peptide nucleic acid.34. The method of aspect 31, wherein the inhibitory nucleic acid inhibits CTLA-4 transcription or protein translation.35. The method of aspect 31, wherein the CRISPR system targets a CTLA-4 gene or a CTLA-4 RNA transcript, or makes epigenetic changes that reduce CTLA-4 expression.36. The method of aspect 35, wherein the CRISPR system comprises Cas9, Casl2a, Casl2d, Casl3a, Casl3b, Casl3d, or a dead Cas9 (dCas9).37. The method of any one of aspects 30-36, wherein the non-human animal model is a rodent or primate.38. The method of aspect 37, wherein the rodent is a mouse.39. The method of any one of aspects 30-38, wherein said measuring inhibition of CTLA-4 comprises measuring inhibition of binding of CTLA-4 to B7.1 and B7.2 antigen- presenting cell ligands.40. The method of any one of aspects 30-39, wherein said measuring inhibition of CTLA-4 comprises measuring proliferation of a Foxp3+ regulatory T cell (Treg).41. The method of any one of aspects 30-40, wherein the level of plasma troponin is measured using an immunoassay or liquid chromatography-tandem mass spectrometry.42. The method of any one of aspects 30-41, wherein the chamber size, the chamber dilatation, the ventricular remodeling, or the area of fibrotic scar tissue is measured by echocardiography, ultrasound, or histology.43. The method of any one of aspects 30-42, wherein the left ventricular ejection fraction is measured by echocardiography, magnetic resonance imaging (MRI), computerized tomography (CT), radionuclide angiography, gated myocardial perfusion single-photon emission computed tomography (SPECT), or gated myocardial perfusion positron emission tomography (PET).44. The method of any one of aspects 30-43, further comprising contacting a cardiomyocyte with the candidate agent; and measuring contractility, calcium (Ca2+)dynamics, extracellular field potential (EFP), pro-arrhythmic properties, or any combination thereof.45. The method of aspect 44, wherein the cardiomyocyte is a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM).46. A composition comprising an inhibitor of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) for use in a method of treating heart disease or ventricular remodeling.47. The composition of aspect 46, further comprising a pharmaceutically acceptable excipient.48. The composition of aspect 46 or 47, further comprising a pharmaceutically acceptable carrier selected from the group consisting of a cream, emulsion, gel, liposome, nanoparticle, or ointment.49. The composition of any one of aspects 46-48, wherein the inhibitor of CTLA-4 is an antibody that specifically binds to CTLA-4.50. The composition of aspect 49, wherein the antibody is ipilimumab or tremelimumab.51. The composition of aspect 49, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a Fvfragment, and a nanobody.52. The composition of any one of aspects 46-51, wherein the inhibitor of CTLA-4 inhibits binding of CTLA-4 to B7.1 and B7.2 antigen-presenting cell ligands.53. The composition of any one of aspects 46-52, wherein the inhibitor of CTLA-4 increases proliferation of a Foxp3+ regulatory T cell (Treg).54. The composition of any one of aspects 46-53, wherein the heart disease is myocardial infarction, myocardial ischemia, myocardial injury, heart failure, atherosclerosis, coronary artery disease, or a cardiomyopathy.55. Use of an inhibitor of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) in the manufacture of a medicament or pharmaceutical composition for treating heart disease in a subject in need thereof.56. The use of aspect 55, wherein the inhibitor of CTLA-4 is an antibody that specifically binds to CTLA-4.57. The use of aspect 56, wherein the antibody is ipilimumab or tremelimumab.58. The use of aspect 56, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a Fvfragment, and a nanobody.59. The use of any one of aspects 55-58, wherein the inhibitor of CTLA-4 inhibits binding of CTLA-4 to B7.1 and B7.2 antigen-presenting cell ligands.60. The use of any one of aspects 55-59, wherein the inhibitor of CTLA-4 increases proliferation of a Foxp3+ regulatory T cell (Treg).61. The use of any one of aspects 55-60, wherein the heart disease is myocardial infarction, myocardial ischemia, myocardial injury, heart failure, atherosclerosis, coronary artery disease, or a cardiomyopathy.EXAMPLES

[0188] As can be appreciated from the disclosure provided above, the present disclosure has a wide variety of applications. Accordingly, the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, dimensions, etc.) but some experimental errors and deviations should be accounted for. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.EXAMPLE 1: HARNESSING IMMUNE CHECKPOINT INHIBITORS FOR CARDI C REPAIR

[0189] Globally, myocardial infarction (MI or heart attack) is a major contributor to mortality and morbidity, accounting for 27% of global deaths in 2019. Limited treatment options primarily focus on symptom management and preventing further cardiac damage, with no effective solutions for heart-specific protection post-MI. Here, we leverage the immune system, specifically modulating immune checkpoints, to treat cardiac ischemia in a new, potentially transformative way. While immune checkpoint inhibitors (ICIs) have been game-changers in oncology, our initial discovery highlights the potential of inhibition of CTLA-4, a specific immune checkpoint, in improving cardiac repair post-MI. After MI, regulated immune cells migrate to the injured heart driving tissue repair and remodeling. Our data suggest that CTLA4 inhibition improves cardiac repair after MI, preserving cardiac function, and reducing infarct size, thereby increasing survival. We aim to bridge the critical gap in current treatment paradigms by not only preventing deterioration but also promoting the repair of heart damage.Studies on immune checkpoints in cardiac irAE underscore the crucial role that immune checkpoints play in cardiovascular biology.

[0190] Checkpoint proteins, such as CTLA4 or programmed death-1 receptor (PD-1) on T cells, and its ligand PD-L1 on tumor cells help keep immune responses in check, negatively regulate the T cell activity (FIG. 1A) (Varricchi et al. (2017) Circulation. 136:1989-1992). ICIs are antibodies that target these proteins that have revolutionized cancer therapy and are now beingused in the front-line treatment of approximately half of all metastatic cancer patients (Haslam et al. (2019) JAMA Netw Open 2(5):el92535). Ipilimumab, the first FDA-approved ICI, targets CTLA4 and maintains the T cell in an activated state, by blocking CTLA-4 from binding with its ligands CD80 / 86 (FIG. IB) (Rulifson et al. (1997) J Immunol. 158:658-665). Comprehensive tumor-infiltrating T cell profiling revealed that anti-PD- 1 predominantly induces the expansion of specific tumor-infiltrating exhausted- like CD8 T cell subsets, while anti-CTLA-4 induces the expansion of an ICOS-i- Th 1 -like CD4 Teffs in addition to engaging exhausted-like CD8 T cells (Wei et al. (2017) Cell 170:1120-1133 ell l7, Chen et al. (2009) Proc Natl Acad Sci U.S.A. 106:2729-2734). Due to systemic effects, patients can develop immune-related adverse events (irAE), including possible life-threatening cardiovascular irAE, like atherosclerosis, myocarditis, and cardiomyopathy. PD-L1 on endothelial cells might protect against atherosclerosis by inhibiting T cell activity and cytokine production, promoting regulatory T-cell differentiation, and inducing T cell exhaustion (Wang et al. (2018) Jama Oncol. 4: 1792-1792). In the heart, IC downregulates the immune response to protect against cardiac damage by reducing T-cell activity and migration. These cardiovascular complications underscore the crucial role that immune checkpoints play in cardiovascular biology.The role of CTLA4 in eardiac repair and remodeling.

[0191] To further investigate the roles of ICIs in MI, male wide-type animals were randomized for treatment with anti-PD-1 (BioCell, #BE0146), anti-PD-Ll (#BE0361), or anti- CTLA-4 (#BE0164) antibodies or an IgG control twice a week and MI surgeries were performed after 2 doses of treatment (FIG. 3A). Kaplan-Meier overall survival analysis showed that mice treated with a monoclonal anti-CTLA4 antibody have significantly better 7 days survival compared with IgG isotype-treated littermates (FIG. 3B). Death events were defined as mice found dead from wall rupture. In addition, the anti-CTLA4 treated animals exhibited a significant improvement in post-injury cardiac function, as well as a decreased chamber dilatation, compared to the IgG isotype-treated and sham animals (FIGS. 3C-3E). The percentage of the infarct area post-MI D28 was measured by a parasternal long-axis view of echocardiography and demonstrated that the area of fibrotic scar tissue was significantly smaller in the anti-CTLA4 treated animals compared to IgG isotype-treated animals (FIG. 3F). Taken together these observations demonstrate that CTLA4 inhibition leads to not only better survival and improved cardiac function but also to a decreased scar size.EXAMPLE 2: SINGLE-CELL RNA-SEQUENCING

[0192] To assess whether immune checkpoints play regulatory roles in myocardial injury, we successfully utilized a mouse acute MI model in C57BL / 6 wild-type mice. In sham-operated animals, the suture was placed under the artery and removed without ligation. Hearts were harvested at 3, 7, and 21 days after ischemic injury, and the fibrotic scar tissue in the injured region remote to the area of injury was dissected from the same heart for RNA-seq (FIG. 2A) to quantify temporal changes in gene expression.

[0193] To understand the immune landscape and subsequent cardiac remodeling modulated by CTLA4 inhibition post-MI, in parallel, single-cell RNA-sequencing (scRNA-seq) during cardiac ischemia is performed to provide detailed transcriptomic profiling of distinct cell populations, emphasizing pathways linked to T-cell regulation, macrophage polarization, and fibroblast differentiation. Key genes and signaling pathways can be validated at the protein level by western blotting and multiplex flow cytometry. By synthesizing data from both protein and transcriptomic dimensions, we aspire to provide a comprehensive understanding of the multifaceted interactions and regulatory networks activated within the cardiac tissue upon CTLA4 inhibition during post-ischemic repair.

[0194] Representative imagery demonstrates our proficiency in performing scRNA-seq analyses on mouse cardiac tissues, illustrating a predominance of fibroblasts, macrophages, and endothelial cells, as highlighted in the UMAP clustering (FIG. 5). For heart analysis, the nonmyocyte cell fraction of ischemic and border zone are collected and disassociated from anti- CTLA4, and IgG control-treated mice 7 days post-MI (n > 4 in each group). Sorted live cells are counted on a hemocytometer before being loaded into a 1 Ox Genomics single cell 3 ’ v3.1 platform for an estimated recovery of 8000 cells per sample. Libraries are synthesized in adherence to the manufacturer’ s guidelines, with adaptations as previously delineated, targeting a sequence read depth of 50,000 per cell. Furthermore, unsupervised cell clustering is used with SingleR and the FindAllMarkers function in Seurat. Cell types, proportions, and gene expression patterns, signaling pathway enrichment of each cluster are analyzed. Single-cell trajectories are determined using the Monocle package and gene expression distribution maps are identified with predominantly differentially expressed genes to predict the potential biological processes of Treg cell expansion, fibroblast differentiation, and macrophage polarization. In addition, CellphoneDB and CellChat are used to build an intercellular network of potential ligand-receptor interactions and study intercellular communication with a focus on T cells, macrophages, and fibroblasts (Armingol et al. (2021 ) Nat Rev Genet. 22:71 -88; Hao et al. (2021) Front. Genet. 12).EXAMPLE 3: UPREGUL TION OF CTLA4 LIG NDS CD80 / 86 IN ACUTE MI is SIGNIFICANTLY CORRELATED WITH POOR CARDIAC FUNCTION

[0195] Our data indicate that the upregulation of CTLA4 ligands CD80 / 86 after MI is associated with poor cardiac function, whereas CTLA4 inhibition leads to improved early postinfarct cardiac repair, increased survival, better preservation of cardiac function, and reduced cardiac infarct size. Intriguingly, these effects are not replicated with other ICIs, such as inhibitors of PD-1 and PD-L1, highlighting the distinct impact of each immune checkpoint signaling. These findings underscore the potential therapeutic value of modulating the CTLA4 / CD80 / 86 axis in post-ischemic cardiac repair.

[0196] As shown in FIG. 2A, mRNA expression of CD80 / CD86 (CTLA4 ligands) was significantly upregulated in the ischemic area compared to the remote area 3 days following MI in the ischemic area compared to the remote area. However, mRNA expression of PD-L1 (PD-1 ligand) was not significantly regulated in the ischemic and non-ischemic areas. Furthermore, I isolated the cardiomyocytes from ischemic heart and border zone by Langendorf and verified the expression of these immune checkpoint ligands by RT-qPCR. Similarly, mRNA expression of CD80 / 86 was significantly upregulated in the cardiomyocytes of ischemic and border zones, compared with non-ischemic areas or sham hearts (FIG. 2B). In addition, cardiac function of was determined by echocardiography and correlation analysis was conducted with the expression level of these IC ligands. Surprisingly, higher expression of CD80 / 86 in cardiomyocytes is significantly correlated with poor left ventricular ejection fraction (FIG. 2C). Among the diverse set of IC ligands, RNA-sequencing data shows CD80 / 86 signaling is significantly regulated (FIG. 2D). Taken together, these data demonstrate that CTLA4 ligands CD80 / 86 are robustly induced early after acute ischemic cardiac injury, and their high expression levels are significantly correlated with poor LV function. These findings suggest that the CTLA4 / CD80 / 86 axis may be a potential therapeutic target for restoring myocardial homeostasis after cardiac injury.EXAMPLE 4; CARDIAC IMMUNE CELL PROFILING

[0197] To investigate the role of CTLA4 in maintaining peripheral homeostasis, wildtype C57BL / 6 mice were treated with anti-CTLA4 (200 pg / mouse) and Abatacept (300 pg / mouse) twice a week for two weeks. Peripheral blood mononuclear cells (PBMCs) were isolated, and multiplex flow cytometry analyses were performed to assess the percentage of various immune cell populations. Our findings revealed no significant changes in the percentages of Grl-i- neutrophils, CD11C+ dendritic cells, CD68+ macrophages, CD19+ B cells, as well as CD4+, CD8+, and NK1.1+ T cells following anti-CTLA4 treatment (FIG. 4A). However, a significant increase in the number of peripheral Foxp3+ regulatory T cells was observed after two weeks ofanti-CTLA4 treatment in blood (FIG. 4B). We also analyzed the cell populations two days post- MI. Surprisingly, we observed a significant 3-fold increase in the number of peripheral Foxp3+ Tregs two days post-MI (FIG. 4C). Notably, peripheral Foxp3+ Tregs were not changed in anti- PD-1 or anti-PD-Ll treated animals two days post-MI (data not shown). Cardiac multiplex flow cytometry reveals increased infiltration of cardiac Foxp3+ Tregs (FIG. 4). In conclusion, our data demonstrate that blockade of CTLA4 leads to an increase in the number of Foxp3+ Tregs in both the peripheral blood and injured heart. Next, we will comprehensively profile and quantify immune cell-infiltration and the released cytokines and chemokines within the cardiac tissue, focusing particularly on proinflammatory e.g., TNFa, IL- I p, IL-6, anti-inflammatory markers TNFp, IL4, IL10, and reparative immune cell populations e.g., Foxp3+ Treg cells via multiplex flow cytometry, immunochemistry, and multiplex cytokine assay. Understanding the dynamics of immune cell migration and activity under CTLA-4 modulation will shed light on its role in shaping the cardiac immune microenvironment post-injury.EXAMPLE 5: EXAMINATION OF FIBROSIS AND CARDIAC DAMAGE BIOMARKERS

[0198] Standard histological methods are employed such as Masson's trichrome staining to assess the extent of fibrosis in the cardiac tissue post-MI. Correlating fibrotic regions with CTLA-4 modulation reveals the potential of CTLA-4 inhibitors to minimize adverse cardiac remodeling post-MI. Fibrosis markers, including collagens and matrix metalloproteinases (MMPs) are measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR) to ascertain extracellular matrix deposition in the injured myocardium. Cardiac damage biomarkers are also examined using a comprehensive multi-plex blood ELISA assay of conventional cardiac biomarkers, including troponins (I and T), creatine kinase-myocardial band (CK-MB), and amino-terminal pro B-type natriuretic peptide (NT-proBNP). By establishing a direct relationship between biomarker concentrations and the therapeutic effects of CTLA4 modulation, we can potentially streamline the future diagnostic and therapeutic stratagems for patients post-MI.EXAMPLE 6; TREG-MEDIATED CARDIOPROTECTIVE MECHANISM OF CTLA4 INHIBITION POST-MI

[0199] Myocardial injury leads to an activation of both the innate and adaptive immune systems which, in turn, drives tissue repair and returns myocardial homeostasis5,6. The early phase witnesses an influx of immune cells like neutrophils, macrophages, monocytes, and T cells, which are crucial in clearing cell debris resulting from injury. However, the regulation of inflammation can promote cardiac repair if well-regulated but can also be detrimental if mismanaged5,6. CTLA-4 is predominantly expressed in Foxp3+ Tregs and is induced in conventional T cells upon their activation7. CD4+ T cells, especially Foxp3+ Tregs, have been proven to promote myocardial repair by regulating inflammation and promoting tissue repair7-9. Tregs also protect cardiomyocytes from apoptosis, modulate macrophage differentiation and fibroblast activation10 11. Tregs present in the heart, which are mainly thymus-derived and recruited from circulation, show active local proliferation with !L-33 / sST2 promoting their expansion12 13.

[0200] Our studies using CTLA4 inhibitor treatment showed an increase in the number of peripheral Foxp3+ Tregs. No significant changes in other immune cell types like Grl-i- neutrophils, CD11C+ dendritic cells, CD68+ macrophages, CD19+ B cells, as well as CD4+, CD8+, or NK1. 1+ T cells were observed. This trend continued after MI, and we observed a 3-fold increase in the number of peripheral Foxp3+ Tregs two days post-ML Cardiac multiplex flow cytometry further revealed increased infiltration of cardiac Foxp3+ Tregs. In conclusion, our data demonstrate that CTLA4 inhibition leads to a significant increase in the number of Foxp3+ Tregs in both the peripheral blood and the injured heart.

[0201] We are further investigating the intricate molecular dynamics of CTLA-4 inhibition, emphasizing its influence on the expansion and function of the Treg population. CTLA- 4 and their ligands CD80 / CD86 have been demonstrated to play critical roles in the generation, maintenance, and function of Tregs15 16. Studies have shown that surprisingly CTLA-4 deficient mice have an enlarged population of peripheral Foxp3+ Tregs suggesting that CTLA-4 in Tregs acts as an intrinsic brake on its proliferation7 17,18. An increase of Tregs in peripheral blood with maintained suppressive function was observed in cancer patients following anti-CTLA-4 treatment1920. The role of CTLA-4 in modulating Treg has been shown to range from intracellular mechanisms that regulate TCR signaling to extracellular interactions that include modulation of CD80 / CD86 and transendocytosis21,22.EXAMPLE 7: OPTIMIZATION OF CTLA-4 INHIBITOR DOSAGE FOR ENHANCED THERAPEUTIC EFFICACY

[0202] Objective: To determine the optimal dosage of CTLA-4 inhibitor that maximizes therapeutic benefits whilst mitigating the risk of adverse effects. The chosen dosage will undergo rigorous testing in pre-clinical MI models to assess its therapeutic benefits and safety profile. Rationale: Recent studies suggest that inhibiting CTLA-4 not only increases the count of active effector CD4+ T cells but also promotes the presence of CD4+ Foxp3+ Tregs in a dose-dependent fashion. Notably, the growth of CD4+ Foxp3+ Tregs is more evident at lower antibody dosages, whereas the surge in effector T cells is more pronounced at higher dosages. The bolstered Foxp3+ Tregs, post-treatment, continue to proliferate and maintain their suppressive function14.

[0203] Research Design: Dose-response experiments are performed to identify the exact dosage of CTLA-4 inhibitor that provides the best Treg therapeutic response without sacrificing safety. We are testing a spectrum of dosages (25 pg / mouse, 100 pg / mouse. and 400 ug / mouse) and various dosing schedules (1 dose, 3 doses, 8 doses) for a well-rounded assessment. Once the optimal dosage is identified for the Treg expansion, it will be rigorously evaluated within the pre- clinical MI model. This will involve analyzing the therapeutic benefits derived and outlining any potential risks. To holistically evaluate the CTLA-4 inhibitor's therapeutic potential and safety, we are using a multifaceted method. A Kaplan- Meier survival analysis is used in tracking survival rates, particularly noting wall rupture-induced mortality. Echocardiographic measurements are used to evaluate post-MI cardiac function, chamber size, and infarct size, focusing on the parasternal long-axis view to determine the extent of the fibrotic scar. Additionally, cardiac damage is evaluated by quantifying plasma troponin levels using an ELISA. A CTLA-4 inhibitor dosage is selected that optimizes therapeutic benefits while ensuring safety.EXAMPLE 8: IN VITRO CELL-BASED MODELS OF CARDIOTOXICITY

[0204] To establish a cell-based model of cardiotoxicity, we treated cardiac cell lines with drugs, in a range of concentrations over a 24-hour, 48-hour and 72 hour-period. Three distinct cell types were used: hiPSC-CM (human induced pluripotent stem cells differentiated to cardiomyocytes using protocols as described previously (Lian, X., et al. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt / beta-catenin signaling under fully defined conditions. Nat Protoc 8, 162-175 (2013); Kleinsorge, M. & Cyganek, L. Subtype- Directed Differentiation of Human iPSCs into Atrial and Ventricular Cardiomyocytes. STAR Protoc 1, 100026 (2020); herein incorporated by reference in their entireties), AC 16 transformed human ventricular cardiomyocytes (Davidson, M.M., et al. Novel cell lines derived from adult human ventricular cardiomyocytes. J Mol Cell Cardiol 39, 133-147 (2005), herein incorporated by reference in its entirety), and HL-1 immortalized mouse atrial cardiomyocytes (Claycomb, W.C., et al. HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. Proc Natl Acad Sci USA. 95(6), 2979-2984 (1998); White, S.M., Constantin, P.E. & Claycomb, W.C. Cardiac physiology at the cellular level: use of cultured HL-1 cardiomyocytes for studies of cardiac muscle cell structure and function. Am J Physiol Heart Circ Physiol 286, H823-829 (2004)). CCK8 Cell Viability is evaluated using annexin V (AxV) with propidium iodide (PI) staining for early apoptosis and cell stress (Vermes, I., Haanen, C., Steffens-Nakken, H. & Reutelingsperger, C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluoresceinlabelled Annexin V. J Immunol Methods 184, 39-51 (1995), herein incorporated by reference in its entirety).

[0205] To test a functional readout of treatment, monolayers of hiPSC-CM, plated on multi-electrode arrays, are studied using extracellular field potential (EFP) recordings. Experiments are performed on the Nanion CardioExcyte96 system from Nanion Technologies (Miinchen, Germany), a high-throughput screening platform which allows for the simultaneous recording of cell impendence (as a marker of cellular contractility) and extracellular field potentials (EFP, a measure of electrophysiologic activity). The CardioExcyte96 platform has been used to study the effect of pharmaceutical compounds for pro- arrhythmic properties in hiPSC- CMs (Doerr, L., et al. New easy-to-use hybrid system for extracellular potential and impedance recordings. J Lab Autom 20, 175-188 (2015); Ziegler, R., Hausermann, F., Kirchner, S. & Polonchuk, L. Cardiac Safety of Kinase Inhibitors - Improving Understanding and Prediction of Liabilities in Drug Discovery Using Human Stem Cell-Derived Models. Front Cardiovasc Med 8, 639824 (2021); herein incorporated by reference in their entireties).

[0206] To further expand on pharmaceutical compounds-induced stress on cardiomyocytes, we are assessing contractility and calcium (Ca2+)dynamics using a custom optimized multicell Calcium and Contractility System (lonOptix) (Wust, R.C., et al. Rapid frequency-dependent changes in free mitochondrial calcium concentration in rat cardiac myocytes. J Physiol 595, 2001-2019 (2017); herein incorporated by reference in its entirety). All cell groups are cultured at low confluency to enable the detection of Ca2+signals at the single-cell level. Our system allows simultaneous data acquisition from multiple cells ensuring high throughput. Fluorescent signals of Ca2+are measured using the ratiometric Ca2+indicator, Fura- 2-acetoxymethyl ester (Furo-2AM, Ex: 360 / 380 nm, Em: 510 nm; 1 pM; abeam), with 1 Hz field electrical stimulation (Martinez, M., Martinez, N.A. & Silva, W.I. Measurement of the Intracellular Calcium Concentration with Fura-2 AM Using a Fluorescence Plate Reader. Bio Protoc 7, e2411 (2017); herein incorporated by reference in its entirety). Properties of Ca2+dynamics, including Ca2+amplitude, time-to-peak, Ca2+reuptake decay constant (r) are compared among different groups. Contractility is evaluated by measuring the shortening and re-lengthening using “cell length recording model”. Acute pharmacological interventions are applied through a perfusion system.Literature Citations:

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[0209] 3. McNichols B, Spratt JR, George J, Rizzi S, Manning EW, Park K. CoronaryArtery Bypass: Review of Surgical Techniques and Impact on Long-Term Revascularization Outcomes. Cardiol Ther. 2021;10:89-109. doi: 10.1007 / s40119-021-00211-z

[0210] 4. Abubakar M, Masood MF, Javed I, Adil H, Faraz MA, Bhat RR, Fatima M,Abdelkhalek AM, Buccilli B, Raza S, et al. Unlocking the Mysteries, Bridging the Gap, and Unveiling the Multifaceted Potential of Stem Cell Therapy for Cardiac Tissue Regeneration: A Narrative Review of Current Literature, Ethical Challenges, and Future Perspectives. Cureus. 2023;15:e41533. doi: 10.7759 / cureus.41533

[0211] 5. Rurik JG, Aghajanian H, Epstein JA. Immune Cells and Immunotherapy forCardiac Injury and Repair. Circ Res. 2021;128:1766-1779. doi: 10.1161 / CIRCRES AH A.121.318005

[0212] 6. Saparov A, Ogay V, Nurgozhin T, Chen WCW, Mansurov N, Issabekova A,Zhakupova J. Role of the immune system in cardiac tissue damage and repair following myocardial infarction. Inflamm Res. 2017;66:739-751. doi: 10.1007 / s00011-017-1060-4

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[0214] 8. Schmidt EM, Wang CJ, Ryan GA, Clough LE, Qureshi OS, Goodall M, AbbasAK, Sharpe AH, Sansom DM, Walker LSK. CTLA-4 Controls Regulatory T Cell Peripheral Homeostasis and Is Required for Suppression of Pancreatic Islet Autoimmunity. J Immunol. 2009;182:274-282. doi: DOI 10.4049 / jimmunol.182.1.274

[0215] 9. Weirather J, Hofmann UD, Beyersdorf N, Ramos GC, Vogel B, Frey A, Ertl G,Kerkau T, Frantz S. Foxp3+ CD4+ T cells improve healing after myocardial infarction by modulating monocyte / macrophage differentiation. Circ Res. 2014;115:55-67. doi: 10.1161 / CIRCRES AHA.115.303895

[0216] 10. Tang TT, Yuan J, Zhu ZF, Zhang WC, Xiao H, Xia N, Yan XX, Nie SF, Liu J,Zhou SF, et al. Regulatory T cells ameliorate cardiac remodeling after myocardial infarction. Basic Res Cardiol. 2012;107:232. doi: 10.1007 / s00395-01 1-0232-6

[0217] 11. Xia N, Lu Y, Gu M, Li N, Liu M, Jiao J, Zhu Z, Li J, Li D, Tang T, et al. AUnique Population of Regulatory T Cells in Heart Potentiates Cardiac Protection FromMyocardial Infarction. Circulation. 2020;142: 1956-1973. doi: 10.1161 / CIRCULATIONAHA. 120.046789

[0218] 12. Kuswanto W, Burzyn D, Panduro M, Wang KK, Jang YC, Wagers AJ, BenoistC, Mathis D. Poor Repair of Skeletal Muscle in Aging Mice Reflects a Defect in Local, Interleukin-33-Dependent Accumulation of Regulatory T Cells. Immunity. 2016;44:355-367. doi: 10.1016 / j.immuni.2016.01.009

[0219] 13. Rieckmann M, Delgobo M, Gaal C, Buchner L, Steinau P, Reshef D, Gil-CruzC, Horst ENT, Kircher M, Reiter T, et al. Myocardial infarction triggers cardioprotective antigenspecific T helper cell responses. J Clin Invest. 2019;129:4922-4936. doi: 10.1172 / JCI123859

[0220] 14. Kavanagh B, O'Brien S, Lee D, Hou Y, Weinberg V, Rini B, Allison JP, SmallEJ, Fong L. CTLA4 blockade expands FoxP3+ regulatory and activated effector CD4+ T cells in a dose-dependent fashion. Blood. 2008;112: 1175-1183. doi: 10.1182 / blood-2007-ll-125435

[0221] 15. Tang Q, Henriksen KJ, Boden EK, Tooley AJ, Ye J, Subudhi SK, Zheng XX,Strom TB, Bluestone JA. Cutting edge: CD28 controls peripheral homeostasis of CD4+CD25+ regulatory T cells. J Immunol. 2003;171:3348-3352. doi: 10.4049 / jimmunol. 171.7.3348

[0222] 16. Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T, Miyara M, Fehervari Z,Nomura T, Sakaguchi S. CTLA-4 control over Foxp3+ regulatory T cell function. Science. 2008;322:271-275. doi: 10. 1126 / science. 1160062

[0223] 17. Takahashi T, Tagami T, Yamazaki S, Uede T, Shimizu J, Sakaguchi N, MakTW, Sakaguchi S. Immunologic self-tolerance maintained by CD25(+)CD4(+) regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4. J Exp Med. 2000;192:303- 310. doi: 10.1084 / jem.192.2.303

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[0229] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of treating heart disease in a subject, the method comprising administering a therapeutically effective amount of an inhibitor of cytotoxic T-lymphocyte- associated protein 4 (CTLA-4) to the subject.

2. The method of claim 1, wherein the inhibitor of CTLA-4 is an antibody that specifically binds to CTLA-4.

3. The method of claim 2, wherein the antibody is ipilimumab or tremelimumab.

4. The method of claim 2, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a Fvfragment, and a nanobody.

5. The method of any one of claims 1-4, wherein the heart disease is myocardial infarction, myocardial ischemia, myocardial injury, heart failure, atherosclerosis, coronary artery disease, and cardiomyopathies.

6. The method of any one of claims 1-5, wherein the inhibitor of CTLA-4 is administered intravenously or intra-arterially.

7. The method of any one of claims 1-5, wherein the inhibitor of CTLA-4 is administered locally to damaged heart tissue or a site of cardiac ischemia or infarction.

8. The method of any one of claims 1-7, wherein multiple therapeutically effective doses of the inhibitor of CTLA-4 are administered to the subject.

9. The method of claim 8, wherein the inhibitor of CTLA-4 is administered according to a daily dosing regimen or intermittently.

10. The method of any one of claims 1 -9, wherein the inhibitor of CTLA-4 is administered after a myocardial infarction.

11. The method of any one of claims 1-9, wherein the inhibitor of CTLA-4 is administered prophylactically to protect against or delay or prevent myocardial infarction.

12. The method of any one of claims 1-11, wherein the subject has cardiovascular disease.

13. The method of any one of claims 1-12, wherein treatment with the inhibitor of CTLA-4 increases left ventricular ejection fraction, reduces chamber dilatation, reduces ventricular remodeling, reduces area of fibrotic scar tissue, reduces plasma troponin levels, and / or increases survival compared to in absence of the treatment.

14. The method of any one of claims 1-13, wherein treatment with the inhibitor of CTLA-4 increases proliferation of Foxp3+ regulatory T cells compared to in absence of the treatment.

15. The method of any one of claims 1-14, wherein the subject is human.

16. A method of treating ventricular remodeling in a subject, the method comprising administering a therapeutically effective amount of an inhibitor of cytotoxic T-lymphocyte- associated protein 4 (CTLA-4) to the subject.

17. The method of claim 16, wherein the inhibitor of CTLA-4 is administered prophylactically or therapeutically after myocardial infarction, myocardial ischemia, myocardial injury, atherosclerosis, or heart failure to prevent, decrease, or delay ventricular remodeling.

18. The method of claim 16 or 17, wherein the ventricular remodeling comprises changes in left ventricular (LV) geometry, mass, and / or volume.

19. The method of any one of claims 16-18, wherein the inhibitor of CTLA-4 is an antibody that specifically binds to CTLA-4.

20. The method of claim 19, wherein the antibody is ipilimumab or tremelimumab.

21. The method of claim 19, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a Fvfragment, and a nanobody.

22. The method of any one of claims 16-21, wherein the inhibitor of CTLA-4 inhibits binding of CTLA-4 to B7.1 and B7.2 antigen-presenting cell ligands.

23. The method of any one of claims 16-22, wherein the inhibitor of CTLA-4 stimulates proliferation of a regulatory T cell (Treg).

24. The method of claim 23, wherein the Treg is a Foxp3+Treg.

25. The method of any one of claims 16-24, wherein the subject has a myocardial infarction, myocardial ischemia, myocardial injury, heart failure, atherosclerosis, coronary artery disease, or a cardiomyopathy.

26. The method of any one of claims 16-25, wherein the inhibitor of CTLA-4 is administered intravenously or intra-arterially.

27. The method of any one of claims 16-25, wherein the inhibitor of CTLA-4 is administered locally to damaged heart tissue or a site of cardiac ischemia or infarction.

28. The method of any one of claims 16-27, wherein multiple therapeutically effective doses of the inhibitor of CTLA-4 are administered to the subject.

29. The method of claim 28, wherein the inhibitor of CTLA-4 is administered according to a daily dosing regimen or intermittently.

30. A method of screening for an inhibitor of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) for treating heart disease, the method comprising:(a) contacting CTLA-4 with a candidate agent;(b) measuring inhibition of CTLA-4 by the candidate agent to determine if the candidate agent is an inhibitor of CTLA-4;(c) administering the candidate agent to a mammalian non-human animal before, during, or after performing left anterior descending artery (LAD) ligation on the mammalian non-human animal, if the candidate agent is determined to be an inhibitor of CTLA-4; and(d) measuring left ventricular ejection fraction, chamber size, chamber dilatation, ventricular remodeling, area of fibrotic scar tissue, survival, level of plasma troponin, or any combination thereof in the mammalian non-human animal after said performing the LAD, wherein increased left ventricular ejection fraction, reduced chamber dilatation, reduced ventricular remodeling, reduced area of fibrotic scar tissue, increased survival, reduced level of plasma troponin, or any combination thereof, compared to in absence of said administering the candidate agent indicates that the candidate agent is useful for treating heart disease.

31. The method of claim 30, wherein the candidate agent is a small molecule, a peptide, a protein, a peptoid, an aptamer, an antibody that specifically binds to CTLA-4, an antibody mimetic, an inhibitory nucleic acid, or a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system.

32. The method of claim 31, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a Fvfragment, and a nanobody.

33. The method of claim 31, wherein the inhibitory nucleic acid is selected from the group consisting of a small interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a Piwi-interacting RNA (piRNA), a small nuclear RNA (snRNA), an antisense oligonucleotide, and a peptide nucleic acid.

34. The method of claim 31, wherein the inhibitory nucleic acid inhibits CTLA-4 transcription or protein translation.

35. The method of claim 31, wherein the CRISPR system targets a CTLA-4 gene or a CTLA-4 RNA transcript, or makes epigenetic changes that reduce CTLA-4 expression.

36. The method of claim 35, wherein the CRISPR system comprises Cas9, Casl2a, Casl 2d, Casl3a, Casl3b, Cas l3d, or a dead Cas9 (dCas9).

37. The method of any one of claims 30-36, wherein the non-human animal model is a rodent or primate.

38. The method of claim 37, wherein the rodent is a mouse.

39. The method of any one of claims 30-38, wherein said measuring inhibition of CTLA-4 comprises measuring inhibition of binding of CTLA-4 to B7.1 and B7.2 antigen- presenting cell ligands.

40. The method of any one of claims 30-39, wherein said measuring inhibition of CTLA-4 comprises measuring proliferation of a Foxp3+ regulatory T cell (Treg).

41. The method of any one of claims 30-40, wherein the level of plasma troponin is measured using an immunoassay or liquid chromatography-tandem mass spectrometry.

42. The method of any one of claims 30-41, wherein the chamber size, the chamber dilatation, the ventricular remodeling, or the area of fibrotic scar tissue is measured by echocardiography, ultrasound, or histology.

43. The method of any one of claims 30-42, wherein the left ventricular ejection fraction is measured by echocardiography, magnetic resonance imaging (MRI), computerized tomography (CT), radionuclide angiography, gated myocardial perfusion single-photon emission computed tomography (SPECT), or gated myocardial perfusion positron emission tomography (PET).

44. The method of any one of claims 30-43, further comprising contacting a cardiomyocyte with the candidate agent; and measuring contractility, calcium (Ca2+)dynamics, extracellular field potential (EFP), pro-arrhythmic properties, or any combination thereof.

45. The method of claim 44, wherein the cardiomyocyte is a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM).

46. A composition comprising an inhibitor of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) for use in a method of treating heart disease or ventricular remodeling.

47. The composition of claim 46, further comprising a pharmaceutically acceptable excipient.

48. The composition of claim 46 or 47, further comprising a pharmaceutically acceptable carrier selected from the group consisting of a cream, emulsion, gel, liposome, nanoparticle, or ointment.

49. The composition of any one of claims 46-48, wherein the inhibitor of CTLA-4 is an antibody that specifically binds to CTLA-4.

50. The composition of claim 49, wherein the antibody is ipilimumab or tremelimumab.

51. The composition of claim 49, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a Fvfragment, and a nanobody.

52. The composition of any one of claims 46-51, wherein the inhibitor of CTLA-4 inhibits binding of CTLA-4 to B7.1 and B7.2 antigen-presenting cell ligands.

53. The composition of any one of claims 46-52, wherein the inhibitor of CTLA-4 increases proliferation of a Foxp3+ regulatory T cell (Treg).

54. The composition of any one of claims 46-53, wherein the heart disease is myocardial infarction, myocardial ischemia, myocardial injury, heart failure, atherosclerosis, coronary artery disease, or a cardiomyopathy.

55. Use of an inhibitor of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) in the manufacture of a medicament or pharmaceutical composition for treating heart disease in a subject in need thereof.

56. The use of claim 55, wherein the inhibitor of CTLA-4 is an antibody that specifically binds to CTLA-4.

57. The use of claim 56, wherein the antibody is ipilimumab or tremelimumab.

58. The use of claim 56, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a F(ab) fragment, a F(ab’)2 fragment, a Fvfragment, and a nanobody.

59. The use of any one of claims 55-58, wherein the inhibitor of CTLA-4 inhibits binding of CTLA-4 to B7.1 and B7.2 antigen-presenting cell ligands.

60. The use of any one of claims 55-59, wherein the inhibitor of CTLA-4 increases proliferation of a Foxp3+ regulatory T cell (Treg).

61. The use of any one of claims 55-60, wherein the heart disease is myocardial infarction, myocardial ischemia, myocardial injury, heart failure, atherosclerosis, coronary artery disease, or a cardiomyopathy.

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