RU2842423C2 - Optimization of bag3 gene therapy - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to a method for diagnosing a patient with cardiac disease, comprising identifying in a patient's sample at least one genetic variant of Bcl2-associated athanogen 3 (BAG3) as compared to a reference nucleic acid sequence BAG3. Also disclosed is a method of treating a patient with cardiac disease, wherein the patient has at least one genetic variant of Bcl2-associated athanogen 3 (BAG3) as compared to the control nucleic acid sequence BAG3, and also disclosed is a method of identifying a patient with heart disease with a worse prognosis, including screening a sample from a patient for the presence of a nucleotide variant (NV) of insert type in reading frame of Bcl2-associated athanogen 3 (BAG3) compared to a control nucleic acid sequence BAG3. Invention also relates to the use of a kit comprising one or more probes, conjugated with a detectable tag to identify variants of BCL2-associated athanogen 3 (BAG3) in a biological sample.

EFFECT: invention is effective for diagnosing a patient with cardiac disease.

19 cl, 8 dwg, 2 tbl, 1 ex

Description

RELATED APPLICATIONS

[0001] This application claims priority based on U.S. Provisional Patent Application No. 62/682,404, filed June 8, 2018. The entire contents of that application are incorporated herein by reference, including all text, tables, sequence listing, and drawings.

STATE SUPPORT

[0002] This invention was made with federal support from Grant Nos. RO1 HL123093 and HL 091799-01 issued by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF INVENTION

[0003] Embodiments of the invention relate to identifying genetic variants of BAG3 in individuals of African descent that negatively impact outcome in patients with cardiac diseases, such as non-ischemic or ischemic dilated cardiomyopathy, by modulating BAG3 function. Compositions include agents that normalize the expression and/or function of Bcl2-associated anthanogen 3 (BAG3).

PRIOR ART

[0004] Heart failure (HF) secondary to dilated cardiomyopathy (DCM) affects approximately 2.5 million Americans age 20 and older._ ADDIN EN.CITE _ ADDIN EN.CITE.DATA ___1_ The epidemiology of DCM varies by race and ethnicity, with African Americans having the highest incidence and prevalence of HF and a predominance of idiopathic dilated cardiomyopathy (IDCM)._HYPERLINK \l "_ENREF_2" \o "Yancy, 2000 #285"_2_,_HYPERLINK \l "_ENREF_3" \o "Loehr, 2008 #286"_3_ This contrasts with patients of European descent, who have the highest incidence of DCM secondary to coronary artery disease._ ADDIN EN.CITE _ ADDIN EN.CITE.DATA ___4-7_ Consistent with the epidemiology of IDCM in African Americans, IDCM is more common and the age at first diagnosis is substantially lower in sub-Saharan Africa than in the United States or Europe._HYPERLINK \l "_ENREF_8" \o "Sliwa, 2008 #293"_8_,_HYPERLINK \l "_ENREF_9" \o "Bibbins-Domingo, 2009 #294"_9_

[0005] The increased prevalence of DCM in African Americans has been attributed to a diverse set of medical and sociological factors, including environmental ¹⁰ factors and a higher burden of cardiovascular risk factors: diabetes, hypertension, cholesterol, smoking status, and ventricular hypertrophy. ¹¹ Some observations, however, suggest that the increased incidence of DCM may also be attributable to genetic risk factors. For example, HF hot spots have been found in geographic regions throughout sub-Saharan Africa. ¹² There is no strong correlation between the presence of hypertension and DCM in sub-Saharan Africans. ¹³ Truncation variants in the TTN gene, the gene most commonly associated with DCM,14 are more common in women of African descent with postpartum cardiomyopathy than in women of European descent with postpartum cardiomyopathy._ ADDIN EN.CITE <EndNote><Cite><Author>Ware</Author><Year>2016</Year><RecNum>300</RecNum><DisplayText><style face="superscript">15</style></DisplayText><record><rec-number>300</rec-number><foreign-keys><key app="EN"db-id="90rfwftdmvpx5sewfdqpa0pkwaz5rd5zx9ts"timestamp="1515438336">300</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Ware,JS</author><author>Seidman,JG</author><author>Arany,Z.</author></authors></contributors><titles><title>Shared Genetic Predisposition in Peripartum and Dilated Cardiomyopathies</title><secondary-title>N Engl J Med</secondary-title><alt-title>The New England journal of medicine</alt-title></titles><periodical><full-title>N Engl J Med</full-title><abbr-1>The New England journal of medicine</abbr-1></periodical><alt-periodical><full-title>N Engl J Med</full-title><abbr-1>The New England journal of medicine</abbr-1></alt-periodical><pages>2601-2</pages><volume>374</volume><number>26</number><keywords><keyword>Cardiomyopathies/*genetics</keyword><keyword>Cardiomyopathy, Dilated/*genetics</keyword><keyword>Connectin/*genetics</keyword><keyword>Female</keyword><keyword>*Genetic Predisposition to Disease</keyword><keyword>Humans</keyword><keyword>*Mutation</keyword><keyword>*Peripartum Period</keyword><keyword>Pregnancy</keyword><keyword>Pregnancy Complications, Cardiovascular/*genetics</keyword></keywords><dates><year>2016</year><pub-dates><date>Jun 30</date></pub-dates></dates><isbn>1533-4406 (Electronic)&#xD;0028-4793 (Linking)</isbn><accession-num>27355546</accession-num><urls><related-urls><url>http://www.ncbi.nlm.nih.gov/pubmed/27355546</u rl></related-urls></urls><electronic-resource-num>10.1056/NEJMc1602671</electronic-resource-num></record></Cite></EndNote>_15_

ESSENCE

[0006] Identification of genetic variants in BAG3 (Bcl2-associated anthanogen 3) has been associated with a prevalence of non-ischemic or ischemic dilated cardiomyopathy (DCM) and DCM outcomes in individuals of African descent. Patients with mutations were found to have a significantly increased risk of death or hospitalization due to heart failure.

[0007] Thus, in certain embodiments, a method of treating a patient with a cardiac disease, wherein said patient has at least one nucleotide variant (NV) of a Bcl2-associated anthanogen 3 (BAG3) in-frame insertion type compared to a BAG3 reference nucleic acid sequence, comprises administering to the patient a therapeutically effective amount of an agent, wherein the agent modulates the expression or amount of BAG3 molecules, proteins or peptides thereof in a target cell or target tissue.

[0008] In certain embodiments, the in-frame insertion encodes an amino acid. In certain embodiments, the in-frame insertion encodes a non-polar amino acid. In certain embodiments, the in-frame insertion comprises a three-nucleotide insertion that adds an alanine at position 160 (p.Ala160dup, 10:121429647 A/AGCG; rs139438727).

[0009]

[0010] In certain embodiments, a method for diagnosing and treating a patient having a cardiac disease comprises identifying at least one genetic variant of Bcl2-associated athanogene 3 (BAG3) in a patient sample compared to a BAG3 nucleic acid reference sequence, wherein detection of the identified variants predicts whether an increase in BAG3 levels is therapeutic for the patient, and administering to a patient in whom such a variant is identified a therapeutically effective amount of an agent, wherein the agent modulates the expression or amount of BAG3 molecules, proteins, or peptides in a target cell or target tissue compared to a normal control. Genetic variants in BAG3, found almost exclusively in individuals of African descent, were not causative of the disease, but adversely affected the outcome of patients with non-ischemic or ischemic dilated cardiomyopathy through modulation of BAG3 function.

[0011] In certain embodiments, a method for diagnosing and treating a patient having a cardiac disease comprises identifying in a patient sample at least one genetic variant of Bcl2-associated anthanogen 3 (BAG3) compared to a BAG3 nucleic acid reference sequence; and administering to a patient in whom such variant is identified a therapeutically effective amount of an agent, wherein the agent modulates the expression or amount of BAG3 molecules, proteins, or peptides, in a target cell or target tissue, compared to a normal control.

[0012] In certain embodiments, the genetic variant is an in-frame insertion. In certain embodiments, the in-frame insertion encodes an amino acid. In certain embodiments, the in-frame insertion encodes a non-polar amino acid. In certain embodiments, the in-frame insertion comprises a three-nucleotide insertion that adds an alanine at position 160 (p.Ala160dup, 10: 121429647 A/AGCG; rs139438727).

[0013] In certain embodiments, a method of treating an individual at risk for or suffering from heart failure comprises administering to the individual a pharmaceutical composition comprising a therapeutically effective amount of at least one agent that modulates the expression or amount of a BCL2-associated anthanogen 3 (BAG3) molecule, wherein the individual has at least one genetic variant of BAG3 compared to a BAG3 reference nucleic acid sequence, wherein said genetic variant of BAG3 is an in-frame insertion, and wherein the agent comprises an expression vector encoding a BCL2-associated anthanogen 3 (BAG3) molecule.

[0014] In certain embodiments, the genetic variant is an in-frame insertion. In certain embodiments, the in-frame insertion encodes an amino acid. In certain embodiments, the in-frame insertion encodes a non-polar amino acid. In certain embodiments, the in-frame insertion comprises a three-nucleotide insertion that adds an alanine at position 160 (p.Ala160dup, 10: 121429647 A/AGCG; rs139438727).

[0015] In certain embodiments, the genetic variant is an in-frame insertion, deletion, substitution, or combination thereof single nucleotide variant (SNV). In certain embodiments, the SNVs include: p.Pro63Ala (10: 121429369 C/G; rs133031999); p.His83Gln (10: 151331972; rs151331972); Pro380Ser (10: 121436204 C/T; rs144692954); Ala479Val (10: 121436502 C/T, rs34656239), or combinations thereof. In some embodiments, the in-frame insertion encodes an amino acid. In some embodiments, the in-frame insertion encodes a non-polar amino acid. In some embodiments, the in-frame insertion encodes an alanine at position 160.

[0016] In certain embodiments, a means for treating a patient identified as having a genetic variant in the BCL2-associated anthanogene 3 (BAG3) gene, wherein the genetic variant is a single nucleotide variant (SNV) comprising an in-frame insertion, in-frame deletion, truncation, substitution, or combinations thereof.

[0017] In certain embodiments, the agent modulates the expression or amount of BCL2-associated anthanogen 3 (BAG3) molecules, proteins, or peptides in a target cell or target tissue, compared to a normal control, and comprises an expression vector expressing the BAG3 protein or active fragments thereof, oligonucleotides, or combinations thereof.

[0018] In certain embodiments, a method of treating an individual at risk for or suffering from heart failure includes identifying BAG3 variants in a biological sample that predict whether increasing BAG3 levels is therapeutic for the subject, and administering to a patient identified with such a variant a therapeutically effective amount of an agent. In certain embodiments, the agent modulates the expression or amount of BAG3 molecules, proteins, or peptides in a target cell or target tissue compared to a normal control.

[0019] In certain embodiments, a method for identifying a patient with a cardiac disease with a worse prognosis comprises screening a sample from the patient for the presence of a nucleotide variant (NV) of the in-frame insertion type of Bcl2-associated anthanogen 3 (BAG3) compared to a BAG3 reference nucleic acid sequence, wherein detection of the nucleotide variant (NV) of the in-frame insertion type of BAG3 indicates a worse prognosis for the patient with the cardiac disease.

[0020] In certain embodiments, a method for identifying a patient at risk for cardiac disease comprises screening a sample from the patient for the presence of a nucleotide variant (NV) of the in-frame insertion type of Bcl2-associated anthanogen 3 (BAG3) compared to a BAG3 reference nucleic acid sequence, wherein detection of the nucleotide variant (NV) of the in-frame insertion type of BAG3 indicates that the patient is at risk for cardiac disease.

[0021] In certain embodiments, the in-frame insertion encodes an amino acid. In certain embodiments, the in-frame insertion encodes a non-polar amino acid. In certain embodiments, the in-frame insertion includes a three-nucleotide insertion that adds an alanine at position 160 (p.Ala160dup, 10: 121429647 A/AGCG; rs139438727).

[0022] In certain embodiments, a method of identifying a patient at risk for cardiac disease comprises screening a sample from the patient for the presence of a Bcl2-associated anthanogene 3 (BAG3) genetic variant, wherein the BAG3 genetic variant comprises p.Ala160dup (10: 121429647 A/AGCG; rs139438727); p.Pro63Ala (10: 121429369 C/G; rs133031999); p.His83Gln (10: 151331972; rs151331972); Pro380Ser (10: 121436204 C/T; rs144692954); Ala479Val (10: 121436502 C/T, rs34656239) or their combinations.

[0023] In certain embodiments, a kit comprising one or more probes conjugated to a detectable label for identifying BCL2-associated anthanogen 3 (BAG3) variants in a biological sample. Detection of certain variants allows for prediction of benefit from treatment for an individual.

[0024] All genes, gene names, and gene products described herein are intended to correspond to homologs of any species to which the compositions and methods described herein are applicable. Thus, the terms include, but are not limited to, genes and gene products from humans and mice. It should be understood that when a gene or gene product from a particular species is described, the description is intended by way of example only, and should not be interpreted as limiting unless the context in which it appears clearly indicates otherwise. Thus, for example, a description of genes or gene products herein that in some embodiments refers to mammalian nucleic acid and amino acid sequences is intended to cover homologous and/or orthologous genes and gene products from other animals, including, but not limited to, other mammals, fish, amphibians, reptiles, and birds. In certain embodiments, the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides, and proteins are human.

[0025] Other aspects are described below.

[0026] Definitions

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar to or equivalent to those described herein can be used in the practice of testing the present invention, specific materials and methods are described herein. The following terminology will be used in the description and claims of the present invention.

[0028] The singular articles are used herein to designate one or more (i.e., at least one) of the grammatical objects of the article. By way of example, "an element" means one element or more than one element. Thus, the description "a cell," for example, includes a plurality of cells of the same type. Furthermore, to the extent that the terms "including," "includes," "having," "has," "with," or variations thereof are used in either the detailed description or the claims, such terms are intended to be inclusive in a manner similar to the term "comprising."

[0029] As used herein, the terms "comprising," "comprises," or "includes," and variations thereof, with respect to specified or described elements of a feature, composition, device, method, process, system, etc. are intended to be inclusive or non-limiting, permitting additional elements, thereby indicating that the specified or described feature, composition, device, method, process, system, etc. includes those specified elements, or their equivalents where appropriate, and other elements that may be included and still fall within the scope/definition of the specified feature, composition, device, method, process, system, etc.

[0030] "Approximately," as used herein when referring to a measurable quantity such as an amount, a time duration, etc., is intended to include variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the stated value, as long as such variations are suitable for implementing the disclosed methods. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, within 5-fold, as well as within 2-fold. Where specific values are described in the application and claims, unless otherwise stated, the term "approximately" should be construed as being within the acceptable error range for the specific value.

[0031] As used herein, "BAG3", "BAG3 molecules", "BCL2-associated anthanogen 3 (BAG3) genes", "BCL2-associated anthanogen 3 (BAG3) molecules" include all family members, mutants, cDNA sequences, alleles, fragments, species, coding and non-coding sequences, sense and antisense polynucleotide strands, etc. (HGNC (939) Entrez Gene (9531) Ensembl (ENSG00000151929) OMIM (603883) UniProtKB (O95817)). Likewise, “BAG3”, “BAG3 molecules”, “BCL2-associated athanogen 3 (BAG3) molecules” also refer to BAG3 polypeptides or fragments thereof, proteins, variants, derivatives, etc. The term “molecule” thus includes both nucleic acid sequences and amino acid sequences of BAG3.

[0032] As used herein, "biological samples" include solid samples and biological fluid samples. Biological samples used in the present invention may include cells, protein or membrane extracts of cells, biological fluids such as ascites fluid or brain fluid (e.g., cerebrospinal fluid). Examples of solid biological samples include, as possible examples, samples taken from tissue of the central nervous system, bone, breast, kidney, cervix, endometrium, head/neck, gallbladder, parotid gland, prostate, pituitary gland, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid, heart, lung, bladder, adipose tissue, lymph node, uterus, ovary, adrenal gland, testicles, tonsils, thymus and skin, or samples taken from tumors. Examples of "biological fluid samples" include, but are not limited to, blood, serum, semen, prostate fluid, seminal fluid, urine, feces, saliva, sputum, mucus, bone marrow, lymph, and tears.

[0033] As used herein, "heart disease" refers to any type of heart disease, including heart failure, heart muscle disease, cardiomyopathy, hypertrophic cardiomyopathy, dilated cardiomyopathy, atherosclerosis, coronary artery disease, non-ischemic heart disease, ischemic heart disease, myocarditis, viral infection, wounds, hypertensive heart, valvular disease, congenital heart disease, myocardial infarction, congestive heart failure, arrhythmias, diseases leading to cardiac remodeling, etc. Heart disease may be caused by any cause, such as damage to cardiac tissue, or loss of contractility (as may be demonstrated by a decrease in ejection fraction). Cardiac injury or disorder characterized by inadequate cardiac function includes any impairment or absence of normal cardiac function or the presence of abnormal cardiac function. Abnormal cardiac function may result from disease, injury, and/or aging. As used herein, "abnormal cardiac function" includes morphological and/or functional abnormalities of cardiomyocytes, the aggregate of cardiomyocytes, or the heart itself. Non-limiting examples of morphological and functional abnormalities include physical deterioration and/or death of cardiomyocytes, abnormal patterns of cardiomyocyte growth, abnormalities in the physical communication between cardiomyocytes, insufficient or excessive production of a substance or substances by cardiomyocytes, failure of cardiomyocytes to produce the substance or substances they normally produce, and transmission of electrical impulses in an abnormal manner or at abnormal times. More general abnormalities include dyskinesias, decreased ejection fraction, changes observed on echocardiography (eg, dilation), ECG changes, changes in exercise tolerance, decreased capillary perfusion, and changes observed on angiography. Abnormal cardiac function is observed in many disorders, including, for example, nonischemic or ischemic heart disease, such as angina pectoris, myocardial infarction, chronic ischemic heart disease, hypertension, cardiopulmonary disease (cor pulmonale), valvular heart disease, such as rheumatic fever, mitral valve prolapse, mitral annular calcification, carcinoid heart disease, infective endocarditis, congenital heart disease, myocardial disease, such as myocarditis, dilated cardiomyopathy, hypertensive cardiomyopathy, cardiac disorders that lead to congestive heart failure, and cardiac tumors, such as primary sarcomas and secondary tumors. Cardiac injury also includes wounds, such as stab wounds; biological (e.g. viral; autoimmune diseases) or chemical (e.g. chemotherapy, drugs); surgery; transplantation, etc.

[0034] As used herein, the phrase "diagnosing" refers to classifying a disease or symptom, determining the severity of a disease, monitoring the progression of a disease, predicting the outcome of a disease and/or the prospects for recovery. The term "detection" may also optionally include any of the above. Diagnosis of a disease of the present invention may be accomplished by determining the level of a polynucleotide or polypeptide of the present invention in a biological sample obtained from an individual, where the determined level may be correlated with a predisposition to the disease, the presence or absence of the disease. It should be noted that a "biological sample obtained from an individual" may also optionally include a sample that has not been physically removed from the individual.

[0035] As used herein, the phrase "diagnostic" means determining the presence or nature of a pathological condition. Diagnostic assays vary in sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (the "true positive" rate). Diseased individuals who are not detected by the assay are "false negatives." Individuals who are not diseased and who test negative are called "true negatives." The "specificity" of a diagnostic assay is 1 minus the number of false positives, where a "false positive" is defined as the proportion of non-disease individuals who test positive. Although a particular diagnostic assay may not provide a definitive diagnosis of a condition, it is sufficient if it provides a positive indication that aids in diagnosis.

[0036] “Effective amount,” as used herein, means an amount that provides a therapeutic or prophylactic benefit.

[0037] "Coding" refers to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as templates for the synthesis of other polymers and macromolecules in biological processes having either a specific nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a specific amino acid sequence and the biological properties derived therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is typically listed in a sequence listing, and the non-coding strand used as a template for transcription of genes or cDNA may be referred to as encoding a protein or other product of the gene or cDNA.

[0038] As used herein, the term "expression" is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter.

[0039] "Expression vector" refers to a vector containing a recombinant polynucleotide containing control sequences operably linked to a nucleotide sequence to be expressed. The expression vector contains a sufficient number of cis-acting elements for expression; other elements for expression may be supplied by the host cell or an in vitro expression system. Expression vectors include all vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that contain a recombinant polynucleotide.

[0040] By "inhibitory nucleic acid" is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, which, when introduced into a mammalian cell, results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Typically, an inhibitory nucleic acid comprises at least a portion of a target nucleic acid molecule or an ortholog thereof, or comprises at least a portion of a complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids described herein.

[0041] "Isolated" means altered or removed from a natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from coexisting materials in their natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-natural environment, such as, for example, a host cell.

[0042] "Isolated nucleic acid" refers to a segment or fragment of nucleic acid that has been separated from the sequences that flank it in its natural state, i.e., a fragment of DNA that has been removed from the sequences that normally adjoin the fragment, i.e., the sequences adjacent to the fragment in the genome in which it occurs naturally. The term also refers to nucleic acids that have been substantially purified from other components that naturally accompany the nucleic acid, i.e., RNA or DNA, or proteins that naturally accompany it in a cell. The term thus includes, for example, recombinant DNA that is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of prokaryotes or eukaryotes, or that exists as a separate molecule (i.e. as cDNA or genomic or cDNA fragment produced by PCR or digested with restriction enzymes) independently of other sequences. It also includes recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence, complementary DNA (cDNA), linear or circular oligomers or polymers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, their substituted and alpha-anomeric forms, peptide nucleic acids (PNA), locked nucleic acids (LNA), thiophosphate, methylphosphonate, and the like.

[0043] A nucleic acid sequence may be "chimeric", i.e., comprised of different regions. In the context of the present invention, "chimeric" compounds are oligonucleotides that comprise two or more chemical regions, such as a DNA region/regions, an RNA region/regions, a PNA region/regions, etc. Each chemical region consists of at least one monomeric unit, i.e., a nucleotide. These sequences typically comprise at least one region in which the sequence has been modified to exhibit one or more desired properties.

[0044] As used herein, the term "kit" refers to any delivery system for delivering materials. With respect to reaction assays, such delivery systems include systems that enable reaction means (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and/or auxiliary materials (e.g., buffers, written instructions for performing the assay, etc.) to be stored, transported, or delivered from one location to another. For example, kits include one or more packages (e.g., boxes) containing the appropriate reaction means and/or auxiliary materials. As used herein, the term "fragmented kit" refers to a delivery system that includes two or more separate containers, each containing a portion of all components. The containers may be delivered together or separately to the intended recipient. For example, a first container contains an enzyme for use in the assay, and a second container contains oligonucleotides. The term "fragmented kit" is intended to include, but is not limited to, kits containing analyte specific reaction means (ASRs) regulated under section 520(e) of the Federal Food, Drug, and Cosmetic Act. In fact, any delivery system consisting of two or more separate containers, each containing a portion of a total set of components, is included within the term "fragmented kit." In contrast, "combination kit" refers to a delivery system containing all of the assay components in a single container (e.g., a single box containing each of the desired components). The term "kit" includes both fragmented and combination kits.

[0045] The term "target nucleic acid" sequence refers to a nucleic acid (often obtained from a biological sample) to which an oligonucleotide is designed to specifically hybridize. Either the presence or absence of the target nucleic acid is to be determined, or the amount of the target nucleic acid is to be quantified. The target nucleic acid has a sequence that is complementary to the nucleic acid sequence of the corresponding oligonucleotide that is targeted to the target. The term "target nucleic acid" may refer to a specific subsequence of a larger nucleic acid to which the oligonucleotide is directed, or to a general sequence (e.g., a gene or mRNA). The difference in usage will be apparent from the context.

[0046] In the context of the present invention, the following abbreviations are used for commonly occurring nucleic bases: “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0047] Unless otherwise specified, a "nucleotide sequence encoding" an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding" a protein or RNA may also include introns to the extent that a nucleotide sequence encoding a protein may, in some version, contain intron(s).

[0048] “Parenteral” administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrathoracic injection or infusion methods.

[0049] The terms "patient" or "individual" or "subject" are used interchangeably herein and refer to a mammal being treated, with a human patient being an example. In some cases, the methods of the invention find use in experimental animals, in veterinary medicine, and in the development of animal models for disease, including, but not limited to, rodents, including mice, rats, and hamsters, and primates.

[0050] The term "percent sequence identity" or the term "sequence identity" refers to the degree of identity between any given query sequence and the sequence of an individual.

[0051] The terms "pharmaceutically acceptable" (or "pharmacologically acceptable") refer to molecular structures and compositions that do not produce adverse, allergic, or other undesirable reactions when administered to an animal or human as needed. As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial, isotonic, and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants, and the like that can be used as a carrier for a pharmaceutically acceptable substance.

[0052] The term "polynucleotide" refers to a chain of nucleotides, also known as a "nucleic acid" or "nucleic acid sequence" and includes, but is not limited to, all nucleic acid sequences that are produced by any methods available in the art, such as natural and synthetic nucleic acids, complementary DNA (cDNA), linear or circular oligomers or polymers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), closed nucleic acids (LNA), thiophosphate, methylphosphonate, and the like. A nucleic acid sequence may be "chimeric", i.e., comprised of different regions. In the context of the present invention, "chimeric" compounds are oligonucleotides that comprise two or more chemical regions, such as a DNA region/regions, an RNA region/regions, a PNA region/regions, etc. Each chemical region consists of at least one monomeric unit, i.e. a nucleotide. These sequences typically comprise at least one region in which the sequence has been modified to exhibit one or more desired properties.

[0053] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and without limitation, contain the maximum number of amino acids that can comprise a protein or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art as peptides, oligopeptides, and oligomers, for example, and longer chains, which are generally referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and others. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0054] The term "transfected" or "transformed" or "transduced" refers to the method by which an exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. A transfected/transformed/transduced cell includes the primary individual cell and its progeny.

[0055] "Treat" a disease as a term is used herein to mean to reduce the incidence of at least one symptom or disease or disorder experienced by an individual. Treating a disease or disorder includes killing the virus.

[0056] "Treatment" is an intervention performed with the purpose of preventing the development of or changing the pathology or symptoms of the disorder. Thus, "treatment" refers to both therapeutic treatment and prophylactic or preventive measures. "Treatment" can also be referred to as palliative care. Treatment is needed both for those who already have the disorder and for those in whom the disorder is to be prevented. Thus, "treatment" of a condition, disorder, or disease includes: (1) eradication of a virus; (2) prevention or delay of the onset of clinical symptoms of the condition, disorder, or disease developing in a human or other mammal that may be affected by the condition, disorder, or disease or is predisposed to the condition, disorder, or disease but does not yet experience or exhibit clinical or subclinical symptoms of the condition, disorder, or disease; (3) suppression of the condition, disorder, or disease, i.e., arresting, reducing, or delaying the progression of the disease or its recurrence (in the case of maintenance treatment) or at least one clinical or subclinical symptom thereof; or (4) alleviation of disease, i.e., causing regression of a condition, disorder, or disease or at least one of its clinical or subclinical symptoms. The benefit to the person being treated is statistically significant or at least perceptible to the patient or physician.

[0057] The term "variant", when used in relation to a polynucleotide sequence, may include a polynucleotide sequence related to a wild-type gene. This definition may also include, for example, an "allelic", "splice", "species" or "polymorphic" variant. A splice variant may have significant identity with a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternative splicing of exons during mRNA processing. The corresponding polypeptide may be characterized by additional functional domains or the absence of domains. Species variants are polynucleotide sequences that vary from one species to another. Variants of wild-type gene products are particularly useful in the invention. Variants may result from at least one mutation of a nucleic acid sequence and may result in a change in mRNA or in polypeptides, the structure or function of which may or may not be changed. Any given natural or recombinant gene may have none, one, or more allelic forms. Common mutational changes that give rise to variants are generally attributed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone or in combination with others, one or more times in a given sequence.

[0058] A "vector" is a composition of matter that contains an isolated nucleic acid and that can be used to deliver the isolated nucleic acid into a cell. Examples of vectors include, but are not limited to, linear polynucleotides, polynucleotides linked to ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or virus. The term is also intended to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acid into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and the like.

[0059] Ranges: Throughout this description, various aspects of the invention may be presented in range format. It should be understood that a description in range format is provided merely for convenience and brevity and should not be considered an inflexible limitation on the scope of the invention. Thus, a description of a range should be considered to specifically disclose all possible subranges, as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0060] Where any nucleic acid sequence or amino acid sequence is specifically referenced by a Swiss Prot or GENBANK accession number, the sequence is incorporated herein by reference. Information associated with the accession number, such as the identification of the signal peptide, extracellular domain, transmembrane domain, promoter sequence, and translation start point, is also incorporated herein by reference in its entirety.

BRIEF DESCRIPTION OF DRAWINGS

[0061] FIG. 1 is a schematic representation of BAG3 showing protein binding domains and single non-synonymous variant sites, a single three-nucleotide insertion, and double heterozygous non-synonymous variants. Boxes indicate conserved amino acids across multiple mammalian species. The WW domain interacts with the PXXP region to modify the three-dimensional structure of BAG3 and is also involved in chaperone-mediated selective autophagy (CASA); IPV motifs interact with the small heat shock proteins HspB8 and HspB6 to support macroautophagy; the PXXP region facilitates binding to the Src homology 3 (SH3) domain to promote malignant cell migration and metastasis and binds the dynein complex to promote intracellular protein transport to perinuclear aggresomes; and the BAG domain interacts with Bcl-2 to inhibit apoptosis.

[0062] FIGS. 2A-2C are Kaplan-Meier curves showing event-free survival in patients with or without the BAG3 genetic variant. FIG. 2A shows event-free survival in all patients with or without the BAG3 variant. FIG. 2B shows event-free survival in patients with non-ischemic HF with or without the BAG3 variant. FIG. 2C shows event-free survival in patients with ischemic dilated cardiomyopathy. Statistical significance is indicated for each comparison. The number of patients in each group is indicated on the x-axis.

[0063] FIG. 3A shows representative confocal images from AC16 cells that were transfected with an empty plasmid or a plasmid containing wild-type BAG3 or c.187C>G+c.1138C>T (p.Pro63Ala+p.Pro380Ser), c.249C>A (p.His83Gln), c.474_476dupGGC (p.Ala160dup), or c.1436C>T (p.Ala479Val) and cotransfected with Adv-RFP-GFP-LC3. Red dots represent increased LC3 in autophagolysosomes in which GFP was suppressed by increased acidity following fusion of lysosomes and autophagosomes. FIG. 3B shows quantification of images from 8 to 42 cells per condition. FIG. 3C is a representative confocal image of AC16 cells that were stained with Annexin-V (green) and propidium iodide (red) to distinguish viable cells from apoptotic cells. Apoptotic cells appear green, late apoptotic and/or necrotic cells appear red, and nonviable cells appear green and red. FIG. 3D is a quantification of images from 200 cells per condition, representing the number of cells that were neither green nor red.

[0064] FIG. 4A is a representative Western blot of protein isolated from hearts of individuals with severe left ventricular dysfunction secondary to idiopathic dilated cardiomyopathy (n=23) or ischemic heart disease (n=16) or non-failure controls (n=4). Samples obtained from hearts found to contain the BAG3 variant are indicated by red squares numbered a through c. FIG. 4B shows quantification of multiple Western blots. FIG. 4C shows quantification of qPCR for BAG3 in hearts of individuals with ischemic dilated cardiomyopathy with heart failure (n=13), iCDCM (n=18), and healthy hearts (n=4) with GAPDH as an internal control.

[0065] Figures 5A-5C show representative Western blots of BAG3 levels in human heart with heart failure (Figure 5A), quantification of multiple Western blots (Figure 5B), and quantification of mRNA levels in human heart with heart failure (Figure 5C). The data show that in patients without a BAG3 mutation and with idiopathic dilated cardiomyopathy or ischemic cardiomyopathy, BAG3 levels are consistently lower than in healthy human hearts. In the case of an in-frame insertion of A160Adup, BAG3 levels are also low. In contrast, BAG3 levels are actually elevated in both P63A+P380S (double heterozygote with variants in cis) and Ala479Val, suggesting that addition of exogenous BAG3 via gene therapy may not have a beneficial effect on this genotype.

[0066] Fig. 6A, 6B are graphs showing cardiac function with BAG3 haploinsufficiency and WtBAG3 restoration. To assess the functional effects of common (>1%) genetic variants of BAG3, mice were infected with BAG3 haploinsufficiency secondary to a deletion on the BAG3 allele using adeno-associated virus (AAV) serotype 9, resulting in transfection of either wild-type BAG3 or a mutated form of BAG3. Because mice with a deletion of one BAG3 allele had half the amount of BAG3 found in wild-type mice, normal BAG3 levels were exceeded upon AAV-BAG3 infection and ameliorated the left ventricular dysfunction found in mice lacking one BAG3 allele (cBAG3 +/-). The same mice were injected with the AAV9 mutant allele. Mice underwent echocardiogram every two weeks to determine left ventricular function. FIG. 6A shows serial measurements of left ventricular function after injection of AAV9-GFP (control) into cBAG3+/+ or cBAG3+/− mice. cBAG3+/+ mice demonstrated normal contractility in the presence of AAV9-GFP, whereas cBAG3+/− mice demonstrated a progressive decline in left ventricular function, consistent with an earlier report from our group. FIG. 6B shows the effects of AAV9-BAG3 challenge in cBAG3+/− mice compared to normal cBAG3+/+ mice. Here, we show for the first time that restoration of normal BAG3 levels with AAV9-BAG3 can ameliorate the decline in BAG3 levels observed in cBAG3+/− mice, which lack one BAG3 allele.

[0067] FIGS. 7A, 7B are graphs showing cardiac function with p.Pro63Ala+Pro380Ser expression. To assess the functional effects of the common p.Pro63Ala+Pro380Ser genetic variant, mice were infected with AAV9-BAG3-WT or AAV9-BAG3-63/380. FIG. 7A shows the effect of aAV9-BAG3-63/380 on left ventricular function in cBAG3 +/- mice compared to cBAG3 +/+ mice (control). Injection of AAV9-BAG3-63/380 resulted in a significant decrease in left ventricular function, a decrease that was greater than that caused by deletion of the BAG3 allele alone. FIG. 7B shows that injection of cBAG3-63/380 into a normal BAG3 (+/+) genotype mouse results in left ventricular function that is worse than that of a wild-type mouse, but significantly better than that of a cBAG3 +/- mouse injected with AAV9-BAG3-63/380. Together, these results strongly support the hypothesis that administration of AAV9-BAG3-WT to mice carrying the cBAG3-63/380 mutation is likely to result in a predominantly negative effect and exacerbate the decline in left ventricular function. Thus, the main conclusion is that patients undergoing BAG3 gene therapy should have their BAG3 gene sequenced to optimize therapy.

[0068] FIGS. 8A, 8B are graphs showing cardiac function with p.Ala479Val expression. To assess the functional effects of Ala479Val, AAV9-BAG3-Ala479Val was injected into the retro-orbital space. FIG. 8A: Unlike injection with AAV9-BAG3-63/380, retro-orbital injection of AAV9-BAG3-A479V did not cause adverse effects in mice with the normal genotype (BAG3 +/+) or with the 479 mutation (A479V). FIG. 8B: Similarly, injection of cBAG3-A479V, unlike injection with AAV9-BAG3-63/380, did not adversely affect left ventricular function. Thus, it is unlikely that the use of A479V will cause a predominantly negative effect when treating patients with heart failure and low BAG3 levels.

DETAILED DESCRIPTION

[0069] The following description of specific embodiments is merely exemplary in nature and is not intended to limit the invention, its application, or its use in any way. Embodiments of the invention may be practiced without the theoretical aspects presented. Furthermore, the theoretical aspects are presented with the understanding that applicants do not seek to be bound by the theory presented.

[0070] The invention is based in part on the discovery that the presence of unique genetic variants in BAG3, found almost exclusively in African Americans, predicts an almost 2-fold greater risk of adverse outcome. In addition, the importance of assessing the pathogenicity of genetic variants and, where possible, their association with protein levels in the human heart is demonstrated. Thus, BAG3 mutations play an important role in cardiovascular pathobiology in individuals of African descent.

[0071] Bcl-2 associated anthanogen-3 (BAG3), also known as BCL2-associated anthanogen 3; MFM6; Bcl-2 binding protein Bis; CAIR-1; CAIR-1 docking protein; BAG family chaperone molecular regulator 3; BAG-3; BCL2-binding anthanogen 3; or BIS, is a cytoprotective polypeptide that competes with Hip-1 for binding to HSP 70. The NCBI reference amino acid sequence for BAG3 can be found in Genbank under accession number NP_004272.2; Public GI:14043024. The amino acid sequence of GeneBank accession number NP_004272.2; Public GI:14043024 is provided herein as SEQ ID NO:1. The NCBI reference nucleic acid sequence for BAG3 can be found in Genbank under accession number NM_004281.3 GI:62530382. The nucleic acid sequence of GeneBank accession number NM_004281.3 GI:62530382 is provided as SEQ ID NO: 2. Other amino acid sequences of BAG3 include, for example, but are not limited to, 095817.3 GI:12643665 (SEQ ID NO: 3); EAW49383.1 GI:119569768 (SEQ ID NO: 4); EAW49382.1 GI:119569767(SEQ ID NO: 5); and CAE55998.1 GI:38502170 (SEQ ID NO: 6). The BAG3 polypeptide of the invention may be a variant of the polypeptide described herein, provided that it retains functionality.

[0072] Compositions

[0073] Genetic variants in more than forty genes have been associated with DCM. One such gene encodes BAG3 (Bcl-2 associated anthanogen-3), an evolutionarily conserved protein that is expressed predominantly in the heart, skeletal muscle, and many malignancies. BAG3 has pleiotropic effects in the heart: it inhibits apoptosis, promotes autophagy, mediates excitation-contraction coupling, maintains sarcomere integrity ^17,18 , and has emerged as a major locus for DCM. ¹⁹ Several observations suggest that BAG3 mutations may be prevalent in African Americans: 1) BAG3 genetic variants are most common in isolated populations ²⁰ ; and 2) the genomic aggregation database (gnomAD) shows marked differences between the frequency of BAG3 allelic variants commonly found in individuals of African descent compared with the frequency of BAG3 allelic variants in individuals of European descent. ²¹ Without wishing to be bound by any particular theory, it has been hypothesized that BAG3 variants may contribute to the increased prevalence of IDCM in African Americans. As detailed in the Examples section, this is the first time that a group of functional BAG3 variants, found, with rare exceptions, in individuals of African descent, have been found to be associated with poor outcomes in patients with IDCM and may serve as a precise target for therapeutic intervention.

[0074] Thus, in certain embodiments, a method of diagnosing and treating a patient having a cardiac disease comprises identifying in the patient at least one genetic variant of Bcl2-associated anthanogen 3 (BAG3) compared to a reference BAG3 nucleic acid sequence, wherein detection of the identified variants allows for the prediction of whether an increase in BAG3 levels is therapeutic for the patient, and administering to a patient identified as having such a variant a therapeutically effective amount of an agent, wherein the agent modulates the expression or amount of BAG3 molecules, proteins, or peptides in a target cell or target tissue, compared to a healthy control.

[0075] In certain embodiments, the genetic variant is a single nucleotide variant (SNV) such as an in-frame insertion, deletion, substitution, or combinations thereof. In certain embodiments, the SNVs include: p.Pro63Ala (10:121429369 C/G; rs133031999); p.His83Gln (10:151331972; rs151331972); Pro380Ser (10:121436204 C/T; rs144692954); Ala479Val (10:121436502 C/T, rs34656239), or combinations thereof. In certain embodiments, the in-frame insertion encodes an amino acid. In certain embodiments, the in-frame insertion encodes a non-polar amino acid. In certain embodiments, the in-frame insertion encodes an alanine at position 160.

[0076] In additional embodiments, a method of treating a patient suffering from a cardiac disease, wherein the patient has at least one nucleotide variant of a Bcl2-associated anthanogen 3 (BAG3) in-frame insertion type compared to a BAG3 reference nucleic acid sequence, comprises administering to the patient a therapeutically effective amount of an agent, wherein the agent modulates the expression or amount of BAG3 molecules, proteins, or peptides in a target cell or target tissue.

[0077] In certain embodiments, the in-frame insertion encodes an amino acid. In certain embodiments, the in-frame insertion encodes a non-polar amino acid. In certain embodiments, the in-frame insertion includes a three-nucleotide insertion that adds an alanine at position 160 (p.Ala160dup, 10: 121429647 A/AGCG; rs139438727).

[0078] In some embodiments, a means for treating a patient having IDCM and identified as having a genetic variant in the BCL2-associated anthanogene 3 (BAG3) gene, wherein detection of certain variants is a predictor of whether increasing BAG3 levels is therapeutic for the patient.

[0079] According to certain embodiments, a therapeutic agent for treating diseases associated with BAG3 and related molecules and pathways thereof modulates the expression or amount of BAG3 in a cell in individuals. In some embodiments, the composition comprises BCL2-associated anthanogen 3 (BAG3) nucleic acid sequences, including, but not limited to, cDNA, sense, and/or antisense sequence of BAG3.

[0080] In certain embodiments, the agent comprises one or more gene editing systems or a nuclease system for removing or editing genetic variants in individuals where an increase in BAG3 is not therapeutic or may even be detrimental to the individual.

[0081] Any suitable nuclease system can be used, including, for example, clustered regularly interspaced short palindromic repeats (CRISPR) nucleases, the Argonaute family of endonucleases, zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), meganucleases, and other endo- or exo-nucleases, or combinations thereof. See Schiffer, 2012, J Virol 88(17):8920-8936, incorporated by reference. In some embodiments, the system is an Argonaute nuclease system.

[0082] CRISPR/Cas: In certain embodiments, the gene editing means includes a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas endonuclease (CRISPR/Cas).

[0083] In some embodiments, CRISPR/Cas comprises a catalytically deficient Cas (dCas) protein, orthologs, homologs, mutant variants, or fragments thereof.

[0084] The compositions described herein may include nucleic acids encoding a CRISPR-associated endonuclease, such as Cas9. In bacteria, CRISPR/Cas loci encode an RNA-guided adaptive immune system against mobile genetic elements (viruses, transposons, and conjugative plasmids). Three types (I-III) of CRISPR systems have been identified. CRISPR clusters contain spacers, sequences complementary to preceding mobile elements. CRISPR clusters are transcribed and processed into mature CRISPR RNA (mRNA). The CRISPR-associated endonuclease, Cas9, belongs to the type II CRISPR/Cas system and has strong endonuclease activity for cutting target DNA. Cas9 is guided by a mature cpRNA, which contains approximately 20 base pairs (bp) of a unique target sequence (called a spacer), and a trans-activated small RNA (tracpRNA), which serves as a guide for processing of the pre-cpRNA by ribonuclease III. The cpRNA:tracpRNA duplex guides Cas9 to the target DNA through complementary base pairing between the spacer on the cpRNA and a complementary sequence (called a protospacer) on the target DNA. Cas9 recognizes a trinucleotide (NGG) motif adjacent to the protospacer (PAM) to define the cut site (3rd nucleotide from the PAM). cpRNA and tracрRNA can be expressed separately or engineered into an artificial small guide RNA (sgRNA) fusion via a synthetic stem loop (AGAAAU) to mimic the natural cpRNA:tracрRNA duplex. Such sgRNA as shRNA can be synthesized or transcribed in vitro for direct RNA transfection or expressed from an RNA expression vector with a U6 or H1 promoter, although the cleavage efficiency of the artificial sgRNA is lower than in systems with cpRNA and tracрRNA expressed separately.

[0085] In embodiments, the CRISPR/Cas system may be a Type I, Type II, or Type III system. Non-limiting examples of suitable CRISPR/Cas proteins include Cas9, CasX, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, spCas, eSpCas, SpCas9-HF1, SpCas9-HF2, SpCas9-HF3, SpCas9-HF4, ARMAN 1, ARMAN 4, Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4 and Cu1966.

[0086] Cas9 may be an ortholog. Six smaller Cas9 orthologs have been used, and reports have shown that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with similar efficiency to SpCas9, but is more than 1 kilobase shorter.

[0087] In addition to the described wild-type and variant Cas9 endonucleases, embodiments of the invention also include CRISPR systems, including recently developed “enhanced specificity” S. pyogenes Cas9 (eSpCas9) variants that dramatically reduce off-target cleavage. These variants are engineered with alanine substitutions to neutralize positively charged sites in the groove that interacts with the off-target DNA strand. The purpose of this modification is to reduce the interaction of Cas9 with the off-target strand, thereby promoting re-hybridization between the target and off-target strands. The effect of this modification is to require tighter Watson-Crick pairing of the gRNA and the target DNA strand, which limits off-target cleavage (Slaymaker, IM et al. (2015) DOI: 10.1126/science.aad5227).

[0088] In some embodiments, the compositions utilize three variants that exhibit the best cleavage efficiency and the least off-target activity: SpCas9 (K855A), SpCas9 (K810A/K1003A/R1060A) (also known as eSpCas9 1.0), and SpCas9 (K848A/K1003A/R1060A) (also known as eSPCas9 1.1). The invention is in no way limited to these variants, but also encompasses all Cas9 variants (Slaymaker, I.M. et al. (2015)). The present invention also relates to another type of Cas9 variant with increased specificity, a “high-fidelity” spCas9 variant (HF-Cas9). Examples of high-fidelity variants include SpCas9-HF1 (N497A/R661A/Q695A/Q926A), SpCas9-HF2 (N497A/R661A/Q695A/Q926A/D1135E), SpCas9-HF3 (N497A/R66116A/Q926A/L169A), SpCas9-HF4 (N497A/R661A/Q695A/Q926A/Y450A). Also included are all SpCas9 variants containing all possible single, double, triple and quadruple combinations of N497A, R661A, Q695A, Q926A or any other substitutions (Kleinstiver, B.P. et al., 2016, Nature. DOI: 10.1038/nature16526).

[0089] As used herein, the term "Cas" includes all Cas molecules containing variants, mutants, orthologs, high-fidelity variants, etc.

[0090] In one embodiment, the endonuclease is derived from a Type II CRISPR/Cas system. In other embodiments, the endonuclease is derived from a Cas9 protein and includes Cas9, CasX, CasY.1, CasY.2, CasY.3, CasY.4, CasY.5, CasY.6, spCas, eSpCas, SpCas9-HF1, SpCas9-HF2, SpCas9-HF3, SpCas9-HF4, ARMAN 1, ARMAN 4, mutants, variants, high-fidelity variants, orthologs, analogs, fragments, or combinations thereof. Cas9 protein can originate from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacreria, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermoprorionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina. Also included are Cas9 proteins encoded by the genomes of nanoarchaeum ARMAN-1 (Candidatus Micrarchaeum acidiphilum ARMAN-1) and ARMAN-4 (Candidatus Parvarchaeum acidiphilum ARMAN-4), CasY (Kerfeldbacteria, Vogelbacteria, Komeilibacteria, Katanobacteria), CasX (Planctomycetes, Deltaproteobacteria).

[0091] Generally, CRISPR/Cas proteins contain at least one RNA recognition and/or RNA binding domain. The RNA recognition and/or RNA binding domain interacts with the guide RNA. CRISPR/Cas proteins may also include nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNase domains, protein-protein interaction domains, dimerization domains, and other domains. Active DNA-targeting CRISPR-Cas utilize 2-4 nucleotide protospacer adjacent motifs (PAMs) located near target sequences to recognize self versus non-self. ARMAN-1 has a strong preference for the "NGG" PAM. Cas9 also uses two separate transcripts, CRISPR RNA (cpRNA) and trans-activating CRISPR RNA (tracрRNA), for RNA-guided DNA cleavage. The putative tracрRNA was found in close proximity to the ARMAN-1 and ARMAN-4 CRISPR-Cas9 systems.

[0092] Embodiments of the invention also include a novel class 2 CRISPR-Cas system found in the genomes of two bacteria isolated from groundwater and sediment samples. This system includes Cas1, Cas2, Cas4, and a protein of approximately 980 amino acids designated CasX. The high conservation (68% protein sequence identity) of this protein in two organisms belonging to different phyla, Deltaproteobacteria and Planctomycetes, suggests a recent transfer between the phyla. The CRISPR repeats associated with each CasX have highly similar repeats (86% identity) of 37 nucleotides (nt), spacers of 33-34 nt, and a putative tracрRNA between the Cas operon and the CRISPR repeats. Using distant homology detection and protein modeling, we identified a RuvC domain near the C-terminus of CasX, with an organization reminiscent of that found in type V CRISPR-Cas systems. The remainder of the CasX protein (630 N-terminal amino acids) showed no similarity to any known proteins, suggesting that it is a novel class 2 effector. The combination of tracрRNA and individual Cas1, Cas2, and Cas4 proteins is unique among type V systems, and phylogenetic analysis shows that the Cas1 of the CRISPR-CasX system is distant from any other known type V protein. Furthermore, CasX is significantly smaller than any known type V protein: 980 amino acids, compared to the typical size of approximately 1200 amino acids for Cpf1, C2c1, and C2c3 (Burstein, D. et al., 2016 above).

[0093] In some embodiments, the BAG3 nucleic acid sequence comprises at least about 50% sequence identity to a wild-type BAG3 sequence or its cDNA sequence. In other embodiments, the BAG3 nucleic acid sequence comprises at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a wild-type BAG3 sequence or its cDNA sequence.

[0094] In some embodiments, the BAG3 nucleic acid sequence further comprises one or more mutations, substitutions, deletions, variants, or combinations thereof.

[0095] In some embodiments, the homology, sequence identity, or complementarity between a BAG3 nucleic acid sequence comprising one or more mutations, substitutions, deletions, variants, or combinations thereof and a native or wild-type or cDNA BAG3 sequence is between about 50% and about 60%. In some embodiments, the homology, sequence identity, or complementarity is between about 60% and about 70%. In some embodiments, the homology, sequence identity, or complementarity is between about 70% and about 80%. In some embodiments, the homology, sequence identity, or complementarity is between about 80% and about 90%. In some embodiments, the homology, sequence identity, or complementarity is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0096] In cases where the variants detected in a sample predict that an increase in the level or activity of BAG3 would be therapeutic, the individual is administered an agent containing BAG3 molecules. In one embodiment, the expression vector encodes a BCL2-associated anthanogen 3 (BAG3) gene or its cDNA sequences or modified sequences thereof. In one embodiment, the expression vector encodes a nucleic acid sequence comprising at least about 50% sequence identity with the wild-type BCL2-associated anthanogen 3 (BAG3) sequences or its cDNA. In other embodiments, the nucleic acid sequence comprises at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with the wild-type BCL2-associated anthanogen 3 (BAG3) sequences or its cDNA.

[0097] A suitable nucleic acid-based delivery system comprises a viral vector, typically a sequence from at least one of an adenovirus, an adenovirus-associated virus (AAV), a helper-dependent adenovirus, a retrovirus, or a hemagglutinating Japanese virus-liposome complex (HVJ). As a specific example, the viral vector comprises a strong eukaryotic promoter operably linked to a polynucleotide, such as a cytomegalovirus (CMV) promoter.

[0098] If desired, the polynucleotides of the invention can also be used with a microdelivery vehicle, such as cationic liposomes and adenoviral vectors. For a review of methods for producing liposomes, targeting, and delivering contents, see Mannino and Gould-Fogerite, BioTechniques, 6: 682 (1988). See also, Felgner and Holm, Bethesda Res. Lab. Focus, 11 (2): 21 (1989) and Maurer, R. A., Bethesda Res. Lab. Focus, 11 (2): 25 (1989).

[0099] Replication-defective adenoviral recombinant vectors can be prepared according to known methods. See, Quantin, et al., Proc. Natl. Acad. Sci. USA, 89:2581-2584 (1992); Stratford-Perricadet, et al., J. Clin. Invest., 90:626-630 (1992); and Rosenfeld, et al., Cell, 68:143-155 (1992).

[0100] Another delivery method is the use of single-stranded DNA producing vectors that can produce BAG3 intracellularly, such as in cardiac tissue. See, for example, Chen et al., BioTechniques, 34: 167-171 (2003), which is incorporated herein by reference in its entirety.

[0101] Expression of BAG3 can be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression. In some embodiments, the promoter is a tissue-specific promoter. Of particular interest are muscle-specific promoters, and more particularly, cardiac-specific promoters. These include the myosin light chain 2 promoter (Franz et al. (1994) Cardioscience, Vol. 5 (4): 235–43; Kelly et al. (1995) J. Cell Biol., Vol. 129 (2): 383–396), the alpha-actin promoter (Moss et al. (1996) Biol. Chem., Vol. 271 (49): 31688–31694), and the troponin 1 promoter (Bhaysar et al. (1996) Genomics, Vol. 35 (1): 11–23); Na+/Ca2+ exchange protein promoter (Barnes et al. (1997) J. Biol. Chem., Vol. 272 (17): 11510–11517), dystrophin promoter (Kimura et al. (1997) Dev. Growth Differ., Vol. 39 (3): 257–265), alpha7-integrin promoter (Ziober and Kramer (1996) J. Bio. Chem., Vol. 271 (37): 229–15–22), brain natriuretic peptide promoter (LaPointe et al. (1996) Hypertension, Vol. 27 (3Pt2): 715–22), and alpha B-crystallin/small heat shock protein promoter (Gopal-Srivastava (1995) J. Mol. Cell. Biol., Vol. 15 (12): 7081–7090), the alpha-myosin heavy chain promoter (Yamauchi-Takihara et al. (1989) Proc. Natl. Acad. Sci. USA, Vol. 86 (10): 3504–3508), and the ANF promoter (LaPointe et al. (1988) J. Biol. Chem., Vol. 263 (19): 9075–9078).

[0102] Yeast-based expression systems can also be used according to the invention to express BAG3. For example, to mention only two, the non-fusion vector pYES2 (with cloning sites XbaI, SphI, Shol, NotI, GstXI, EcoRI, BstXI, BamH1, SacI, KpnI and HindIII; Invitrogen) or the fusion pYESHisA, B, C (cloning sites XbaI, SphI, Shol NotI, BstXI, EcoRI, BamH1, SacI, KpnI and HindIII, N-terminal peptide purified with ProBond resin and digested with enterokinase; Invitrogen) can be used according to the invention. A yeast-based two-hybrid expression system can be prepared according to the invention.

[0103] One example of a delivery system is a recombinant viral vector that includes one or more polynucleotides, such as about one polynucleotide. A typical viral vector used in the methods of the invention has a pfu (plaque forming units) of about 10 ⁸ to about 5 x 10 ¹⁰ pfu. In embodiments in which the polynucleotide is to be administered with a non-viral vector, it will often be useful to use from about 0.1 nanogram to about 4000 micrograms, such as from about 1 nanogram to about 100 micrograms.

[0104] In some embodiments, the vector is an adenovirus-associated viral vector (AAV), such as AAV9. The term "AAV vector" means a vector derived from an adeno-associated serotype, including, but not limited to, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, and AAV-8. The AAV may have one or more wild-type AAV genes deleted in whole or in part, such as the rep and/or cap genes, but retaining functional flanking ITR sequences. Despite the high degree of homology, different serotypes have different tissue tropisms. The receptor for AAV1 is unknown; however, AAV1 is known to transduce skeletal and cardiac muscle more efficiently than AAV2. Since most studies have been performed using a pseudotype in which vector DNA flanked by the AAV2 ITR is packaged into capsids of alternative serotypes, it is apparent that the biological differences are related to the capsid rather than the genomes. Recent data indicate that DNA expression cassettes packaged into AAV1 capsids are at least 1 log10 more efficient in transducing cardiomyocytes than those packaged into AAV2 capsids. In one embodiment, the viral delivery system is an adeno-associated viral delivery system. Adeno-associated virus can be serotype 1 (AAV1), serotype 2 (AAV2), serotype 3 (AAV3), serotype 4 (AAV4), serotype 5 (AAV5), serotype 6 (AAV6), serotype 7 (AAV7), serotype 8 (AAV8), or serotype 9 (AAV9).

[0105] Some skilled in the art have circumvented some of the limitations of the adenovirus-directed interface by using adenovirus "hybrid" viruses that incorporate desirable properties of adenovirus as well as other types of viruses as a way to create unique vectors with highly specialized properties. For example, chimeras of viral vectors without adenovirus and adeno-associated virus (AAV). These aspects of the invention do not deviate from the scope of the invention described herein.

[0106] Nucleic acids encoding the BAG3 proteins of the invention can be delivered to cardiac muscle by methods known in the art. For example, cardiac cells of a large mammal can be transfected by a method that includes dilating a blood vessel of the coronary circulation by administering a vasodilator to said mammal prior to and/or simultaneously with administration of the nucleic acids. In some embodiments, the method includes administering nucleic acids into a blood vessel of the coronary bed in vivo, wherein the nucleic acids are infused into the blood vessel for a period of at least about three minutes, wherein the coronary circulation is not isolated or is substantially isolated from the systemic circulation of the mammal, and wherein the nucleic acids transfect cardiac cells of the mammal.

[0107] In some embodiments, the individual may be a human, an experimental animal such as a rat or mouse, a domestic animal such as a dog, cow, sheep, pig, or horse, or a non-human primate such as a monkey. The individual may have a cardiac disorder such as heart failure, ischemia, myocardial infarction, congestive heart failure, arrhythmia, transplant rejection, and the like. In one embodiment, the individual suffers from heart failure. In another specific embodiment, the individual suffers from arrhythmia. In one embodiment, the individual is a human. For example, the individual is between the ages of 18 and 65. In another embodiment, the individual is a non-human animal.

[0108] In one embodiment, the individual has heart failure or is at risk for heart failure, such as nonischemic cardiomyopathy, mitral valve regurgitation, ischemic cardiomyopathy, or aortic stenosis or regurgitation.

[0109] In some embodiments, transfection of a cardiac cell with nucleic acid molecules encoding a BAG3 protein or a BAG3 protein fused to an effector domain increases the fractional shortening of the lateral ventricle. In some embodiments, the mammal is a human and the disease is congestive heart failure. In some embodiments, transfection of the cardiac cells increases the fractional shortening of the lateral ventricle, as measured at about 4 months after said infusion, by at least 25% compared to the fractional shortening of the lateral ventricle prior to infusion of the polynucleotide. In some embodiments, transfection of the cardiac cells results in an improvement in a cardiac function indicator selected from the group consisting of BAG3 protein expression, fractional shortening, ejection fraction, cardiac output, ventricular relaxation time constant, and regurgitant volume.

[0110] Treatment can be assessed by assessing the effect of treatment on a parameter related to contractility. For example, sarcoplasmic reticulum Ca2+-ATPase activity or intracellular Ca2+ concentration can be measured. In addition, force production by the heart or cardiac tissue can be measured using the methods described in Strauss et al., Am. J. Physiol., 262: 1437-45, 1992, the contents of which are incorporated herein by reference.

[0111] Modified Nucleic Acid Sequences: It should be understood that the present invention is not limited by the nature of the nucleic acid used. The nucleic acid may be DNA or RNA and may exist in double-stranded, single-stranded, or partially double-stranded form.

[0112] Nucleic acids useful in the present invention include, by way of example and without limitation, oligonucleotides and polynucleotides such as antisense DNA and/or RNA; ribozymes; shRNA; inhibitory nucleic acids; DNA for gene therapy; viral fragments, including viral DNA and/or RNA; chimeric DNA and/or RNA; mRNA; plasmids; cosmids; genomic DNA; cDNA; gene fragments; various structural forms of DNA, including single-stranded DNA, double-stranded DNA, supercoiled DNA and/or triple-helix DNA; Z-DNA; and the like. Nucleic acids can be prepared by any conventional methods typically used to prepare nucleic acids in large quantities. For example, DNA and RNA can be synthesized chemically using commercially available reagents and synthesizers by methods well known in the art (see, e.g., Gait, 1985, Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, England)). RNA can be produced in high yield by in vitro transcription using plasmids such as the pGEM™ T or SP65 vector (Promega Corporation, Madison, Wis.).

[0113] Thus, some nucleic acid sequences of the present invention are chimeric nucleic acid sequences. "Chimeric nucleic acid sequences" or "chimeras" in the context of the present invention comprise two or more chemically distinct regions, each of which consists of at least one nucleotide. These sequences typically comprise at least one region of modified nucleotides that confers one or more advantageous properties (such as, for example, increased nuclease resistance, increased cellular uptake, increased binding affinity for a target).

[0114] The chimeric nucleic acid sequence of the invention can be formed as combined constructs of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics. Such compounds are also referred to in the art as hybrids or gapmers. Primary U.S. patents that comprise the production of such hybrid structures include, but are not limited to, U.S. Patent Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is incorporated herein by reference.

[0115] Specific examples of some modified nucleic acid sequences provided for in this invention include those that contain modified backbones, such as with thiophosphates, phosphotriesters, methylphosphonates, short chain alkyl or cycloalkyl intersugar linkages, or short chain heteroatom or heterocyclic intersugar linkages. In some embodiments, modified oligonucleotides include oligonucleotides with a phosphorothioate backbone and oligonucleotides with a heteroatom backbone, _CH2 -NH-O- _CH2 , CH,--N( _CH3 )--O-- _CH2 [known as a methylene (methylimino) or MMI backbone], _CH2 --O--N( _CH3 )-- _CH2 , CH2-- _N (CH3)--N( _{CH3 )} -- _CH2 , and O--N( _CH3 )-- _CH2 -- _CH2 backbones, wherein the natural phosphodiester backbone is represented as O--P--O--CH. The amide backbones described in De Mesmaeker et al. Acc. Chem. Res. 1995, 28:366-374 are also included herein. In some embodiments, nucleic acid sequences having morpholine backbone structures (Summerton and Weller, U.S. Patent No. 5,034,506), a peptide nucleic acid (PNA) backbone wherein the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone, wherein the nucleobases bind directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al. Science, 19912541497). The nucleic acid sequences may also include one or more substituted sugar groups. The nucleic acid sequences may also contain sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.

[0116] Examples of modified oligonucleotide backbones include, but are not limited to, backbones containing thiophosphates, chiral thiophosphates, phosphodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates containing 3'-aminophosphoramidate and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters and boranophosphates with conventional 3'-5' linkages, their 2'-5'-linked analogs, and those having reverse polarity where adjacent pairs of nucleoside units are linked from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms.

[0117] Examples of modified oligonucleotide backbones that do not contain a phosphorus atom have backbones formed by short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatom or heterocyclic internucleoside linkages. These include backbones that have morpholine linkages (derived in part from the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimine and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones having mixed N, O, S, and _CH2 component parts.

[0118] Nucleic acid sequences may also include, additionally or alternatively, modifications or substitutions of a nucleobase (often referred to in the art simply as a "base"). As used herein, "unmodified" or "natural" nucleotides include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleotides include nucleotides that are infrequently or transiently found in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidines, especially 5-methylcytosine (also referred to as 5-methyl-2'deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC, and gentobiosyl HMC, as well as synthetic nucleotides, such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenes, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine and 2,6-diaminopurine. (Kornberg, A., DNA Replication, W.H. Freeman & Co., San Francisco, 1980, pp 75-77; Gebeyehu, G., (1987) et al. Nucl. Acids Res. 15:4513). A "universal" base known in the art, such as inosine, may be included.

[0119] Another modification includes chemically linking to the oligonucleotide one or more groups or conjugates that enhance the activity or cellular uptake of the oligonucleotide. Such groups include, but are not limited to, lipid groups such as a cholesterol group, a cholesteryl group, cholic acid, a thioester such as hexyl-5-tritylthiol, an aliphatic chain such as dodecanediol or undecyl residues, a phospholipid such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or polyethyleneglycol chain, or adamantaneacetic acid. Nucleic acid sequences containing lipophilic groups and methods for producing such oligonucleotides are known in the art, for example, U.S. Patent Nos. 5,138,045, 5,218,105, and 5,459,255.

[0120] It is not necessary that all positions in a given nucleic acid sequence be uniformly modified, and in fact more than one of the above modifications may be included in a single nucleic acid sequence or even within a single nucleoside within such a sequence. The present invention also relates to oligonucleotides that are chimeric oligonucleotides as defined above.

[0121] In another embodiment, a BAG3 nucleic acid molecule of the present invention is conjugated to another moiety, including, but not limited to, basic nucleotides, polyether, polyamine, polyamides, peptides, carbohydrates, lipid, or polyhydrocarbon compounds. Those skilled in the art will appreciate that these molecules can be linked to any one or more nucleotides, including multiple positions in the nucleic acid molecule, by a sugar, basic, or phosphate group.

[0122] In another embodiment, the BAG3 nucleic acid sequences comprise one or more nucleotides substituted with closed nucleic acids (LNAs). The nucleic acid sequences modified with LNAs may be similar in size to the parent or native sequence, or may be larger or smaller. Such LNA-modified oligonucleotides may comprise less than about 70%, or less than about 60%, or less than about 50% LNA monomers, and their sizes range from about 1 to 25 nucleotides.

[0123] Antisense BAG3 Oligonucleotides: In another embodiment, it may be desirable to reduce the expression of BAG3 containing one or more variants that may be predictive of a therapeutic outcome of a cell or patient by having the oligonucleotides modulate the expression of BAG3, such as transcriptional regulatory elements.

[0124] In one embodiment, the oligonucleotide comprises at least five consecutive bases complementary to a nucleic acid sequence, wherein the oligonucleotide specifically hybridizes to modulate the expression of BAG3 in vivo or in vitro. In another embodiment, the oligomeric compounds of the present invention also include variants in which another base is present at one or more nucleotide positions in the compound. For example, if the first nucleotide is adenosine, variants can be made that contain thymidine, guanosine, or cytidine at that position. This can be done at any of the positions in the oligonucleotide. These compounds are then tested using the methods described herein to determine their ability to inhibit the expression of a target nucleic acid.

[0125] In some embodiments, the homology, sequence identity, or complementarity between the oligonucleotide and the target is between about 50% and about 60%. In some embodiments, the homology, sequence identity, or complementarity is between about 60% and about 70%. In some embodiments, the homology, sequence identity, or complementarity is between about 70% and about 80%. In some embodiments, the homology, sequence identity, or complementarity is between about 80% and about 90%. In some embodiments, the homology, sequence identity, or complementarity is about 90%, about 92%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0126] In another embodiment, the oligonucleotide comprises combinations of thiophosphate internucleotide linkages and at least one internucleotide linkage selected from the group consisting of alkyl phosphonate, phosphorodithioate, alkyl phosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and/or combinations thereof.

[0127] In another embodiment, the oligonucleotide optionally comprises at least one modified nucleobase comprising peptide nucleic acid molecules, locked nucleic acid (LNA), analogs, derivatives, and/or combinations thereof.

[0128] An oligonucleotide is capable of specifically hybridizing when binding of the compound to a target nucleic acid interferes with the normal function of the target nucleic acid, resulting in loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target nucleic acid sequences under conditions under which specific binding is required. Such conditions include, for example, physiological conditions in the case of in vivo assays or therapeutic treatments, and conditions under which assays are performed in the case of in vitro assays.

[0129] An oligonucleotide, whether DNA, RNA, chimeric, substituted, etc., is capable of specifically hybridizing when binding of the compound to a target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, rendering it unusable, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target nucleic acid sequences under conditions in which specific binding is required, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, and in the case of in vitro assays, under the conditions in which the assays are performed.

[0130] The specificity and sensitivity of antisense oligonucleotides are also being evaluated by those skilled in the art for therapeutic purposes. Antisense oligonucleotides have been used as therapeutic groups in the treatment of disease states in animals and humans. Antisense oligonucleotides have been safely and effectively administered to humans, and numerous clinical trials are currently underway. Thus, it has been established that oligonucleotides can be useful therapeutic agents that can be customized for use in treatment regimens for the treatment of cells, tissues, and animals, particularly humans.

[0131] In embodiments of the present invention, oligomeric oligonucleotides, in particular oligonucleotides, bind to target nucleic acid molecules and modulate the expression of molecules encoded by a BAG3 gene containing one or more variants, which either benefits from an increase in the level of BAG3, or in the case of a deletion, substitution or some other mechanism, where the agent used will be therapeutically useful for this particular individual. The DNA functions that must be interfered with include, for example, replication and transcription. The RNA functions that must be interfered with include all vital functions such as, for example, translocation of RNA to the site of protein translation, translation of protein from RNA, splicing of RNA to form one or more mRNA species and catalytic activity in which RNA may be involved or facilitated by RNA. The functions can be enhanced or blocked depending on the desired functions.

[0132] Oligonucleotides include antisense oligomeric compounds, antisense oligonucleotides, oligonucleotides with an external guide sequence (EGS), alternative splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of a target nucleic acid. As such, these compounds can be administered in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds.

[0133] Targeting an oligonucleotide to a specific nucleic acid molecule in the context of the present invention may be a multi-step process. The process typically begins with the identification of a target nucleic acid whose function is to be modulated. This target nucleic acid may be, for example, a cellular gene (or mRNA transcribed from a gene) whose expression is associated with a particular disorder or disease state.

[0134] The targeting process typically also includes determining at least one target region, segment, or site within the target nucleic acid so that an antisense interaction occurs that results in a desired effect, such as modulation of expression. In the context of the present invention, the term "region" is defined as a portion of a target nucleic acid that has at least one identifiable structure, function, or characteristic. Within the regions of the target nucleic acids are segments. "Segments" are defined as smaller portions or subregions of regions within the target nucleic acid. "Sites," as used herein, are defined as positions within the target nucleic acid.

[0135] In another embodiment, antisense oligonucleotides bind to coding and/or non-coding regions of a target polynucleotide and modulate the expression and/or function of the target molecule.

[0136] In another embodiment, antisense oligonucleotides bind to natural antisense polynucleotides and modulate the expression and/or function of a target molecule. An example of a "function" may be a function that inhibits a negative transcriptional regulator, thereby allowing for increased expression of a desired molecule, such as, for example, BAG3.

[0137] In another embodiment, antisense oligonucleotides bind to sense polynucleotides and modulate the expression and/or function of a target molecule.

[0138] In embodiments of the invention, oligonucleotides bind to the antisense strand of a specific target. The oligonucleotides are at least 5 nucleotides in length and can be synthesized such that each oligonucleotide targets overlapping sequences, such that the oligonucleotides are synthesized to cover the entire length of the target polynucleotide. Targets also include coding and non-coding regions.

[0139] According to the present invention, antisense compounds include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double-stranded RNA interference (RNAi) compounds such as siRNA compounds and other oligomeric compounds that hybridize to at least a portion of a target nucleic acid and modulate its function. As such, they can be DNA, RNA, DNA-like, RNA-like, or mixtures thereof, or can be mimetics of one or more of the above. These compounds can be single-stranded, double-stranded, circular or hairpin, oligomeric compounds and can contain structural elements such as internal or terminal bulges, mismatches or loops. Antisense compounds are typically prepared in a linear form, but they may be linked or otherwise prepared to be circular and/or branched. Antisense compounds may include constructs such as, for example, two strands hybridizing to form fully or partially double-stranded compounds, or a single strand with sufficient self-complementarity to allow hybridization and formation of fully or partially double-stranded compounds. The two strands may be linked internally, leaving free 3' or 5' ends, or may be linked to form a continuous hairpin structure or loop. The hairpin structure may contain an overhang at either the 5' or 3' end, forming a continuation of the single-stranded type. Double-stranded compounds may optionally include overhangs at the ends. Further modifications may include conjugate groups attached to one of the ends, at selected positions along the nucleobase, at sugar positions, or at one of the internucleoside linkages. Alternatively, the two strands may be linked via a non-nucleic acid group or a linker group. When formed from only one strand, dsRNA may take the form of a self-complementary hairpin-type molecule that is duplicated to form a duplex. Thus, dsRNA may be fully or partially double-stranded. Specific modulation of gene expression can be achieved by stable expression of dsRNA hairpins in transgenic cell lines, but in some embodiments, gene expression is increased. When formed from two strands or a single strand that takes the form of a self-complementary hairpin-type molecule and is duplicated by itself to form a duplex, the two strands (or duplex-forming regions of the single strand) are complementary RNA strands that pair according to the Watson-Crick rules.

[0140] In another embodiment, the desired oligonucleotides or antisense compounds include at least one of: antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides containing modified linkages, interference RNA (RNAi), small interfering RNA (siRNA); microinterfering RNA (miRNA); small temporary RNA (stRNA); or short hairpin RNA (shRNA); small RNA (RNAa)-induced gene activation; small activating RNA (maRNA), or combinations thereof.

[0141] dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation" or RNA. dsRNAs targeting gene promoters induce potent transcriptional activation of associated genes. RNA has been demonstrated in human cells using a synthetic dsRNA termed "small activating RNA" (sRNA).

[0142] Small double-stranded RNA (dsRNA) can also act as small activating RNA (maRNA). Without being bound by theory, by targeting sequences in gene promoters, maRNAs will induce expression of the target gene in a phenomenon referred to as dsRNA-induced transcriptional activation (RNAa).

[0143] In some embodiments, the ribonucleic acid sequence is specific for regulatory segments of the genome that control transcription of BAG3. Thus, a candidate therapeutic agent may be a dsRNA that activates expression of BAG3 in a cell and that is administered to a patient in need of treatment.

[0144] Peptides: In another embodiment, the BAG3 peptide is encoded by a nucleic acid comprising wild-type BCL2-associated anthanogen 3 (BAG3) sequences, chimeric sequences thereof, or cDNA sequences thereof. The peptide may also be a synthetic BCL2-associated anthanogen 3 (BAG3) peptide.

[0145] It should be understood that the peptide sequences of BCL2-associated anthanogen 3 (BAG3) molecules are not limited to native sequences or their cDNA sequences. Those skilled in the art will appreciate that conservative amino acid substitutions may be made that, although they alter the primary sequence of the protein or peptide, typically do not alter its function. Conservative amino acid substitutions typically include substitutions in the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, phenylalanine, tyrosine.

[0146] Conservative substitutions can also be made based on the types of amino acids: aliphatic (valine, isoleucine, leucine, and alanine); charged (aspartic acid, glutamic acid, lysine, arginine, and histidine); aromatic (phenylalanine, tyrosine, and tryptophan); and sulfur-containing (methionine and cysteine). The polypeptide sequences have at least about 68% identity, at least about 70% identity, or at least about 71% identity to the BCL2-associated anthanogen 3 (BAG3) nucleic acid sequence or its cDNA sequence.

[0147] Determining the percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm. For example, a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877). This algorithm is included in the NBLAST and XBLAST programs of Altschul, et al. (1990, J. Mol. Biol. 215:403–410), and can be accessed, for example, at the National Center for Biotechnology Information (NCBI) World Wide Web site at the Uniform Resource Locator http://blast(dot)ncbi(dot)nlm(dot)nih(dot)gov/blast.cgi/. BLAST nucleotide searches can be performed with the NBLAST program (designated "blastn" on the NCBI Web site) using the following parameters: gap penalty=5; gap extension penalty=2; mismatch penalty=3; match reward=1; expectation value of 10.0; and segment length=11 to obtain nucleotide sequences homologous to the nucleic acid described herein. Protein BLAST searches can be performed with the XBLAST program or the NCBI "blastp" program using the following parameters: expectation of 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to the protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described by Altschul et al. (1997, Nucleic Acids Res. 25: 3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used to perform iterative searches that detect distant relationships between molecules and relationships between molecules that have the same pattern. When using the BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. Exact matches are generally considered when calculating percent identity.

[0148] Embodiments of the invention also include polynucleotides encoding fusion proteins comprising a BCL2-associated anthanogen 3 (BAG3) polypeptide operably fused, directly or indirectly through a peptide linker, to a second polypeptide sequence. Linker sequences are well known in the art. In one embodiment, the fusion protein comprises a BAG3 polypeptide or a BAG3 polypeptide operably fused to a detectable moiety, such as a reporter polypeptide, wherein the reporter polypeptide is fused to the N- or C-terminal end of the BAG3 polypeptide, directly or indirectly. Exemplary reporter polypeptides include luciferase (LUC), green fluorescent protein (GFP), and GFP derivatives.

[0149] Fusion proteins comprising a BAG3 polypeptide or fragment thereof may be linked to other types of polypeptides, in addition to the reporter polypeptide or instead of the reporter polypeptide. These additional polypeptides may be any amino acid sequence useful for purification, identification, and/or therapeutic or prophylactic use of the peptide. In addition, the additional polypeptide may be a signal peptide, or a targeting peptide, etc.

[0150] In some cases, other additions, substitutions, or deletions may enhance the stability of the polypeptide (including, but not limited to, resistance to proteolytic degradation) or increase the affinity of the polypeptide for its corresponding receptor, ligand, and/or binding proteins. In some cases, other additions, substitutions, or deletions may increase the solubility of the polypeptide. In some embodiments, sites for substitution with a naturally encoded or non-natural amino acid in addition to another site for inclusion of a non-natural amino acid are selected to increase the solubility of the polypeptide after expression in recombinant host cells. In some embodiments, the polypeptides contain another addition, substitution, or deletion that modulates affinity for the associated ligand, binding proteins, and/or receptor, modulates (including, but not limited to, increases or decreases) receptor dimerization, stabilizes dimers, modulates circulating half-life, modulates release or bioavailability, facilitates clearance, improves or alters a particular route of administration. Similarly, a polypeptide with a non-natural amino acid may include a chemical or enzymatic cleavage sequence, a protease cleavage sequence, reactive groups, antibody binding domains (including, but not limited to, FLAG or poly-His) or other affinity-based sequences (including, but not limited to, FLAG, poly-His, GST, etc.) or linked molecules (including, but not limited to, biotin) that enhance detection (including, but not limited to, GFP), purification, transport across tissues or cell membranes, release or activation of a prodrug, size reduction, or other properties of the polypeptide.

[0151] The methods and compositions described herein include inserting one or more unnatural amino acids into a polypeptide. The one or more unnatural amino acids can be included at one or more specific positions that do not disrupt the activity of the polypeptide. This can be accomplished by making "conservative" substitutions, including, but not limited to, substituting hydrophobic amino acids for unnatural or naturally occurring hydrophobic amino acids, bulky amino acids for unnatural or naturally occurring bulky amino acids, hydrophilic amino acids for unnatural or naturally occurring hydrophilic amino acids, and/or adding an unnatural amino acid at a position that is not required for activity.

[0152] A number of biochemical and structural approaches can be used to select desired sites for substitution of an unnatural amino acid in a polypeptide. Any position in the polypeptide chain is suitable for selection involving unnatural amino acids, and the selection can be based on rational design or random selection for any or some specific desired purpose. The selection of desired sites may be based on obtaining a polypeptide with unnatural amino acids (which may or may not be further modified) having any desired property or activity, including, but not limited to, agonists, superagonists, partial agonists, inverse agonists, antagonists, receptor binding modulators, receptor activity modulators, binding partner binding modulators, binding partner activity modulators, binding partner conformation modulators, dimer or multimer formation, lack of change in activity or properties compared to a natural molecule, or manipulation of any physical or chemical property of the polypeptide, such as solubility, aggregation, stability. For example, locations in a polypeptide necessary for the biological activity of the polypeptide may be identified using methods including, but not limited to, point mutation analysis, alanine scanning, or homologue scanning. Residues other than those determined to be critical for biological activity by methods including, but not limited to, alanine scanning or homolog scanning mutagenesis may be good candidates for substitution with unnatural amino acids, depending on the desired activity of the polypeptide. Alternatively, sites determined to be critical for biological activity may also be good candidates for substitution with an unnatural amino acid, again depending on the desired activity required for the polypeptide. Another alternative would be to make sequential substitutions at each position in the polypeptide chain with an unnatural amino acid and observe the effect on the activity of the polypeptide. Any means, methods, or techniques for selecting a position for substitution with an unnatural amino acid in any polypeptide are suitable for use in the methods, means, and compositions described herein.

[0153] Candidate Screening Agents and Assays

[0154] The compositions described herein may also be used in the fields of drug discovery and target testing. The present invention encompasses the use of the nucleic acid sequences and peptides described herein in drug discovery efforts to elucidate relationships that exist between Bcl-2-associated anthanogene 3 (BAG3) polynucleotides and a disease, phenotype, or condition. The method comprises detecting in a sample from a patient at least one Bcl2-associated anthanogene 3 (BAG3) genetic variant as compared to a BAG3 nucleic acid reference sequence; and administering to a patient identified as having a BAG3 genetic variation a therapeutically effective agent, wherein the agent modulates the expression or amount of BCL2-associated anthanogene 3 (BAG3) molecules, proteins, or peptides thereof in a target cell or target tissue as compared to a normal control. In some embodiments, the genetic variant is a single nucleotide variant (SNV) such as an in-frame insertion, deletion, substitution, or combination thereof. In some embodiments, the SNV comprises: p.Pro63Ala (10: 121429369 C/G; rs133031999); p.His83Gln (10: 151331972; rs151331972); Pro380Ser (10: 121436204 C/T; rs144692954); Ala479Val (10: 121436502 C/T, rs34656239), or combinations thereof. In some embodiments, the in-frame insertion encodes an amino acid. In some embodiments, the in-frame insertion encodes a non-polar amino acid. In some embodiments, the in-frame insertion encodes an alanine at position 160.

[0155] Screening assays of the invention suitably include animal models, cellular systems, and non-cellular systems. The nucleic acid and peptide sequences set forth herein are used to identify agents of therapeutic interest, such as by screening libraries of compounds or otherwise identifying compounds of interest using any of a variety of drug screens or methods for assaying or synthesizing new compounds. The gene, allele, fragment, or oligopeptide used in such screening may be free in solution, attached to a solid support, coated on a cell surface, or located intracellularly. The measurements are performed as described in detail in the following Examples section. In embodiments, candidate agents are screened to identify agents that modulate BAG3 translation.

[0156] The assays may be in vitro or in vivo formats. In vitro formats of interest include cell-based formats in which contact occurs, for example, by introducing a substrate into a medium, such as an aqueous medium, in which the cell is present. In yet other embodiments, the assay may be performed in vivo, using a multicellular organism that includes a cell. Contacting a targeting vector encoding a nucleic acid sequence described herein with a target cell/s may be accomplished using any convenient protocol. In embodiments where the target cells are present as part of a multicellular organism, such as an animal, the vector is conveniently administered (e.g., administered, fed, etc.) to the multicellular organism, such as a whole animal, wherein the administration may be systemic or localized, such as directly to a specific tissue/tissues and/or organ(s) of the multicellular organism.

[0157] Multicellular organisms of interest include, but are not limited to, insects, vertebrates such as avian species such as chicken; mammals including rodents such as mice, rats; ungulates such as pigs, cows, horses; dogs, cats, primates such as monkeys, apes, humans; and the like. As such, target cells of interest include, but are not limited to, insect cells, vertebrate cells, in particular avian cells such as chicken cells; mammalian cells including mice, pigs, ungulates, sheep, horses, rats, dogs, cats, monkeys, and human cells; and the like.

[0158] In certain embodiments, the disclosed methods are conducted in a high-throughput (HT) format. In HT embodiments of the present invention, a plurality of different cells are examined or tested simultaneously. By testing simultaneously is meant that each of the cells in the plurality is tested at substantially the same time. Generally, the number of cells that are tested simultaneously in the disclosed HT methods ranges from about 10 to 10,000, typically from about 100 to 10,000, and in some embodiments from 1,000 to 5,000. A variety of high-throughput screening assays for determining the activity of a candidate agent are known in the art and can be readily adapted to the present invention, including assays that are described, for example, in Schultz (1998) Bioorg Med Chem Lett 8: 2409-2414; Fernandes (1998) Curr Opin Chem Biol 2: 597-603; as well as those described in U.S. Patent No. 6,127,133; which are incorporated herein by reference.

[0159] In embodiments, the detectable group is conjugated to the agent of interest, wherein the detectable group comprises a luminescent group, a chemiluminescent group, a fluorescent group, a bioluminescent group, an enzyme, a natural or synthetic group.

[0160] Candidate agents: The methods can be practiced with any test compounds as candidate agents. Test compounds useful in practicing the method of the invention can be obtained using any of a variety of approaches in a combinatorial library known in the art, including biological libraries, spatially accessible solid-phase or liquid-phase parallel libraries, synthetic library methods requiring deconvolution, a one-bead-one-compound library, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide compound libraries, non-peptide oligomer compound libraries, or small molecule compound libraries (Lam, 1997, Anticancer Drug Des. 12: 145).

[0161] Examples of methods for synthesizing molecular libraries can be found in the art, for example, in: DeWitt et al., 1993, Proc. Natl. Acad. Sci. USA 90:6909-6913; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426; Zuckermann et al., 1994, J. Med. Chem. 37:2678-2685; Cho et al., 1992, Science 261:1303-1305; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2059-2061; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33:2061-2064; and Gallop et al., 1994, J. Med. Chem. 37:1233–1251.

[0162] Compound libraries can be presented in solution (e.g., Houghten, 1992, Bio/Techniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), in bacteria (U.S. Patent No. 5,223,409), in spores (U.S. Patent Nos. 5,571,698; 5,403,484; and 5,223,409), in plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89:1865-1869), or phages (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al., 1990, Proc. Natl. Acad. Sci. USA 87:6378-6382; and Felici, 1991, J Mol. Biol. 222:301-310).

[0163] Commercially available libraries that can be screened include, but are not limited to, the TimTec Natural Product Library (NPL), NPL-640, and the TimTec NDL-3000 Library. Libraries containing compounds modeled on polyamines (i.e., polyamine analogs) can also be screened.

[0164] In certain embodiments, the candidate agent is a small molecule ligand or a large molecule ligand. By small molecule ligand is meant a ligand of about 50 to about 10,000 Daltons in size, typically about 50 to about 5,000 Daltons or larger, typically about 100 to about 1,000 Daltons. By large molecule is meant a ligand of about 10,000 Daltons or larger in molecular weight.

[0165] The method can be practiced iteratively using different concentrations of the test candidate and/or different testing conditions, such as the duration of the reaction time. The test candidates identified by the method can be further tested by standard methods in the art to check for specificity, dose response, in vivo efficacy, etc. The test candidates can serve as lead compounds for the development of additional test candidates.

[0166] Based on any information available to a person skilled in the art, a prototype compound or agent may be expected to have therapeutic activity. For example, a prototype agent may be expected to have therapeutic activity based on information contained in a Physician's Handbook. Additionally, as a non-limiting example, a compound may be expected to have therapeutic activity based on a clinician's experience, the structure of the compound, structure-activity relationship data, EC ₅₀ , assay data, IC ₅₀ assay data, animal or clinical studies, or any other basis or combination of such bases.

[0167] A therapeutically active compound or agent is an agent that has therapeutic activity, including, for example, the ability of the agent to elicit a specific response when administered individually or tested in vitro. Therapeutic activity includes treating a disease or condition by any amount, including both prophylactic treatment and treatment that ameliorates the condition. Treating a disease or condition can include improving the disease or condition by any amount, including preventing, ameliorating, and eliminating the disease or condition. Therapeutic activity can be against any disease or condition, including, in one embodiment, against any disease or disorder associated with damage by reactive oxygen products. Any method that can assess the therapeutic activity of a compound can be used to determine therapeutic activity. For example, both in vivo and in vitro methods can be used, including, for example, clinical evaluation, EC ₅₀ and IC ₅₀ assays, and dose response curves.

[0168] Candidate compounds for use with the assay of the present invention or identified by the assay of the present invention as useful pharmacological agents may be pharmacological agents already known in the art or variations thereof, or may be compounds previously unknown to have any pharmacological activity. Candidate compounds may be natural or developed in a laboratory. Candidate compounds may include one diastereomer, more than one diastereomer, or one enantiomer, or more than one enantiomer.

[0169] The candidate compounds can be isolated from microorganisms, animals, or plants, for example, they can be produced recombinantly, or they can be synthesized using chemical methods known in the art. If desired, the candidate compounds of the present invention can be prepared using any of a variety of combinatorial libraries known in the art, including, but not limited to, biological libraries, spatially accessible solid-phase or liquid-phase parallel libraries, synthetic library methods requiring deconvolution, a one-bead-one-compound library, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries. The other four approaches are applicable to polypeptide libraries, non-peptide oligomer libraries, or small molecule libraries and are examples of the approaches in the present invention. See Lam, Anticancer Drug Des. 12: 145-167 (1997).

[0170] In an embodiment, the present invention provides a method for identifying a candidate compound as a suitable prodrug. A suitable prodrug includes any prodrug that can be identified by the methods of the present invention. Any method obvious to one of skill in the art can be used to identify a candidate compound as a suitable prodrug.

[0171] In another aspect, the present invention relates to methods of screening candidate compounds for suitability as therapeutic agents. Screening for suitability of therapeutic agents may include evaluating one, more, or more criteria relating to a compound that may affect the ability of the compound to be a therapeutic agent. Factors such as, for example, efficacy under controlled conditions, safety, efficacy in clinical settings, retention, localization, tissue selectivity, degradation, or intracellular stability may be considered. In an embodiment, a method of screening a candidate compound for suitability as a therapeutic agent is provided, wherein the method comprises providing a candidate compound identified as a suitable prodrug, determining the therapeutic activity of the candidate compound, and determining the intracellular stability of the candidate compound. Intracellular stability may be measured by any method known to those skilled in the art, for example, using a radioactive marker, heavy isotope labeling, or LCMS.

[0172] When screening compounds for suitability as therapeutic agents, the intracellular stability of the candidate compound is assessed. In one embodiment, the agents are assessed for their ability to modulate the translation of the compositions described herein over a period of time in response to the candidate therapeutic agent.

[0173] In another embodiment, soluble and/or membrane forms of the compositions described herein, such as proteins, mutants thereof, or biologically active portions thereof, can be used in assays for screening candidate agents. When membrane-bound forms of the protein are used, it may be desirable to use a solubilizer. Examples of such solubilizers include nonionic detergents such as n-octyl glucoside, n-dodecyl glucoside, n-dodecyl maltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, TRITON™ X-100, TRITON™ X-114, THESIT™, isotridecyl poly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propanesulfonate.

[0174] A cell-free assay may also be used, which comprises preparing a reaction mixture that comprises BAG3 molecules (nucleic acids or peptides) containing a bioluminescent group and a test compound under conditions and time periods that allow measurement of translational and/or transcriptional activity over time and concentrations of test agents.

[0175] Microarrays: Identification of nucleic acid or peptide sequences capable of binding to a target molecule can be achieved by immobilizing a nucleic acid library on a substrate surface such that each unique nucleic acid is positioned at a specific position, forming an array. Typically, the immobilized nucleic acid or peptide library is exposed to a biomolecule or candidate agent under conditions that promote binding of the biomolecule to the nucleic acids or peptides. The array is then analyzed by the methods described herein to determine which nucleic acid sequences or peptides are bound to the biomolecule. Such biomolecules can carry a predetermined label for use in detecting the location of bound nucleic acids or peptides.

[0176] An assay using an immobilized array of BAG3 nucleic acid sequences can be used to determine the sequence of an unknown nucleic acid; single nucleotide polymorphism (SNP) analysis; analysis of BAG3 gene expression patterns from a particular species, tissue, cell type, etc.; gene identification; etc.

[0177] In embodiments, oligonucleotides or longer fragments derived from any of the BAG3 polynucleotide sequences can be used as targets in a microarray. The microarray can be used to monitor the identity and/or expression level of a large number of gene transcripts and genes simultaneously to identify genes with which target genes or their product interact and/or to assess the effectiveness of candidate therapeutics for regulating gene expression products that mediate, for example, neurological disorders. This information can be used to determine gene function, as well as to develop and monitor the activity of therapeutics.

[0178] Microarrays can be prepared, used, and analyzed using methods known in the art (see, e.g., Brennan et al., 1995, U.S. Patent No. 5,474,796; Schena et al., 1996, Proc. Natl. Acad. Sci. USA 93: 10614-10619; Heller et al., 1997, Proc. Natl. Acad. Sci. USA 94: 2150-2155; and Heller et al., 1997, U.S. Patent No. 5,605,662). In other embodiments, the microarray comprises BAG3 peptides or other desired molecules that can be analyzed to identify a candidate agent.

[0179] Another embodiment of a method for screening candidate agents for treating or preventing a cardiac disease or disorder includes contacting a sample with a candidate therapeutic agent and measuring the effects that the agent has on the target. For example, the agent may regulate the expression of BAG3, and then the agent may be further studied for any possible therapeutic effects (monitoring an increase or decrease parameter, such as expression). The abnormal expression condition may be caused by a pathology such as heart failure, disease, cancer, genetic defects, and/or a toxin.

[0180] Antibodies. Useful diagnostic assays may include one or more antibodies that specifically bind BAG3. In some embodiments, the antibody specifically binds to a mutant BAG3. The term "antibody" is used broadly and refers to immunoglobulin-based binding molecules, the term includes conventional antibodies (e.g., tetrameric class G antibodies (e.g., IgG1)), fragments thereof that retain the ability to bind the intended target (e.g., a Fab' fragment), and single-chain antibodies (scFvs). The antibody may be polyclonal or monoclonal and may be produced in human, mouse, rabbit, sheep, or goat cells or from hybridomas derived from these cells. The antibody may be humanized, chimeric, or bispecific.

[0181] Antibodies may take a variety of configurations and include proteins consisting of one or more polypeptides essentially encoded by immunoglobulin genes. Any of a number of antibody structures may be used, including intact antibodies, multimeric antibodies, or antibody fragments or other variants thereof that include functional antigen-binding regions of the antibody. We may use the term "immunoglobulin" synonymously with "antibody." Antibodies may be monoclonal or polyclonal in origin. Regardless of the source of the antibody, suitable antibodies include intact antibodies, such as IgG tetramers with two heavy (H) chains and two light (L) chains, single chain antibodies, chimeric antibodies, humanized antibodies, complementarity determining region (CDR)-grafted antibodies, as well as antibody fragments, such as Fab, Fab', F(ab') ₂ , scFv, Fv, and recombinant antibodies derived from such fragments, such as camel antibodies, microbodies, diabodies, and bispecific antibodies.

[0182] An intact antibody is an antibody that includes an antigen-binding variable region (VH and VL), as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. As is known in the art, the VH and VL regions are further subdivided into regions of hypervariability, called "complementarity determining regions" (CDRs), interspersed with more conserved framework regions (FRs).

[0183] The anti-BAG3 antibody may be of any immunoglobulin class, such as IgA, IgG, IgE, IgD, IgM (as well as their subtypes (e.g., IgG1, IgG2, IgG3, and IgG4)), and the immunoglobulin light chains may be of the kappa or lambda type. Known human immunoglobulin genes include kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes, as well as a variety of immunoglobulin variable region genes.

[0184] The term "antigen-binding portion" of an immunoglobulin or antibody refers generally to the portion of the immunoglobulin that specifically binds to a target, in this case, an epitope comprising amino acid residues on a BAG3 polypeptide. An antigen-binding portion of an immunoglobulin is thus a molecule in which one or more immunoglobulin chains are not of full length, but which specifically binds to a cellular target. Examples of antigen-binding portions or fragments include: (i) a Fab fragment, a monovalent fragment consisting of the VLC, VHC, CL, and CH1 domains; (ii) an F(ab') ₂ fragment, a divalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fv fragment consisting of the VLC and VHC domains of one arm of an antibody, and (v) an isolated CDR having a sufficient framework for specific binding, for example, an antigen-binding portion of a variable region. The antigen-binding portion of the variable region of the light chain and the antigen-binding portion of the variable region of the heavy chain, for example, two fragments of the Fv domains, VLC and VHC, can be joined by recombinant methods with a synthetic linker, which allows them to be created as a single protein chain in which the VLC and VHC regions are paired to form a monovalent molecule (known as a single-chain Fv (scFv). Such scFvs can be a targeting agent of the present invention and are encompassed by the term "antigen-binding portion" of an antibody.

[0185] The "Fv" fragment is the minimum fragment of an antibody that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in close, concovalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. While the six hypervariable regions confer antigen-binding specificity, even a single variable domain (or half of an Fv, containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the entire binding site. To enhance stability, the VH-VL domains can be joined by a flexible peptide linker such as (Gly ₄ Ser) ₃ to form a single chain Fv or scFV antibody fragment, or can be engineered to form a disulfide bond by introducing two cysteine residues into the framework regions to form a disulfide-stabilized Fv (dsFv). Antibody fragments are suitable for use provided that they retain the desired specificity of the full-length antibody and/or sufficient specificity to specifically bind to the BAG3 polypeptide.

[0186] The compositions of the present invention include antibodies that (1) exhibit a threshold level of binding activity; and/or (2) do not significantly cross-react with known related polypeptide molecules. The binding affinity of an antibody can be readily determined by one of skill in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Acad, Sci. 51: 660-672 (1949)).

[0187] In some embodiments, anti-BAG3 antibodies can bind to their target epitopes or decoy mimetics at least 1.5-fold, 2-fold, 5-fold, 10-fold, 100 ^- fold, 10-fold, 10-fold, ¹⁰ -fold, 10 ^- fold, 10 ^- fold, or more for the target anti-BAG3 than for other proteins suspected of having some homology to BAG3.

[0188] In some embodiments, the anti-BAG3 antibodies bind with high affinity to 10^-4M or less, 10^-7M or less, 10^-9M or less or with a subnanomolar affinity (0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 nM or even less). In some embodiments, the binding affinity of the antibody to BAG3 for their respective targets is at least 1×10⁶Ka. In some embodiments, the binding affinity of the antibody to BAG3 for BAG3 is at least 5×10⁶Ka at least 1×10⁷At least 2x10⁷Ka at least 1×10⁸Ka or greater. Antibodies may also be described or defined in terms of their binding affinity to BAG3. In some embodiments, binding affinities include those with a Kd of less than 5×10^-2M, 10^-2M, 5×10^-3M, 10^-3M, 5×10^-4M, 10^-4M, 5×10^-5M, 10^-5M, 5×10^-6M, 10^-6M, 5×10^-7M, 10^-7M, 5×10^-8M, 10^-8M, 5×10^-9M, 5×10^-10M, 10^-10M, 5×10^-11M, 10^-11M, 5×10^-12M, 10 M, 5×10^-13M, 10^-13M, 5×10^-14M, 10^-14M, 5×10^-15M, or 10^-15M, or less.

[0189] In some embodiments, the antibodies do not bind to known related polypeptide molecules; for example, they bind BAG3 but not to known related polypeptides. In some embodiments, the antibodies specifically bind to a mutant BAG3 polypeptide, for example, a BAG3 polypeptide having a ten base pair deletion, but not to a wild-type BAG3 polypeptide. The antibodies can be tested against known related polypeptides to isolate a population of antibodies that specifically bind to BAG3.

[0190] The diagnostic assay of the invention may include simultaneous immunoelectrophoresis, radioimmunoassay (RIA), radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot, inhibition or competition assay, and sandwich assay. Anti-BAG3 antibodies may include a label, which may also be referred to as a reporter or marker (e.g., a detectable marker). A detectable marker may be any molecule that is covalently linked to an anti-BAG3 antibody, or a biologically active fragment thereof, and that allows for the qualitative and/or quantitative assessment of the expression or activity of the labeled peptide. Activity may include biological activity, physicochemical activity, or a combination thereof. Both the shape and position of the detectable marker may vary, so long as the labeled antibody retains biological activity. A variety of different markers may be used, and the choice of a particular marker will depend on the desired application. Labeled antibodies to BAG3 can be used, for example, to assess levels of BAG3 or a BAG3 mutant in a biological sample, such as urine, saliva, cerebrospinal fluid, blood, or a biopsy sample, or to assess the clinical response to a cardiovascular therapeutic agent, such as the BAG3 constructs described above.

[0191] Typical detectable labels that can be conjugated to the agent include radiopaque or contrast agents such as barium, diatrizoate, ethiodized oil, gallium citrate, iocarmic acid, iocetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide, iohexol, iopamidol, iopanoic acid, iopromemic acid, iosefamic acid, ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetrinic acid, iothalamic acid, iyotroxic acid, ioxaglic acid, iothalamic acid, iyotroxic acid, ioxaglic acid, ioxotrizolic acid, ipodate, meglumine, metrizamide, metrizoate, propyliodone and thallium chloride. Alternatively or in addition, the detectable label may be a fluorescent label such as fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine; a chemiluminescent compound selected from the group consisting of luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester; a liposome or dextran; or a bioluminescent compound such as luciferin, luciferase and aecorin.

[0192] Suitable markers include, for example, enzymes, photoaffinity ligands, radioactive isotopes, and fluorescent or chemiluminescent compounds. Methods for introducing detectable markers into peptides are well known in the art. Markers can be added during synthesis or after synthesis. Recombinant anti-BAG3 antibodies or biologically active variants thereof can also be labeled by adding labeled precursors (e.g., radioactively labeled amino acids) to the culture medium in which the transformed cells are grown. In some embodiments, peptide analogs or variants can be used to facilitate the incorporation of detectable markers. For example, any N-terminal phenylalanine residue can be replaced with a closely related aromatic amino acid, such as tyrosine, which can be readily labeled with ¹²⁵ I. In some embodiments, additional functional groups that support efficient labeling can be added to fragments of the anti-BAG3 antibody or biologically active variant thereof. For example, a 3-tributyltinbenzoyl group can be added to the N-terminus of the native structure; subsequent substitution of the tributyltin group with ¹²⁵ I will generate a radiolabeled iodobenzoyl group.

[0193] Any known method can be used to detect such markers, such as positron emission tomography (PET), SPECT imaging, magnetic resonance imaging, x-ray, or ultrasound.

[0194] Diagnostics, treatment, kits

[0195] The compositions of the present document and the compounds of the present invention can be used for diagnosis, treatment and prevention, as well as as research reagents and components of kits.

[0196] The compositions described herein are generally and to varying degrees useful for treating an individual suffering from a cardiac disease or disorder, such as heart failure or dilated cardiomyopathy. "Subject," "patient," or "individual" are used interchangeably. An individual receives effective treatment whenever a clinically beneficial outcome occurs. This may mean, for example, a complete disappearance of the symptoms of the disease, a reduction in the severity of the symptoms of the disease, or a slowing of the progression of the disease. These methods may further include identifying BAG3 variants in a biological sample that predict whether an increase in BAG3 levels is therapeutic for the subject, and administering to a patient identified as having such a variant a therapeutically effective amount of the agent.

[0197] In certain embodiments, the kit includes one or more probes conjugated to a detectable label for identifying BCL2-associated anthanogen 3 (BAG3) variants in a biological sample. Detection of certain variants allows for prediction of benefit from treatment for an individual.

[0198] Cardiovascular disorders associated with the therapeutic and/or prognostic methods of the invention may be disorders that respond to BAG3 modulation or, in the treatment of those individuals who may not respond to BAG3 modulating agents. The compositions of the present invention are not limited to those that act by affecting any particular cellular mechanism. Any form of cardiovascular disorder associated with BAG3 dysregulation is within the scope of the present invention.

[0199] The methods of the invention can be expressed in relation to the production of a medicament. Thus, the invention relates to the use of the means and compositions described herein for the production of a medicament. The compounds described herein are useful in therapeutic compositions and treatment regimens or for the production of a medicament for use in the treatment of diseases or conditions described herein (e.g., a cardiovascular disorder herein).

[0200] Any composition described herein can be administered to any part of the host body for subsequent delivery to a target cell. The composition can be delivered to, without limitation, the brain, cerebrospinal fluid, joints, nasal mucosa, blood, lungs, intestines, muscle tissue, skin, or peritoneal cavity of a mammal. With respect to delivery routes, the composition can be administered by intravenous, intracranial, intraperitoneal, intramuscular, subcutaneous, intramuscular, intrarectal, intravaginal, intrathecal, intratracheal, intradermal, or transdermal injection, by oral or nasal administration, or by gradual perfusion over time. In an additional example, the aerosol preparation can be administered to the host by inhalation.

[0201] The required dosage will depend on the route of administration, the nature of the formulation, the nature of the patient's disease, the patient's size, weight, surface area, age, and sex, other drugs being administered, and the judgment of the treating physician. Suitable dosages are in the range of 0.01-1000 mg/kg. Wide variations in the required dosage are to be expected due to the diversity of cellular targets and the varying efficiencies of different routes of administration. Variations in these dosage levels can be adjusted using standard empirical optimization procedures well known in the art. Administration can be single or multiple (e.g., 2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100-, 150-fold or more). Encapsulation of the compounds in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) can enhance delivery efficiency.

[0202] The duration of treatment with any composition provided herein may be any duration, from one day to the lifespan of the host (e.g., many years). For example, the compound may be administered once a week (e.g., from 4 weeks to many months or years); once a month (e.g., from three to twelve months or for many years); or once a year for a period of 5 years, ten years or more. It should also be noted that the frequency of treatment may be variable. For example, the present compounds may be administered once (or twice, three times, etc.) daily, weekly, monthly or annually.

[0203] An effective amount of any composition provided herein can be administered to an individual in need of treatment. As used herein, the term "effective" refers to any amount that produces a desired response but does not cause significant toxicity in the patient. Such an amount can be determined by assessing the patient's response after administration of a known amount of the particular composition. In addition, the level of toxicity, if any, can be determined by assessing clinical symptoms before and after administration of a known amount of the particular composition. It should be noted that the effective amount of a particular composition administered to a patient can be adjusted according to the desired result, as well as the patient's response and level of toxicity. Significant toxicity can vary for each individual patient and depends on a variety of factors, including, but not limited to, the patient's disease state, age, and tolerance to side effects.

[0204] Any method known in the art can be used to determine whether a particular response has been elicited. Clinical methods that assess the extent of a particular disease state can be used to determine whether a response has been elicited. The specific methods used to assess the response will depend on the nature of the patient's disease, the patient's age, gender, other drugs administered, and the judgment of the treating physician.

[0205] Concomitant administration of two or more therapeutic agents does not require that the agents be administered at the same time or by the same route, provided that the time periods during which the agents exert their therapeutic effect overlap. Concomitant or sequential administration, such as administration on different days or weeks, is contemplated. The composition may also be administered with another standard therapeutic agent for the treatment of cardiovascular disease. Examples of such diseases or disorders related to them include cardiac disease or disorders, skeletal muscle disease or multiple sclerosis, senile plaques, cerebral amyloid angiopathy, atherosclerosis, glioblastoma, amyloid deposition, neurodegenerative diseases, neurofibrillary tangles, dementia, choriocarcinoma, astrocytoma, amyloidosis, hyperlipidemia, neurodegeneration, neoplastic transformation, prostate cancer, atherosclerotic plaque, obstruction, AIDS, metastasis, myocardial infarction, pulmonary fibrosis, necrosis, shock, melanoma, colorectal carcinoma, genetic predisposition, psoriasis, malignancy, inflammation, glioma, carcinoma, breast cancer, pathology of the nervous system, tumors, prostate carcinoma, vascular diseases, cellular injuries, brain tumors, non-small cell lung carcinoma (NSCLC), hypercholesterolemia. Examples of skeletal muscle diseases include primary (genetic) muscle diseases (e.g. muscular dystrophies and congenital myopathies, metabolic myopathies); acquired diseases (e.g. myositis, toxic myopathy); secondary muscle diseases (e.g. neurogenic atrophy, atrophy from chronic lung disease, heart disease, kidney disease, HIV/AIDS, malignancy, sarcopenia), etc.

[0206] Kits: The present invention further relates to systems and kits (e.g., commercial therapeutic, diagnostic, or research products, reaction mixtures, etc.) that contain one or more or all of the components sufficient, necessary, or useful for practicing any of the methods described herein. These systems and kits may include buffers, detection/imaging components, positive/negative control reagents, instructions, software, equipment, packaging, or other desired components.

[0207] Kits provide useful tools for screening test compounds that can modulate the effects of the compound on a target molecule. Kits can be packaged in any suitable manner to assist research, clinical, and testing laboratories, typically with the various parts in a suitable container along with instructions for use.

[0208] In certain embodiments, the kits may further comprise lipids and/or solvents. In certain embodiments, the kits may further comprise buffers and reagents necessary for the procedure and instructions for performing the assay. In certain embodiments, the kits may further comprise, if necessary, agents for reducing background interference in the test, reagents for positive and negative controls, a device for performing the test, and the like.

[0209] Also provided are kits for determining whether an individual has a mutation in the BAG3 polypeptide, for diagnosing patients with cardiovascular disease or a predisposition to developing cardiovascular disease. The kits can also be used to monitor the effectiveness of agents used to treat cardiovascular disease.

[0210] Introduction of compositions

[0211] The agents identified by the methods described herein can be formulated and the composition of the present invention can be administered in combination with one or more additional active ingredients, pharmaceutical compositions, or other compounds. The therapeutic agents of the present invention can be administered to an animal, such as a mammal, including a human.

[0212] In other embodiments, the pharmaceutical composition comprises at least one or more agents and/or candidate therapeutic agents described herein.

[0213] Pharmaceutical formulations may be intended for administration by the oral route (solid or liquid), parenteral route (intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection), intracardiac route, transdermal route (passive or using iontophoresis or electroporation), transmucosal and systemic routes (nasal, vaginal, rectal, or sublingual), or inhalation routes of administration, or using biodegradable inserts, and may be formulated into dosage forms suitable for each route of administration.

[0214] The agents can be formulated in pharmaceutically acceptable carriers or diluents, such as saline or buffered saline. Suitable carriers and diluents can be selected based on the mode and route of administration and standard pharmaceutical practice. Descriptions of typical pharmaceutically acceptable carriers and diluents, as well as formulations, can be found in Remington's Pharmaceutical Sciences, a standard text in the field, and in USP/NF. Other substances can be added to the composition to stabilize and/or preserve the composition.

[0215] The composition of the invention can be administered to animals by any conventional route. The composition can be administered directly to the target site, such as by surgical delivery to an internal or external target site or by catheter delivery to a site accessible to a blood vessel. Other delivery methods, such as liposomal delivery or diffusion from a device impregnated with the composition, are known in the art. The composition can be administered by a single bolus, multiple injections, or continuous infusion (e.g., intravenously). For parenteral administration, the compositions are formulated, for example, in a sterilized, pyrogen-free form.

[0216] The compounds of the present invention can also be administered orally to a patient in a manner such that the drug is sufficient to inhibit bone resorption or to achieve any other therapeutic purpose described herein. Typically, a pharmaceutical composition containing the compound is administered at an oral dose of about 0.1 to about 50 mg/kg, depending on the patient's condition. An exemplary oral dose would be about 0.5 to about 20 mg/kg.

[0217] Intravenous infusion of the compound in 5% dextrose in water or normal saline or a similar formulation with suitable excipients is most effective, although intramuscular bolus injection is also suitable. Typically, the parenteral dose will be from about 0.01 to about 100 mg/kg; such as between 0.1 and 20 mg/kg, so as to maintain the plasma drug concentration at a concentration effective to increase BAG3 expression. The compound can be administered from one to four times daily at a level to achieve a total daily dose of from about 0.4 to about 400 mg/kg/day. The precise amount of a compound of the invention that is therapeutically effective and the route by which such compound is best administered can be readily determined by one of skill in the art by comparing the blood level of the agent with the dose required for therapeutic effect. Prodrugs of the compounds of the present invention can be prepared by any suitable method.

[0218] No unacceptable toxicological effects are expected when the compounds, derivatives, salts, compositions, etc., of the present invention are administered in accordance with the present invention. Compounds of the present invention that may have good bioavailability can be tested in one of several biological assays to determine the concentration of the compound that is necessary to produce a desired pharmacological effect.

[0219] In another embodiment, there is provided a pharmaceutical or veterinary composition comprising one or more identified compounds and a pharmaceutically acceptable carrier. Other active substances that may be considered suitable or appropriate for the treatment or prevention of a disease or condition may also be present.

[0220] The carrier, or if more than one, each carrier, must be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the recipient.

[0221] The compounds defined by the methods herein will be suitable for use in a variety of drug delivery systems described above. Additionally, to increase the in vivo serum half-life of the administered compound, the compounds can be encapsulated, introduced into the lumen of a liposome, formulated as a colloid, or other conventional methods that provide an extended serum half-life for the compounds can be used. Several methods are available for preparing liposomes, as described, for example, in Szoka, et al., U.S. Patent Nos. 4,235,871, 4,501,728, and 4,837,028, each of which is incorporated herein by reference. Additionally, the drug can be administered in a targeted drug delivery system, such as a liposome coated with a tissue-specific antibody. The liposomes will be targeted to and selectively taken up by the organ.

[0222] The formulations include those suitable for rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration, or the formulation is an orally administrable formulation. The formulations may conveniently be presented in unit dosage form, for example, in tablets and in sustained-release capsules, which may be prepared by any methods known in the art of pharmacy.

[0223] Such methods include the step of combining the above-defined active agent with a carrier. Generally, the compositions are prepared by uniformly and intimately combining the active agent with liquid carriers or finely divided solid carriers or both, and then, if necessary, forming a product.

[0224] A compound identified using these methods can be formulated according to known methods for preparing useful compositions whereby the compound is combined in admixture with a pharmaceutically acceptable carrier. Therapeutic formulations are prepared for storage by mixing the active ingredient, having the desired degree of purity, with optional physiologically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™ (ICI Americas Inc., Bridgewater, N.J.), PLURONICS™ (BASF Corporation, Mount Olive, N.J.), or PEG.

[0225] Formulations used for in vivo administration must be sterile and non-pyrogenic. This is readily achieved by filtration through sterile filter membranes prior to or after lyophilization and reconstitution.

[0226] The dosages and desired drug concentrations of the pharmaceutical compositions of the present invention may vary depending on the intended use. Determination of the appropriate dosage or route of administration is well within the skill of the art. Animal experiments provide a reliable guide to determining effective dosages for human therapy. Interspecies scaling of effective dosages can be accomplished following the principles set forth by Mordenti, J. and Chappell, W. "The use of interspecies scaling in toxicokinetics" In Toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, New York 1989, pp. 42-96.

[0227] The oral formulations of the present invention may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active agent; as a powder or granule; as a solution or suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or an oil-in-oil liquid emulsion; or as a bolus, etc.

[0228] For an oral composition (e.g., tablets and capsules), the term "acceptable carrier" includes carriers such as conventional excipients, for example, binding agents, for example, syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (Povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, sucrose and starch; fillers and carriers, for example, corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride and alginic acid; and lubricants such as magnesium stearate, sodium stearate and other metallic stearates, glycerol stearate stearic acid, silicone fluid, talc waxes, oils and colloidal silicon dioxide. Flavoring agents such as peppermint, oil of wintergreen, cherry flavoring, etc. may also be used. It may be desirable to add a coloring agent to make the dosage form easily identifiable. The tablets may also have a coating well known in the art.

[0229] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active agent in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. Tablets may optionally be coated or scored and may be formulated to provide slow or controlled release of the active agent.

[0230] Other formulations suitable for oral administration include lozenges containing the active agent in a flavored base, typically sucrose and acacia or tragacanth; pastilles containing the active agent in an inert base such as gelatin and glycerol, or sucrose and acacia; and mouthwashes containing the active agent in a suitable liquid carrier.

[0231] Parenteral formulations will generally be sterile.

[0232] Dose: An effective dose of a composition of the subject matter of the present invention is administered to an individual in need thereof. A "therapeutically effective amount" or "therapeutic amount" is an amount of a therapeutic composition sufficient to produce a measurable response (e.g., a biologically or clinically significant response in the individual being treated). The response can be measured in a variety of ways, as noted above, such as cytokine profiles, cell types, cell surface molecules, etc. Actual dosage levels of the active ingredients in a composition of the subject matter of the present invention can be varied to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular individual. The dosage level selected will depend on the activity of the therapeutic composition, the route of administration, the combination with other drugs or treatments, the severity of the disease being treated, and the condition and prior medical history of the individual being treated. However, it is recommended that those skilled in the art begin dosing the compound at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. The potency of the composition may vary, and thus the "therapeutically effective amount" may vary. However, using the assay methods described herein, a person skilled in the art can readily assess the potency and efficacy of a candidate compound of the subject matter claimed in the present invention and adjust the treatment regimen accordingly.

[0233] The present invention has been described in detail with reference to some embodiments thereof. However, it should be understood that those skilled in the art, after reviewing this description, can make modifications and improvements within the spirit and scope of the invention.

[0234] All documents cited in this document are herein incorporated by reference. All publications and patent documents cited in this application are hereby incorporated by reference for all purposes to the same extent as if each individual publication or patent document was individually so indicated. By citing various references in this document, applicants do not admit any specific reference to their invention as "prior art."

EXAMPLES

[0235] Example 1: Variants in BAG3 Predict Worse Outcome in African Americans with Cardiomyopathy

[0236] To investigate the association between a BAG3 genetic variant and DCM in subjects of African descent, BAG3 was sequenced from DNA obtained from subjects participating in one of three clinical trials. The results showed that a group of functional BAG3 variants, found with rare exceptions in individuals of African descent, are associated with poor outcomes in patients with DCM and may serve as a precise target for therapeutic intervention.

[0237] Methods

[0238] Patients: Genomic DNA was obtained from 509 African Americans with DCM enrolled in three US clinical trials and whose DNA was banked: 342 patients from the Genetic Risk Assessment of African Americans with Heart Failure (GRAHF) study (cohort A); 109 patients from the Intervention in Myocarditis and Acute Cardiomyopathy-2 (IMAC-2; cohort B); ²⁶ and 58 patients from the Genetic Risk Assessment of Cardiac Events (GRACE) study (cohort C). ^27,28 DNA was also obtained from left ventricular (LV) myocardium in individuals of African descent who underwent heart transplantation at the University of Colorado (cohort D) or the University of Pittsburgh (cohort E). Healthy human cardiac tissue that could not be used for transplantation served as healthy controls. ²⁹ Informed consent was obtained from all patients who contributed DNA or tissue to the study repositories listed above, and protocols were approved by the institutional ethics committee for each participating institution.

[0239] Three reference populations were used. In the first, DNA was sequenced from individuals of African descent with ischemic cardiomyopathy who were included in the AC cohort studies. In the second, BAG3 sequence data were obtained from a cohort of individuals of European descent with familial and sporadic DCM collected at Brigham and Women's Hospital in Boston from individuals of European descent collected from heart failure clinics throughout the United States. ³⁰ In the third, population genetic data were taken from gnomAD (available online), which contains data from 123,136 exome sequences and 15,496 full-length genome sequences from unrelated individuals. Of these, 7,509 whole genomes and 55,860 exomes were from individuals of non-Finnish European descent and 4,368 whole genomes and 7,652 exomes were from individuals of African descent. ²¹

[0240] DNA sequencing and analysis: DNA from the GRAHF study was sequenced at the Genetic Resource Core Laboratory of the McKusick-Nathans Institute for Genetic Medicine at the Johns Hopkins University School of Medicine. Briefly, genomic DNA from each GRAHF cohort patient was amplified by PCR and then sequenced using the fluorescent dideoxy terminator cycle sequencing method. Sequence data were analyzed using Sequencer software, version 5.4.6 (Gene Codes, Ann Arbor, MI). SNVs were identified using the secondary peak naming feature of the software, and all data were manually curated.

[0241] Targeted genotyping was performed to confirm the GRAHF cohort results and to identify SNVs in each of the subsequent cohorts using real-time PCR and SNP-specific reagents. SNVs were selected for confirmatory genotyping and functional analysis if they: 1) had an allele frequency >0.005 in GRAHF; 2) were nonsynonymous; and 3) were more common in individuals of African ancestry than in individuals of European ancestry. To determine whether two BAG3 SNVs in a single sample were located in cis, the BAG3 locus was amplified, cloned into a plasmid, and subjected to Sanger sequencing.

[0242] Western blot analysis of human cardiac tissue: Western blot analysis was performed as previously described. ³¹ Briefly, frozen tissues were homogenized in buffer, centrifuged, and the resulting pellets were resuspended in buffer. Following protein measurement using the Bradford method, protein was fractionated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Membranes were then incubated with antibodies and visualized. Signal intensity was normalized to GAPDH. qPCR was used to quantify mRNAs specific for each BAG3 variant.

[0243] Measurement of autophagic flux and apoptosis: The methods have been described in detail previously. ^18,32 Briefly, wild-type BAG3 was inserted into a plasmid under the control of the CMV promoter, and all SNVs of interest were then generated. The p.160Aladup variant was synthesized by GenScript (Piscataway Township, NJ). Transformed human ventricular cells (AC16) ³³ were cultured at 37°C with 5% CO ₂ in Dulbecco's modified Eagle's medium with 5% FBS to 60-70% confluency. Cells were then transfected with plasmids containing c.null, c.BAG3, c.187C>G+c.1138C>T (p.P63Ala+P380S), c.249C>A, (p.H83Q) c.474_476dupGGC, (p.A160dup) or c.1436C>T (p.A479V) and cotransfected with an autophagy reporter construct: adenovirus-red fluorescent protein-green fluorescent protein-LC3 (Adv-RFP-GFP-LC3). Each transfection was performed for 36 hours, after which cells were subjected to four hours of hypoxia (CO ₂ /1% N ₂ O ₂ ) followed by two hours of reoxygenation or normoxia (5% CO ₂ : 21% O ₂ ). Cells were visualized by confocal microscopy and LC3 puncta were counted as previously described. ³⁴ Expression levels of each plasmid were assessed by Western blotting and were equivalent.

[0244] In a second set of experiments, cells were transfected in the same manner for 36 hours, hypoxic for 12 hours, and then reoxygenated for up to 2 hours. Cells were stained with annexin-V and propidium iodide for 30 minutes and imaged by confocal microscopy. Annexin-V positive cells (apoptosis) appear green, while propidium iodide positive cells (late apoptosis and necrosis) appear red. There were at least 20 cells per field and 10 fields per condition. The investigator was blinded to the experimental group.

[0245] Statistical Analysis: Statistical analyses were performed based on stratification/division by BAG3 SNV (SNV vs. no SNV), ischemia (ischemic vs. nonischemic), and covariate interaction. Continuous variables were assessed using Student's t-test or ANOVA, as appropriate, and categorical variables were assessed using chi-square tests or Fisher's exact tests. For time-to-event analyses, an event was defined as death, transplantation, or hospitalization for heart failure. Survival was estimated using the Kaplan-Meier method; resulting curves were assessed using the log-rank test. Hazard ratios were determined using Cox proportional hazards models. All statistical analyses were performed using SAS® 9.4 (SAS Institute). Statistical significance was defined as p < 0.05. All reported p values are two-sided where applicable and have not been adjusted for multiple comparisons unless otherwise stated.

[0246] Results

[0247] Study subjects: Genomic DNA from 509 African Americans with DCM was obtained from three independent studies: 342 patients from GRAHF (cohort A); ^23,24 109 patients from GRACE (cohort B); ^27,28 and 58 patients from IMAC-2 (cohort C). ²⁶ Patients were excluded from analysis if they had acute myocarditis, perinatal cardiomyopathy, or any potentially reversible cause of DCM. Patients in GRAHF and IMAC-2 were followed to the survival endpoint of death or hospitalization for heart failure. Subjects in GRACE were followed until heart transplantation or death.

[0248] BAG3 Genetic Variants:

[0249] Sanger sequencing of the GRAHF cohort identified 18 variants (Table 1), eight of which were synonymous. Four SNVs met the inclusion criteria for this study: p.Pro63Ala (10: 121429369 C/G; rs133031999); p.His83Gln (10: 151331972; rs151331972); Pro380Ser (10: 121436204 C/T; rs144692954); and Ala479Val (10: 121436502 C/T, rs34656239). A three-nucleotide in-frame insertion was also identified that added an alanine to the protein at position 160 (p.Ala160dup; 10:121429647 A/AGCG). The Sanger results were confirmed by targeted sequencing. Every individual who carried the p.Pro63Ala variant also carried the p.Pro380Ser variant, and the corresponding allele frequencies for these two SNVs in gnomAD were nearly identical, suggesting that the two SNVs are linked in cis. This was confirmed by Sanger sequencing. The characteristics of African-descended heart failure patients with and without the 4 identified BAG3 variants are shown in Table 1. The linked SNVs are denoted as p.Pro63Ala+Pro380Ser. As shown in FIG. 1, the residues (His83, Ala160, Pro380, and Ala479) affected in the BAG3 variants are highly conserved across mammals.

[0250] The prevalence of the four BAG3 variants in 402 patients with IDCM in cohorts A, B, and C (42 of 402 patients; 10.45%) was greater than the prevalence of the variants in patients with ischemic heart failure (9 of 107 subjects; 8.41%), adjusting for multiple alleles; however, the difference was not statistically significant. (Table 2) Similarly, the proportion of patients in cohorts A, B, and C with IDCM and one of the four BAG3 variants was not significantly different from the proportion of patients with a BAG3 variant in the sum of the corresponding gnomAD dataset for each variant (9.06%). In contrast, the proportion of subjects in the three cohorts who had a BAG3 variant (10.02%) was significantly (p < 0.0001) higher than the prevalence of the four BAG3 variants among the over 60,000 subjects of European descent in gnomAD. The European data set (0.020%; p < 0.0001) was significantly larger than the control population of 359 individuals of European descent and IDCM who did not have BAG3 variants (Boston cohort; 0.000% BAG3 variants; p < 0.0001).

[0251] Impact of BAG3 Variants on Heart Failure Outcomes: We next sought to determine whether the presence of any of the four BAG3 variants was associated with worse outcome, as reflected in the combined heart failure outcome variable of hospitalization, heart transplant, or death. As seen in FIG. 2A, compared with subjects with both ischemic and nonischemic heart failure who did not carry one of the four BAG3 variants, subjects carrying a BAG3 variant had a significantly (p=0.010) higher incidence of adverse events. Similarly, individuals with nonischemic HF who carried a BAG3 variant had a worse outcome compared with nonischemic patients who did not carry a BAG3 variant. (FIG. 2B; p=0.018) There was a trend toward worse outcome in subjects with ischemic HF and a BAG3 SNV; however, the difference was not statistically significant. (Fig. 2C, p=0.177). Using Cox proportional hazards analysis, we determined that the risk of being a carrier of one of the BAG3 variants having an adverse event was 1906 times higher than for an individual with HF who did not have the BAG3 variant (95% CI: 1.157-3.141, p=0.011).

[0252] Pathogenicity of BAG3 Variants: The effects of BAG3 variants on two primary cellular actions of BAG3, autophagy and apoptosis, were assessed. As shown in FIG. 3A, AC16 cells were transfected with either empty plasmid or plasmid containing wild-type BAG3, c.187C>G+c.1138C>T (p.Pro63Ala+p.Pro380Ser), c.249C>A (p.His83Gln), c.474_476dupGGC (p.Ala160dup), or c.1436C>T (p.Ala479Val), and cotransfected with Adv-RFP-GFP-LC3. When cells were exposed to hypoxia-reoxygenation stress, a significant increase in LC3 was observed in cells that were transfected with either the empty plasmid or wild-type BAG3; however, the autophagic response to hypoxia-reoxygenation was significantly reduced in the four AC16 cells overexpressing each of the BAG3 variants, as no significant increase in LC3 was observed. (Figures 3A and 3B) Furthermore, after hypoxia-reoxygenation, cells transfected with the BAG3 variants showed significantly more annexin-V-positive cells than cells transfected with the plasmid expressing wild-type BAG3. (Figures 3C and 3D) Thus, unlike the plasmid with wild-type BAG3, the BAG3 variants could not prevent hypoxia-induced apoptosis.

[0253] BAG3 Levels in Human Failing Hearts: Western blot analysis of BAG3 levels and qPCR were performed to quantify BAG3 mRNA in failing and healthy hearts from individuals of African descent (Cohorts D and E). As shown in Figs. 4A-4C, BAG3 levels were significantly reduced in hearts harvested from transplant recipients with IDCM (p=0.0001, n=23) or ischemic HF (p<0.0001, n=16) compared to healthy controls (n=4). BAG3 mRNA levels from IDCM hearts (n=18) and from DCM hearts with ischemic DCM (n=13) were not different from BAG3 mRNA levels in controls (n=4). Targeted sequence analysis revealed that one heart contained the p.His83Gln variant, two contained the p.Pro63Ala+Pro380Ser variant, and one contained the p.Ala160dup insertion. BAG3 levels were elevated above normal in two p.Pro63Ala+Pro380 hearts, suggesting that expression of the BAG3 variant overrode the innate regulatory mechanism regulating BAG3 expression. In contrast, BAG3 levels were unaffected in p.His83Gln hearts and could not be measured in p.Ala160dup hearts, making them potential targets for gene therapy.

[0254] Table 1. Characteristics of individuals based on the presence or absence of BAG3 SNVs

Parameter SNV BAG3 (N=51) Without BAG3 SNV (N=458) p-value Age 55.6±13.4 54.5±13.6 0.578 Gender (% male) 58.8 61.1 0.748 Heart rate 72.4±12.9 75.5±3.3 0.119 Systolic pressure 126.6±15.3 124.1±18.5 0.348 Diastolic pressure 76.8±10.9 76.7±11.2 0.930 Initial ejection fraction 30.6±9.5 30.4±10.2 0.918 Change in ejection fraction after 6 months 4.6±9.8 4.3±10.2 0.851 Left ventricular baseline end-diastolic dimension (LVEDD) 6.3±1.3 6.4±1.2 0.594 LVEDD change after 6 months -0.2±1.1 -0.2±1.0 0.897 NYHA III or IV(%) 80.4 80.3 0.983 Implantable defibrillator (ICD) (%) 35.7 34.0 0.896 Diabetes (%) 24.4 37.5 0.083 Preparations: Beta blockers (%) 88.2 82.3 0.287 ACE Inhibitors or ARBs (%) 98.0 92.8 0.234 Aldosterone antagonists (%) 33.3 35.4 0.773 Hydralazine and Isosorbide dinitrate (%) 54.1 44.9 0.292

[0255] Table 2A. BAG3 mutation frequencies by pathology and data source

63/380 Ala160dup His83Gln A479V Uncorrected incidence Corrected for multiple alleles Research and pathology GRAPH Non-ischemic 4/255 or 1.57% 18/249 or 7.23% 9/246 or 3.66% 4/255 or 1.57% 14.02% 13.78% Ischemic 0/87 or 0.00% 6/87 or 6.90% 2/87 or 2.30% 0/87 or 0.00% 9.20% 9.03% GRACE Non-ischemic 1/88 or 1.14% 5/89 or 5.62% 0/89 or 0.00% 1/88 or 1.14% 7.89% 7.75% Ischemic 0/20 or 0.00% 0/20 or 0.00% 1/20 or 5.00% 0/19 or 0.00% 5.00% 4.91% IMAC2 Non-ischemic 0/58 or 0.00% 3/58 or 5.17% 2/58 or 3.45% 1/58 or 1.72% 10.34% 10.16% Combination of all studies Non-ischemic 5/401 or 1.25% 26/396 or 6.57% 11/393 or 2.80% 6/401 or 1.50% 12.11% 11.90% Ischemic 0/107 or 0.00% 6/107 or 5.61% 3/107 or 2.80% 0/106 or 0.00% 8.41% 8.26% European reference data Non-ischemic 0/359 or 0.00% 0/359 or 0.00% 0/359 or 0.00% 0/359 or 0.00% 0.00% 0.00% gnomAD Population Data gnomAD Of African descent 263/12015 or 2.19% 627/11682 or 5.37% 250/12015 or 2.08% 76/12019 or 0.63% 10.27% 10.09% 263/12015 or 2.19% gnomAD of European origin 9/63271 or 0.01% 0/62112 or 0.00% 5/63345 or 0.01% 0/63354 or 0.00% 0.022% 0.022% 9/63271 or 0.01%

[0256] Table 2B: Analysis of Black and Caucasian Patients by BAG3 Mutations (All with Non-Ischemic HF)

Sign Any BAG3 mutation Without BAG3 mutation Total p-value Testing method Any BAG3 mutation by group, n (%) <.0001 Fisher's exact test GRAHF, GRACE, and IMAC2 patients 42 (10.4%) 360 (89.6%) 402 (100.0%) Reference European patients 0 (0.0%) 359 (100%) 359 (100.0%) Total 42 (5.5%) 719 (94.5%) 761 (100.0%)

[0257] Discussion

[0258] BAG3 regulates important cellular functions including protein quality control, apoptosis, and excitation-contraction coupling. ¹⁷ BAG3 levels are decreased in end-stage heart failure and in models of left ventricular dysfunction. The functional significance of BAG3 deficiency was demonstrated by knocking down BAG3 with siRNA that disrupts excitation-contraction coupling, and these BAG3 haploinsufficient mice exhibited significant LV dysfunction, decreased autophagy, and increased apoptosis at 10 weeks of age. ^32,35 In the present study, two common (>1%) non-synonymous SNVs, a common double heterozygote variant and an in-frame three-nucleotide insertion, were identified in individuals of African descent with DCM that were not observed at appreciable levels in individuals of European descent with DCM (0.00%). The results of the present study differ from earlier studies in that they show both a reduction in critical BAG3 function and an exclusivity of the mutations in African Americans.

[0259] Four BAG3 variants were annotated by ClinVar ⁴⁰ as benign (p.Pro63Ala), benign/likely benign (p.Ala160dup; p.Pro380Ser), or “conflicting interpretations of pathogenicity” (p.His83Gln and p.Ala479Val). Statistical analyses of in silico pathogenicity prediction algorithms, improved software, and computational predictions were unreliable for annotation. ⁴¹ Thus, pathogenicity was based on the finding that: 1) they are found in highly conserved regions of BAG3; 2) their function is lost in vitro; 3) they are located in domains that correspond to the functional consequences of the variant; and 4) the four SNVs collectively significantly alter outcome in patients with DCM. However, evidence supporting the pathogenicity of these SNVs would be strengthened by analysis of familial recurrence and segregation ^42,43 .

[0260] The variants identified in the present study have not been previously identified in cohorts of probands with familial DCM. For example, in four studies that identified BAG3 variants in independent case-nulls with familial DCM, fewer than 16 patients were identified as having African ancestry. ^20,44-46 In a large whole-exome array association study, investigators identified a BAG3 locus (c.451T>C, p.Cys151Arg) that was associated with a reduced risk of DCM; however, this variant is common in both Europeans (allele frequency 0.2163) and Africans (allele frequency 0.03) and thus did not meet the criteria for inclusion in the present analysis. ³⁰

[0261] Earlier studies in individuals of European descent support the findings of the present study. BAG3 variants were most common in cohorts of probands that were ethnically or geographically isolated: 15% in null patients recruited from an isolated population in the Gaspésie region of Quebec; ²⁰ 6.7% in a single-center Polish study, ¹⁹ 3.5% in a European population ⁴⁴ and 2.25% (7/311) in a large US study ⁴⁵ .

[0262] The results herein confirm that unique BAG3 variants found almost exclusively in individuals of African descent negatively impact outcomes in patients with IDCM and possibly ischemic DCM. This study of BAG3 genetic variations that are selective for African Americans highlights the importance of considering biological differences as one of several factors driving the phenotypic differences observed in different patient populations.

[0263] Links

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[0264] Equivalents

Those skilled in the art will recognize, or be able to determine using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following claims.

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SEQUENCE LIST

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Thr Gln Pro Asp Lys Gln Cys Gly Gln Val Ala Ala Ala Ala Ala Ala

145 150 155 160

Gln Pro Pro Ala Ser His Gly Pro Glu Arg Ser Gln Ser Pro Ala Ala

165 170 175

Ser Asp Cys Ser Ser Ser Ser Ser Ser Ala Ser Leu Pro Ser Ser Gly

180 185 190

Arg Ser Ser Leu Gly Ser His Gln Leu Pro Arg Gly Tyr Ile Ser Ile

195 200 205

Pro Val Ile His Glu Gln Asn Val Thr Arg Pro Ala Ala Gln Pro Ser

210 215 220

Phe His Gln Ala Gln Lys Thr His Tyr Pro Ala Gln Gln Gly Glu Tyr

225 230 235 240

Gln Thr His Gln Pro Val Tyr His Lys Ile Gln Gly Asp Asp Trp Glu

245 250 255

Pro Arg Pro Leu Arg Ala Ala Ser Pro Phe Arg Ser Ser Val Gln Gly

260 265 270

Ala Ser Ser Arg Glu Gly Ser Pro Ala Arg Ser Ser Thr Pro Leu His

275 280 285

Ser Pro Ser Pro Ile Arg Val His Thr Val Val Asp Arg Pro Gln Gln

290 295 300

Pro Met Thr His Arg Glu Thr Ala Pro Val Ser Gln Pro Glu Asn Lys

305 310 315 320

Pro Glu Ser Lys Pro Gly Pro Val Gly Pro Glu Leu Pro Pro Gly His

325 330 335

Ile Pro Ile Gln Val Ile Arg Lys Glu Val Asp Ser Lys Pro Val Ser

340 345 350

Gln Lys Pro Pro Pro Pro Ser Glu Lys Val Glu Val Lys Val Pro Pro

355 360 365

Ala Pro Val Pro Cys Pro Pro Pro Ser Pro Gly Pro Ser Ala Val Pro

370 375 380

Ser Ser Pro Lys Ser Val Ala Thr Glu Glu Arg Ala Ala Pro Ser Thr

385 390 395 400

Ala Pro Ala Glu Ala Thr Leu Pro Lys Pro Gly Glu Ala Glu Ala Pro

405 410 415

Pro Lys His Pro Gly Val Leu Lys Val Glu Ala Ile Leu Glu Lys Val

420 425 430

Gln Gly Leu Glu Gln Ala Val Asp Asn Phe Glu Gly Lys Lys Thr Asp

435 440 445

Lys Lys Tyr Leu Met Ile Glu Glu Tyr Leu Thr Lys Glu Leu Leu Ala

450 455 460

Leu Asp Ser Val Asp Pro Glu Gly Arg Ala Asp Val Arg Gln Ala Arg

465 470 475 480

Arg Asp Gly Val Arg Lys Val Gln Thr Ile Leu Glu Lys Leu Glu Gln

485 490 495

Lys Ala Ile Asp Val Pro Gly Gln Val Gln Val Tyr Glu Leu Gln Pro

500 505 510

Ser Asn Leu Glu Ala Asp Gln Pro Leu Gln Ala Ile Met Glu Met Gly

515 520 525

Ala Val Ala Ala Asp Lys Gly Lys Lys Asn Ala Gly Asn Ala Glu Asp

530 535 540

Pro His Thr Glu Thr Gln Gln Pro Glu Ala Thr Ala Ala Ala Thr Ser

545 550 555 560

Asn Pro Ser Ser Met Thr Asp Thr Pro Gly Asn Pro Ala Ala Pro

565 570 575

<210> 5

<211> 574

<212> Protein

<213> Homo sapiens

<400> 5

Met Ser Ala Ala Thr His Ser Pro Met Gln Val Ala Ser Gly Asn

1. 5 10 15

Gly Asp Arg Asp Pro Leu Pro Pro Gly Trp Glu Ile Lys Ile Asp Pro

20 25 30

Gln Thr Gly Trp Pro Phe Phe Val Asp His Asn Ser Arg Thr Thr Thr

35 40 45

Trp Asn Asp Pro Arg Val Pro Ser Glu Gly Pro Lys Glu Thr Pro Ser

50 55 60

Ser Ala Asn Gly Pro Ser Arg Glu Gly Ser Arg Leu Pro Pro Ala Arg

65 70 75 80

Glu Gly His Pro Val Tyr Pro Gln Leu Arg Pro Gly Tyr Ile Pro Ile

85 90 95

Pro Val Leu His Glu Gly Ala Glu Asn Arg Gln Val His Pro Phe His

100 105 110

Val Tyr Pro Gln Pro Gly Met Gln Arg Phe Arg Thr Glu Ala Ala Ala

115 120 125

Ala Ala Pro Gln Arg Ser Gln Ser Pro Leu Arg Gly Met Pro Glu Thr

130 135 140

Thr Gln Pro Asp Lys Gln Cys Gly Gln Val Ala Ala Ala Ala Ala Ala

145 150 155 160

Gln Pro Pro Ala Ser His Gly Pro Glu Arg Ser Gln Ser Pro Ala Ala

165 170 175

Ser Asp Cys Ser Ser Ser Ser Ser Ser Ala Ser Leu Pro Ser Ser Gly

180 185 190

Arg Ser Ser Leu Gly Ser His Gln Leu Pro Arg Gly Tyr Ile Ser Ile

195 200 205

Pro Val Ile His Glu Gln Asn Val Thr Arg Pro Ala Ala Gln Pro Ser

210 215 220

Phe His Gln Ala Gln Lys Thr His Tyr Pro Ala Gln Gln Gly Glu Tyr

225 230 235 240

Gln Thr His Gln Pro Val Tyr His Lys Ile Gln Gly Asp Asp Trp Glu

245 250 255

Pro Arg Pro Leu Arg Ala Ala Ser Pro Phe Arg Ser Ser Val Gln Gly

260 265 270

Ala Ser Ser Arg Glu Gly Ser Pro Ala Arg Ser Ser Thr Pro Leu His

275 280 285

Ser Pro Ser Pro Ile Arg Val His Thr Val Val Asp Arg Pro Gln Pro

290 295 300

Met Thr His Arg Glu Thr Ala Pro Val Ser Gln Pro Glu Asn Lys Pro

305 310 315 320

Glu Ser Lys Pro Gly Pro Val Gly Pro Glu Leu Pro Pro Gly His Ile

325 330 335

Pro Ile Gln Val Ile Arg Lys Glu Val Asp Ser Lys Pro Val Ser Gln

340 345 350

Lys Pro Pro Pro Pro Ser Glu Lys Val Glu Val Lys Val Pro Pro Ala

355 360 365

Pro Val Pro Cys Pro Pro Pro Ser Pro Gly Pro Ser Ala Val Pro Ser

370 375 380

Ser Pro Lys Ser Val Ala Thr Glu Glu Arg Ala Ala Pro Ser Thr Ala

385 390 395 400

Pro Ala Glu Ala Thr Leu Pro Lys Pro Gly Glu Ala Glu Ala Pro Pro

405 410 415

Lys His Pro Gly Val Leu Lys Val Glu Ala Ile Leu Glu Lys Val Gln

420 425 430

Gly Leu Glu Gln Ala Val Asp Asn Phe Glu Gly Lys Lys Thr Asp Lys

435 440 445

Lys Tyr Leu Met Ile Glu Glu Tyr Leu Thr Lys Glu Leu Leu Ala Leu

450 455 460

Asp Ser Val Asp Pro Glu Gly Arg Ala Asp Val Arg Gln Ala Arg Arg

465 470 475 480

Asp Gly Val Arg Lys Val Gln Thr Ile Leu Glu Lys Leu Glu Gln Lys

485 490 495

Ala Ile Asp Val Pro Gly Gln Val Gln Val Tyr Glu Leu Gln Pro Ser

500 505 510

Asn Leu Glu Ala Asp Gln Pro Leu Gln Ala Ile Met Glu Met Gly Ala

515 520 525

Val Ala Ala Asp Lys Gly Lys Lys Asn Ala Gly Asn Ala Glu Asp Pro

530 535 540

His Thr Glu Thr Gln Gln Pro Glu Ala Thr Ala Ala Ala Thr Ser Asn

545 550 555 560

Pro Ser Ser Met Thr Asp Thr Pro Gly Asn Pro Ala Ala Pro

565 570

<210> 6

<211> 575

<212> Protein

<213> Homo sapiens

<400> 6

Met Ser Ala Ala Thr His Ser Pro Met Gln Val Ala Ser Gly Asn

1. 5 10 15

Gly Asp Arg Asp Pro Leu Pro Pro Gly Trp Glu Ile Lys Ile Asp Pro

20 25 30

Gln Thr Gly Trp Pro Phe Phe Val Asp His Asn Ser Arg Thr Thr Thr

35 40 45

Trp Asn Asp Pro Arg Val Pro Ser Glu Gly Pro Lys Glu Thr Pro Ser

50 55 60

Ser Ala Asn Gly Pro Ser Arg Glu Gly Ser Arg Leu Pro Pro Ala Arg

65 70 75 80

Glu Gly His Pro Val Tyr Pro Gln Leu Arg Pro Gly Tyr Ile Pro Ile

85 90 95

Pro Val Leu His Glu Gly Ala Glu Asn Arg Gln Val His Pro Phe His

100 105 110

Val Tyr Pro Gln Pro Gly Met Gln Arg Phe Arg Thr Glu Ala Ala Ala

115 120 125

Ala Ala Pro Gln Arg Ser Gln Ser Pro Leu Arg Gly Met Pro Glu Thr

130 135 140

Thr Gln Pro Asp Lys Gln Cys Gly Gln Val Ala Ala Ala Ala Ala Ala

145 150 155 160

Gln Pro Pro Ala Ser His Gly Pro Glu Arg Ser Gln Ser Pro Ala Ala

165 170 175

Ser Asp Cys Ser Ser Ser Ser Ser Ser Ala Ser Leu Pro Ser Ser Gly

180 185 190

Arg Ser Ser Leu Gly Ser His Gln Leu Pro Arg Gly Tyr Ile Ser Ile

195 200 205

Pro Val Ile His Glu Gln Asn Val Thr Arg Pro Ala Ala Gln Pro Ser

210 215 220

Phe His Gln Ala Gln Lys Thr His Tyr Pro Ala Gln Gln Gly Glu Tyr

225 230 235 240

Gln Thr His Gln Pro Val Tyr His Lys Ile Gln Gly Asp Asp Trp Glu

245 250 255

Pro Arg Pro Leu Arg Ala Ala Ser Pro Phe Arg Ser Ser Val Gln Gly

260 265 270

Ala Ser Ser Arg Glu Gly Ser Pro Ala Arg Ser Ser Thr Pro Leu His

275 280 285

Ser Pro Ser Pro Ile Arg Val His Thr Val Val Asp Arg Pro Gln Gln

290 295 300

Pro Met Thr His Arg Glu Thr Ala Pro Val Ser Gln Pro Glu Asn Lys

305 310 315 320

Pro Glu Ser Lys Pro Gly Pro Val Gly Pro Glu Leu Pro Pro Gly His

325 330 335

Ile Pro Ile Gln Val Ile Arg Lys Glu Val Asp Ser Lys Pro Val Ser

340 345 350

Gln Lys Pro Pro Pro Pro Ser Glu Lys Val Glu Val Lys Val Pro Pro

355 360 365

Ala Pro Val Pro Cys Pro Pro Pro Ser Pro Gly Pro Ser Ala Val Pro

370 375 380

Ser Ser Pro Lys Ser Val Ala Thr Glu Glu Arg Ala Ala Pro Ser Thr

385 390 395 400

Ala Pro Ala Glu Ala Thr Pro Pro Lys Pro Gly Glu Ala Glu Ala Pro

405 410 415

Pro Lys His Pro Gly Val Leu Lys Val Glu Ala Ile Leu Glu Lys Val

420 425 430

Gln Gly Leu Glu Gln Ala Val Asp Asn Phe Glu Gly Lys Lys Thr Asp

435 440 445

Lys Lys Tyr Leu Met Ile Glu Glu Tyr Leu Thr Lys Glu Leu Leu Ala

450 455 460

Leu Asp Ser Val Asp Pro Glu Gly Arg Ala Asp Val Arg Gln Ala Arg

465 470 475 480

Arg Asp Gly Val Arg Lys Val Gln Thr Ile Leu Glu Lys Leu Glu Gln

485 490 495

Lys Ala Ile Asp Val Pro Gly Gln Val Gln Val Tyr Glu Leu Gln Pro

500 505 510

Ser Asn Leu Glu Ala Asp Gln Pro Leu Gln Ala Ile Met Glu Met Gly

515 520 525

Ala Val Ala Ala Asp Lys Gly Lys Lys Asn Ala Gly Asn Ala Glu Asp

530 535 540

Pro His Thr Glu Thr Gln Gln Pro Glu Ala Thr Ala Ala Ala Thr Ser

545 550 555 560

Asn Pro Ser Ser Met Thr Asp Thr Pro Gly Asn Pro Ala Ala Pro

565 570 575

<---

Claims (19)

1. A method for diagnosing a patient with a cardiac disease, comprising: identifying in a patient sample at least one genetic variant of Bcl2-associated athanogene 3 (BAG3) compared to a BAG3 nucleic acid reference sequence, wherein said genetic variant comprises a three-nucleotide insertion that adds an alanine at position 160 (p.Ala160dup, 10:121429647 A/AGCG; rs139438727), or a single-nucleotide variant (SNV) of an in-frame insertion comprising: p.Pro63Ala (10:121429369 C/G; rs133031999); p.His83Gln (10:151331972; rs151331972); Pro380Ser (10: 121436204 C/T; rs144692954); Ala479Val (10: 121436502 C/T, rs34656239), or a combination thereof, and where detection of said genetic variant indicates that the heart disease from which the patient suffers can be treated by increasing BAG3 levels. 2. A method of treating a patient with a cardiac disease, wherein the patient has at least one genetic variant of Bcl2-associated athanogene 3 (BAG3) compared to a BAG3 nucleic acid reference sequence, wherein said genetic variant comprises a three-nucleotide insertion that adds an alanine at position 160 (p.Ala160dup, 10:121429647 A/AGCG; rs139438727), or a single-nucleotide variant (SNV) of an in-frame insertion comprising: p.Pro63Ala (10:121429369 C/G; rs133031999); p.His83Gln (10:151331972; rs151331972); Pro380Ser (10: 121436204 C/T; rs144692954); Ala479Val (10: 121436502 C/T, rs34656239), or a combination thereof, and administering to a patient having said genetic variant a therapeutically effective amount of the agent, wherein the agent comprises an expression vector for expressing the BAG3 protein or its biologically active fragment. 3. The method of claim 1, wherein the BAG3 polypeptides encoded by the BAG3 genetic variants negatively affect the outcome in patients with non-ischemic or ischemic dilated cardiomyopathy. 4. The method according to claim 1 or 2, wherein the genetic variant comprises a three-nucleotide insertion that adds an alanine at position 160 (p.Ala160dup, 10:121429647 A/AGCG; rs139438727). 5. The method according to claim 1 or 2, wherein the genetic variant comprises: p.Pro63Ala (10:121429369 C/G; rs133031999); p.His83Gln (10:151331972; rs151331972); Ala479Val (10:121436502 C/T, rs34656239) or combinations thereof. 6. The method according to claim 2, wherein the expression vector comprises a viral vector, a cardiotropic vector, a plasmid, or a yeast vector. 7. The method according to claim 6, wherein the cardiotropic vector comprises an adenovirus vector, an adeno-associated virus vector (AAV), a coxsackievirus vector, a cytomegalovirus vector, an Epstein-Barr virus vector, a parvovirus vector, or hepatitis virus vectors. 8. The method according to claim 7, wherein the expression vector is a cardiotropic pseudotyped viral vector. 9. The method according to claim 1 or 2, wherein the heart disease is heart failure. 10. A method for treating a patient with a cardiac disease, wherein said patient has at least one nucleotide variant (NV) of an in-frame insertion in Bcl2-associated athanogen 3 (BAG3) compared to a BAG3 nucleic acid reference sequence, wherein the in-frame insertion comprises a three-nucleotide reading frame that adds an alanine at position 160 (p.Ala160dup, 10:121429647 A/AGCG; rs139438727), comprising administering to said patient a therapeutically effective amount of an agent comprising an expression vector for expressing the BAG3 protein or a biologically active fragment thereof. 11. A method for identifying a patient with a worse prognosis for a cardiac disease comprising screening a sample from the patient for the presence of a nucleotide variant (NV) of the Bcl2-associated athanogene 3 (BAG3) in-frame insertion type compared to a BAG3 nucleic acid reference sequence, wherein the in-frame insertion type comprises a three-nucleotide reading frame that adds an alanine at position 160 (p.Ala160dup, 10:121429647 A/AGCG; rs139438727), and wherein detection of the nucleotide variant (NV) of the BAG3 in-frame insertion type indicates a worse prognosis for the patient with the cardiac disease. 12. A method for identifying a patient at risk for cardiac disease, comprising screening a sample from the patient for the presence of a nucleotide variant (NV) of the in-frame insertion type of Bcl2-associated athanogene 3 (BAG3) compared to a BAG3 nucleic acid reference sequence, wherein the in-frame insertion type comprises a three-nucleotide reading frame that adds an alanine at position 160 (p.Ala160dup, 10:121429647 A/AGCG; rs139438727), wherein detection of a nucleotide variant (NV) of the in-frame insertion type of BAG3 indicates that the patient is at risk for cardiac disease. 13. A method for identifying a patient at risk for cardiac disease, comprising screening a sample from the patient for the presence of a Bcl2-associated athanogene 3 (BAG3) genetic variant, wherein the BAG3 genetic variant comprises p.Ala160dup (10:121429647 A/AGCG; rs139438727); p.Pro63Ala (10:121429369 C/G; rs133031999); p.His83Gln (10:151331972; rs151331972); Ala479Val (10:121436502 C/T, rs34656239), or combinations thereof. 14. Use of a kit comprising one or more probes conjugated to a detectable label for identifying BCL2-associated athanogene 3 (BAG3) variants in a biological sample, wherein the BAG3 variants comprise one or more of p.Ala160dup (10:121429647 A/AGCG; rs139438727); p.Pro63Ala (10:121429369 C/G; rs133031999); p.His83Gln (10:151331972; rs151331972); or Ala479Val (10:121436502 C/T, rs34656239), for identifying BCL2-associated athanogene 3 (BAG3) variants in a biological sample. 15. The use according to paragraph 14, where the identification of the said variants makes it possible to predict whether or not the patient will benefit from the treatment. 16. A method of treating an individual at risk of or suffering from heart failure, comprising administering to the individual a pharmaceutical composition comprising a therapeutically effective amount of an expression vector for expressing a BAG3 protein or a biologically active fragment thereof, wherein the individual has at least one genetic variant of BAG3 compared to a BAG3 nucleic acid reference sequence, wherein said genetic variant of BAG3 comprises a three-nucleotide insertion that adds an alanine at position 160 (p.Ala160dup, 10:121429647 A/AGCG; rs139438727), or a single-nucleotide variant (SNV) of an in-frame insertion comprising: p.Pro63Ala (10:121429369 C/G; rs133031999); p.His83Gln (10: 151331972; rs151331972); Ala479Val (10: 121436502 C/T, rs34656239). 17. The method of claim 16, wherein the in-frame insertion comprises a three-nucleotide insertion that adds an alanine at position 160 (p.Ala160dup, 10:121429647 A/AGCG; rs139438727). 18. The method of any one of claims 1-9, wherein the genetic variants are not the cause of the disease, but negatively affect the outcome of patients with non-ischemic or ischemic dilated cardiomyopathy. 19. The method according to any one of claims 1-13, 16 or 17, wherein the genetic variant BAG3 comprises a combination of: p.Pro63Ala (10:121429369 C/G; rs133031999) and Pro380Ser (10:121436204 C/T; rs144692954).