Bag3 as target for therapy of heart failure

Abstract

To provide a pharmaceutical composition for administration to a patient, for example a patient having heart failure, a treatment method and a method for identifying a candidate therapeutic agent.SOLUTION: A pharmaceutical composition contains one or more BCL2-associated athanogene 3 (BAG3) molecules, or an agent for modulating the expression of BAG3.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 61 / 934,483, filed January 31, 2014. For purposes of U.S. applications which may claim the benefit of U.S. Provisional Patent Application No. 61 / 934,483, the contents of the earlier filed application are incorporated herein by reference in their entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under Grant No. P01HL091799 awarded by the National Institutes of Health. The U.S. Government may have certain rights in this invention.

[0003] FIELD OF THEINVENTION Aspects of the present invention relate to compositions and methods of treatment for cardiac diseases or disorders, such as heart failure, cardiovascular diseases or disorders, or skeletal muscle diseases associated with expression of Bcl-2 associated anthanogene-3 (BAG3). Assays for identifying novel therapeutic agents are also provided. [Background technology]

[0004] background Heart failure (HF), secondary to systolic dysfunction and cardiac enlargement, affects over 5 million individuals in the United States and is a significant cause of both morbidity and mortality. Approximately 30% of these patients have non-ischemic disease or idiopathic dilated cardiomyopathy (IDC). In the majority of patients with IDC, the causative factor remains undetermined, and emerging evidence suggests that up to 35% of individuals with IDC have an affected first-degree relative (Jefferies JL TJ. Lancet. 2010;375:752-762 (Non-Patent Document 1)) and that IDC may be associated with genetic abnormalities in 20-35% of individuals, leading to the use of the nomenclature familial dilated cardiomyopathy (FDC) (Judge DP et al, Journal of Cardiovascular Translational Research. 2008;1:144-154 (Non-Patent Document 2); Hershberger RE et al., Circulation. Cardiovascular Genetics. 2010;3:155-161 (Non-Patent Document 3)). Indeed, mutations in more than 30 genes have been identified as causative factors (Hershberger RE et al., Circulation. Heart Failure. 2009;2:253-261 (Non-Patent Document 4)), and the most common pattern of inheritance is autosomal dominant with reduced penetrance and variable expressivity (Morales A, Hershberger RE. Current Cardiology Reports. 2013;15:375 (Non-Patent Document 5)).

[0005] Mutations that cause FDC are found in a wide range of protein-coding genes 6However, the majority of mutations that cause FDC occur in genes that code for sarcomeric proteins, or in the complex network of proteins in the Z-disc (Chang AN, Potter JD. Heart Failure Reviews. 2005;10:225-235 (Non-Patent Document 6); Selcen D. Myofibrillar myopathies. Neuromuscular disorders:NMD. 2011;21:161-171 (Non-Patent Document 7)). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Jefferies JL TJ. Lancet. 2010;375:752-762 [Non-Patent Document 2] Judge DP et al, Journal of Cardiovascular Translational Research. 2008;1:144-154 [Non-Patent Document 3] Hershberger RE et al., Circulation. Cardiovascular Genetics. 2010;3:155-161 [Non-Patent Document 4] Hershberger RE et al., Circulation. Heart Failure. 2009;2:253-261 [Non-Patent Document 5] Morales A, Hershberger RE. Current Cardiology Reports. 2013;15:375 [Non-Patent Document 6] Chang AN, Potter JD. Heart Failure Reviews. 2005;10:225-235 [Non-Patent Document 7] Selcen D. Myofibrillar myopathies. Neuromuscular disorders:NMD. 2011;21:161-171 Summary of the Invention

[0007] overview The present invention relates to compositions for modulating the expression of Bcl-2-associated anthanogene-3 (BAG3) molecules, methods for identifying agents for the treatment of cardiac diseases or disorders. In particular, these agents include expression vectors encoding Bcl-2-associated anthanogene-3 (BAG3) molecules, Bcl-2-associated anthanogene-3 (BAG3) nucleic acid sequences, Bcl-2-associated anthanogene-3 (BAG3) peptides, or any other agents that modulate BAG3 expression. Such agents are identified by the methods embodied herein. Conditions to be treated include, for example, heart failure, cardiomyopathy, and the like. In some embodiments, the target tissue is cardiac tissue, such as, for example, myocardium.

[0008] Briefly, the results obtained herein identify rare novel variants in families with familial dilated cardiomyopathy. A general aspect of the present invention relates to the treatment of patients identified as having a variant of the BAG3 molecule.

[0009] Patients with idiopathic dilated cardiomyopathy who do not have mutations in the BAG3 gene have been found to have half the normal levels of BAG3, and the same reduction has been found in the hearts of patients with familial disease and BAG3 mutations. Another general aspect of the invention is the treatment of patients with agents that modulate the expression of the BAG3 molecule, preferably with agents that result in overexpression of the normal BAG3 molecule.

[0010] The results also showed that mice with heart failure due to aortic banding (a commonly used model for heart failure testing) had substantially less BAG3 than normal controls, and this reduction in BAG3 closely resembled that seen in humans.

[0011] The results also showed that when AAV vector (AAV9) was administered in vivo to overexpress BAG3 in the heart, strong overexpression was observed. In another general embodiment, the agent comprises a cardioactive vector expressing a BAG3 molecule. In some embodiments, the vector is an AAV9 vector.

[0012] It was also found that when BAG3 protein was overexpressed using AAV9 vectors in the hearts of mice with heart failure (and low levels of BAG3) secondary to aortic banding, normal left ventricular performance was reconstituted. These results provide evidence that BAG3 levels are reduced in failing mouse hearts and that AAV9 vectors overexpress BAG3 in desired targets in the heart, resulting in a change in function equivalent to normal function.

[0013] [The present invention 1001] 1. A method of treating a patient suffering from or at risk of developing a muscle-related disease or disorder, comprising: Administering to a patient a therapeutically effective amount of an agent, which modulates the expression or amount of a BCL2-associated athanogene 3 (BAG3) molecule, protein, or peptide thereof in a target cell or tissue compared to a normal control. [The present invention 1002] 1001. The method of claim 1001, wherein said agent comprises an expression vector expressing a BAG3 protein or an active fragment thereof, an oligonucleotide, or a combination thereof. [The present invention 1003] 1003. The method of claim 1002, wherein said expression vector further comprises a promoter, said promoter comprising an inducible promoter, a constitutive promoter, a bicistronic promoter, or a tissue-specific promoter. [The present invention 1004] 1001. The method of claim 1001, wherein the expression vector comprises a viral vector, a cardioactive vector, a plasmid, or a yeast vector. [The present invention 1005] 1005. The method of claim 1004, wherein said cardioactive 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 a hepatitis virus vector. [The present invention 1006] 1001. The method of claim 1001, wherein said expression vector is a cardioactive pseudotyped viral vector. [The present invention 1007] The method of claim 1001, wherein said agent comprises a protein or a peptide thereof, a peptidomimetic, a small molecule, an organic or inorganic compound, a synthetic or naturally occurring compound. [The present invention 1008] The method of claim 10, wherein said muscle-related disease or disorder is a cardiac disease or disorder. [The present invention 1009] 1. A method for identifying a candidate therapeutic agent, comprising the steps of: contacting the biological sample or cells with a candidate therapeutic agent; assaying for modulation of expression or amount of BCL2-associated aganocyte gene 3 (BAG3) protein or peptides thereof in the presence or absence of said candidate therapeutic agent, wherein said agent modulates expression of BAG3 protein relative to a normal control; Thereby identifying candidate therapeutic agents. [The present invention 1010] The method of claim 1009, wherein said candidate therapeutic agent increases the expression or amount of BAG3 in the cell compared to a baseline control. [The present invention 1011] The method of the present invention 1009, wherein the assay for quantifying BAG3 or measuring expression of BAG3 comprises an immunoassay, a phosphorylation assay, an enzyme assay, a biological assay, a biochip assay, a blot, a hybridization assay, a cell-based assay, a high-throughput screening assay, a chromatography, a chemical assay, a phage display assay, a lab-on-a-chip, a microfluidic-based assay, a microarray, a microchip, a nanotube-based assay, a colorimetric assay, a spectrophotometric assay, or a combination thereof. [The present invention 1012] 1. A method of diagnosing a patient having a cardiac disease or disorder, comprising: obtaining a biological sample from the patient; Measuring expression of BCL2-associated aganocyte gene 3 (BAG3) polynucleotides, polypeptides, or variants thereof, or quantifying BAG3 polynucleotides, polypeptides, or variants thereof, and / or identifying mutations in the BAG3 molecule, wherein detection of decreased amount or expression of BAG3 compared to healthy controls or detection of mutations in the BAG3 molecule is diagnostic of a cardiac disease or disorder. [The present invention 1013] The method of claim 1012, wherein the mutation comprises a deletion or variant of a BAG3 polynucleotide or polypeptide. [The present invention 1014] The method of any one of claims 10 to 12, wherein the biological sample comprises a body fluid, a peptide, a polypeptide, an oligonucleotide, a polynucleotide, a cell, a tissue, or a combination thereof. [The present invention 1015] The method of the present invention, wherein the step of measuring expression of or quantifying a BCL2-associated aganocyte 3 (BAG3) molecule and / or identifying a mutation in a BAG3 molecule comprises an immunoassay, a phosphorylation assay, an enzyme assay, a biological assay, a biochip assay, a blot, a hybridization assay, a cell-based assay, a high-throughput screening assay, a chromatography, a chemical assay, a phage display assay, a lab-on-a-chip, a microfluidic-based assay, a microarray, a microchip, a nanotube-based assay, a colorimetric assay, a spectrophotometric assay, or a combination thereof. [The present invention 1016] A high throughput screening method for identifying candidate therapeutic agents comprising the steps of: contacting a support surface comprising BCL2-associated aganocyte gene 3 (BAG3) molecules with a candidate therapeutic agent; assaying for modulation of expression of BAG3 compared to a baseline control in the presence or absence of the candidate therapeutic agent, wherein the candidate therapeutic agent increases expression of BAG3; Thereby identifying candidate therapeutic agents. [The present invention 1017] The method of claim 1016, wherein said support surface comprises plastic, glass, beads, fibers, gels, electrochemical detectors, nanotubes, porous strips, paper, matrices, or combinations thereof. [The present invention 1018] The method of the present invention, wherein the assay for measuring expression of BCL2-associated aganocyte gene 3 (BAG3) comprises an immunoassay, an enzymatic assay, a biological assay, a biochip assay, a blot, a hybridization assay, a cell-based assay, a high-throughput screening assay, a chromatography, a chemical assay, a phage display assay, a lab-on-a-chip, a microfluidic-based assay, a microarray, a microchip, a nanotube-based assay, a colorimetric assay, a spectrophotometric assay, or a combination thereof. [The present invention 1019] A pharmaceutical composition comprising a therapeutically effective amount of an agent identified by the method of any one of claims 1009 to 1016. [The present invention 1020] A therapeutic agent that modulates the expression or amount of BCL2-associated aganocyte gene 3 (BAG3) molecules in a cell, in vivo, or in vitro. [The present invention 1021] An expression vector encoding a BCL2-associated aganocyte gene 3 (BAG3) molecule or its complementary sequence. [The present invention 1022] The expression vector of the present invention 1021, wherein said vector is cardioactive. [The present invention 1023] A pharmaceutical composition comprising an expression vector encoding a BCL2-associated aganocyte gene 3 (BAG3) molecule or its complementary sequence. [The present invention 1024] A cell comprising an expression vector encoding a BCL2-associated aganocyte gene 3 (BAG3) molecule or its complementary sequence. [The present invention 1025] 1. A method of treating a subject at risk for or suffering from heart failure, comprising: Administering to a subject a pharmaceutical composition comprising a therapeutically effective amount of at least one agent that regulates the expression or amount of a BCL2-associated aganocyte 3 (BAG3) molecule, wherein the agent comprises an expression vector encoding the BCL2-associated aganocyte 3 (BAG3) molecule. [The present invention 1026] The method of claim 1025, wherein said agent increases expression of BAG3 in a cell or tissue relative to a baseline control. [The present invention 1027] 1. A method for diagnosing or identifying a patient at risk for developing idiopathic dilated cardiomyopathy, comprising: obtaining a biological sample from the patient; Measuring expression of BCL2-associated aganocyte 3 (BAG3) polynucleotide or polypeptide, or quantifying a BAG3 polynucleotide or polypeptide, and / or identifying mutations in a BAG3 molecule, wherein detection of reduced amount or expression of BAG3 compared to a healthy control, or detection of mutations in a BAG3 molecule, is diagnostic of a cardiac disease or disorder. [The present invention 1028] The method of claim 1027, wherein the mutation comprises a deletion or variant of a BAG3 polynucleotide or polypeptide. [The present invention 1029] The method of any one of claims 10 to 12, wherein the biological sample comprises a body fluid, a peptide, a polypeptide, an oligonucleotide, a polynucleotide, a cell, a tissue, or a combination thereof. [The present invention 1030] 1. A method of treating a subject at risk for or suffering from idiopathic dilated cardiomyopathy, comprising: Administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of at least one agent that modulates the expression or amount of a BCL2-associated aganocyte gene 3 (BAG3) molecule. [The present invention 1031] The method of claim 1030, wherein said agent increases the expression of BAG3 in a cell or tissue relative to a normal control. [The present invention 1032] The method of claim 1030, wherein the agent comprises a BCL2-associated aganocyte gene 3 (BAG3) molecule, an oligonucleotide, a polynucleotide, a polypeptide, a peptide, a BAG3 protein, an expression vector encoding a BAG3 protein or an active fragment thereof, or a combination thereof. [The present invention 1033] The method of claim 1032, wherein the expression vector further comprises a promoter, the promoter comprising an inducible promoter, a constitutive promoter, a bicistronic promoter, or a tissue-specific promoter. [The present invention 1034] The method of claim 1032, wherein the expression vector comprises a viral vector, a cardioactive vector, a plasmid, or a yeast vector. [The present invention 1035] The method of claim 1034, wherein the cardioactive 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 a hepatitis virus vector. [The present invention 1036] The method of claim 1034, wherein said expression vector is a cardioactive pseudotyped viral vector. [The present invention 1037] The method of claim 1030, wherein said agent comprises a protein or a peptide thereof, a peptidomimetic, a small molecule, an organic or inorganic compound, a synthetic or naturally occurring compound. [The present invention 1038] A method for modulating expression of a BCL2-associated aganocyte 3 (BAG3) molecule in vitro or in vivo, comprising the steps of: A step of contacting cells in vitro or administering to a patient a therapeutically effective amount of an agent, which modulates the expression or amount of BCL2-associated aganocyte gene 3 (BAG3) molecules, proteins, or peptides thereof in target cells or tissues in vitro or in vivo compared to normal controls. [The present invention 1039] The method of claim 1038, wherein said agent comprises an expression vector expressing a BAG3 protein or an active fragment thereof. [The present invention 1040] The method of claim 1039, wherein the expression vector comprises an oligonucleotide, a polynucleotide, or a combination thereof. [The present invention 1041] The method of claim 1039, wherein the expression vector further comprises a promoter, the promoter comprising an inducible promoter, a constitutive promoter, a bicistronic promoter, or a tissue-specific promoter. [The present invention 1042] The method of claim 1039, wherein the expression vector comprises a viral vector, a cardioactive vector, a plasmid, or a yeast vector. [The present invention 1043] The method of claim 1042, wherein the cardioactive 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 a hepatitis virus vector. [The present invention 1044] The method of claim 1042, wherein said expression vector is a cardioactive pseudotyped viral vector. [The present invention 1045] The method of claim 1030, wherein said agent comprises a protein or a peptide thereof, a peptidomimetic, a small molecule, an organic or inorganic compound, a synthetic or naturally occurring compound. [The present invention 1046] An expression vector comprising an adeno-associated viral vector serotype 9 (AAV9) encoding the BCL2-associated aganocyte gene 3 (BAG3) molecule. [The present invention 1047] 1046. The expression vector of the present invention, wherein said vector is cardioactive and expresses BAG3 in cardiac tissue. [The present invention 1048] The expression vector of the present invention 1046, which regulates the expression of a BAG3 molecule in cardiac tissue. [The present invention 1049] The expression vector of the present invention 1048, wherein the BAG3 protein is overexpressed in cardiac tissue compared to the normal control. Other aspects are described below. [Brief description of the drawings]

[0014] [Figure 1]FIG. 1 is a schematic showing the pedigree of BAG3-associated dilated cardiomyopathy. Males are indicated by boxes. Circles indicate females. Open symbols indicate unaffected individuals, and filled symbols indicate affected individuals. The presence or absence of the 10 nucleotide deletion in BAG3 is indicated by (+) or (-), respectively. Arrows denote probands. Asterisks are used to denote individuals whose DNA was used for whole-exome sequencing. Diagonal lines are used to denote deceased individuals. [Diagram 2] Figure 2A is a schematic diagram showing a sequencing alignment for a 10 nucleotide deletion in BAG3. Figure 2A discloses SEQ ID NOs: 9-49, respectively, in the order of appearance. Figure 2B is a schematic diagram showing representative Sanger sequencing of a deletion in the BAG3 gene of an affected individual. Figure 2B discloses SEQ ID NOs: 50 and 51, respectively, in the order of appearance. [Diagram 3] Figures 3A and 3B show low levels of BAG3 expression in patients with heart failure. Representative Western blots of BAG3 and GAPDH levels in non-failing (NF) and failing (F) human hearts are shown. The graph shows quantification of BAG3 protein levels in non-failing and failing human hearts. Values ​​are normalized to levels of GAPDH to account for variation in protein loading. Horizontal lines indicate the mean and standard error of the mean. Statistical analysis was performed using unpaired t-tests with Welch's correction for unequal variances. [Figure 4] Measurements of ejection fraction are shown in sham operated wild type mice, wild type mice injected with the AAV9-BAG3 construct, mice that underwent aortic banding (and developed heart failure), and mice that were ligated and had heart failure but were injected with the AAV9-BAG3 construct. As shown, BAG3 infusion normalized LV function in temporal correlation with BAG3 protein expression (approximately 5-6 weeks after infusion). [Diagram 5]Western blot showing BAG3 levels in patients. Shown are 3 lanes of "control" and 3 lanes of "patient". This is the affected patient IV-1. The "control" lane is from a non-failing human heart, i.e. a normal human heart, obtained at the time of tissue harvest that could not be used for transplantation due to size and tissue type incompatibility with the available recipients. All of the "patient" lanes shown were from the same patient, IV-1. They were obtained from pieces of his heart explanted at the time he underwent heart transplantation. The results show that the reduction in BAG3 levels is comparable to that seen in patients with non-familial heart failure. [Figure 6] Figures 6A-D show BAG3 levels in failing mouse hearts: Figure 6A is a graph showing heart weight vs. body weight; Figure 6B is a graph showing contractility; Figure 6C is a graph showing BAG3 protein levels; Figure 6D is an immunoblot analysis of BAG3 protein levels. [Figure 7-1] Figures 7A-7F show hemodynamic indexes and BAG3 levels in porcine hearts after balloon occlusion: Figure 7A is a graph showing ejection fraction; Figure 7B is a graph showing shortening fraction; Figure 7C is a graph showing end-diastolic volume; Figure 7D is a graph showing end-systolic volume. [Figure 7E] 1 is a graph showing BAG3 protein levels. [Figure 7F] Immunoblot analysis of BAG3 protein levels. [Figure 8] The NCBI reference amino acid sequence for BAG3 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Detailed Description The present invention is based, in part, on the inventor's discovery of a novel BAG3 mutation in a family with adult-onset familial dilated cardiomyopathy (FDC). More specifically, the inventor found that a novel 10-nucleotide deletion segregated in all affected individuals. In addition, the inventor also found that BAG3 protein levels were significantly reduced in hearts from unrelated patients with end-stage heart failure compared to non-failing controls. Furthermore, the inventor showed that administration of an AAV vector expressing BAG3 restored normal ventricular function in a mouse model of heart failure. Thus, the present invention features compositions that increase expression of BAG3, methods of making such compositions, and methods of using such compositions to treat subjects, i.e., patients suffering from dilated cardiomyopathy. Also featured are methods and compositions for the diagnosis of heart failure, such as idiopathic dilated cardiomyopathy (IDC).

[0016] Bcl-2-associated anthanogene-3 (BAG3), also known as BCL2-associated athanogene 3; MFM6; Bcl-2 binding protein Bis; CAIR-1; docking protein CAIR-1; BAG family molecular chaperone regulator 3; BAG-3; BCL2-associated athanogene 3; or BIS, is a cytoprotective polypeptide that competes with Hip-1 for binding to HSP70. BAG3 function is shown in FIG. 8, and the mechanism for BAG3 involvement in cardiomyocyte function is shown in FIG. 9. The NCBI reference amino acid sequence of BAG3 can be found in Genbank under accession number NP_004272.2; public GI:14043024. We set forth the amino acid sequence of Genbank Accession No. NP_004272.2; Public GI:14043024 as SEQ ID NO:1, shown in Figure 10. The NCBI reference nucleic acid sequence of BAG3 can be found in Genbank under Accession No. NM_004281.3 GI:62530382. We set forth the nucleic acid sequence of Genbank Accession No. NM_004281.3 GI:62530382 as SEQ ID NO:2. Other BAG3 amino acid sequences include, but are not limited to, for example, O95817.3 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 polypeptides of the present invention may be variants of the polypeptides described herein, so long as they retain functionality.

[0017] Provided herein are vectors containing a nucleic acid encoding a BAG3 polypeptide.

[0018] Novel BAG3 mutations were identified in families with adult-onset FDC.BAG3 protein levels were significantly decreased in unrelated patients with non-familial IDC, providing evidence that altered levels of BAG3 protein are involved in the progression of HF.

[0019] The embodiments relate to compositions that modulate the expression of Bcl-2-associated anthanogene-3 (BAG3) in vivo or in vitro. Modulation of BAG3 in patients in need of such treatment includes patients with cardiac disease or disorder, such as heart failure, or musculoskeletal disease or disorder. The embodiments also relate to identifying novel compounds or agents that modulate BAG3 expression using assays that measure BAG3 expression.

[0020] Some aspects of the present invention are described below with reference to illustrative application examples. It should be understood that numerous specific details, associations, and methods are described to provide a thorough understanding of the present invention. However, those skilled in the art will readily recognize that the present invention can be implemented without one or more of the specific details or with other methods. The present invention is not limited by the described order of acts or events, since some acts may occur in different orders and / or simultaneously with other acts or events. Moreover, not all described acts or events are required to perform the methodology according to the present invention.

[0021] Aspects of the invention may be practiced without the theoretical aspects presented. Moreover, the theoretical aspects are presented with the understanding that applicants do not seek to be bound by the presented theories.

[0022] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species to which the compositions and methods disclosed herein are applicable. Thus, these terms include, but are not limited to, genes and gene products from human and mouse. When genes or gene products from a particular species are disclosed, it is understood that this disclosure is intended to be illustrative only and is not to be interpreted as limiting unless the context clearly indicates otherwise. Thus, for example, in some embodiments, for genes disclosed herein with respect to mammalian nucleic acid and amino acid sequences, it is intended to include homologous and / or orthologous genes and gene products from other animals, including, but not limited to, other mammals, fish, amphibians, reptiles, and birds. In a preferred embodiment, the gene or nucleic acid sequence is human.

[0023] definition The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the present invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms unless the context clearly indicates otherwise. Furthermore, when the terms "including", "includes", "having", "has", "with" or variations thereof are used in either the detailed description and / or the claims, such terms are intended to include the same manner as the term "comprising".

[0024] As used herein, with respect to defined or described elements of an item, composition, apparatus, method, process, system, etc., the terms "comprising," "comprise," or "comprised," and variations thereof, are meant to be inclusive or open-ended, allowing for additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those identified elements, i.e., appropriate equivalents thereof, and that other elements may be included and still be within the scope / definition of the defined item, composition, apparatus, method, process, system, etc.

[0025] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or more than 1 standard deviation, according to the practice in the art. Or, "about" can mean within 20%, preferably within 10%, more preferably within 5%, and even more preferably within 1% of a given value. Or, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of the value, preferably within 5 times the value, and more preferably within 2 times the value. When a particular value is described in the specification and claims, unless otherwise stated, the term "about" should be considered to mean within an acceptable error range for the particular value.

[0026] "Optional" or "optionally" means that the subsequently described circumstance may or may not occur, such that the description includes instances in which the circumstance occurs and instances in which it does not occur.

[0027] The term "expression vector" as used herein refers to a vector that contains a nucleic acid sequence that codes for at least a portion of a gene product that can be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules, siRNA, ribozymes, etc. Expression vectors can contain various control sequences, which represent the nucleic acid sequences necessary for the transcription, and possibly the translation, of the functionally linked coding sequence, particularly in the host organism. In addition to the control sequences that control transcription and translation, vectors and expression vectors can contain nucleic acid sequences that also perform other functions.

[0028] "Recombinant viral vector" refers to a viral vector that contains one or more heterologous gene products or sequences. Because many viral vectors exhibit size limitations related to packaging, heterologous gene products or sequences are typically introduced by replacing one or more portions of the viral genome. Such viruses can be replication-defective and require that the deleted functions are provided in trans during viral replication and encapsidation (e.g., by using a helper virus or packaging cell line that has gene products required for replication and / or encapsidation). Modified viral vectors in which delivered polypeptides are carried outside the viral particle have also been described (see, e.g., Curiel, DT, et al., PNAS 88:8850-8854, 1991).

[0029] With respect to a specified nucleic acid, "encoding" or "encoded" or "encodes" means containing information for translation into a specified protein. A nucleic acid that encodes a protein may include non-translated sequences (e.g., introns) within the translated region of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in a cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, an amino acid sequence is encoded by a nucleic acid using the "universal" genetic code.

[0030] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to the promoter / regulator sequence. In some instances, this sequence may be a core promoter sequence, and in other instances, this sequence may also include enhancer sequences and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue-specific manner.

[0031] A "constitutive promoter" is a promoter that drives expression of a gene operably linked to it in a continuous manner in a cell. By way of example, a promoter that drives expression of a cellular housekeeping gene would be considered to be a constitutive promoter.

[0032] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a living cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0033] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a living cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0034] As used herein, the terms "BAG3", "BAG3 molecule", "BCL2-associated athanogene 3 (BAG3) gene", "BCL2-associated athanogene 3 (BAG3) molecule" include all family members, mutations, cDNA sequences, alleles, fragments, species, coding and non-coding regions, sense and antisense polynucleotide strands, and the like. Similarly, "BAG3", "BAG3 molecule", "BCL2-associated athanogene 3 (BAG3) molecule" also refers to BAG3 polypeptides or fragments, proteins, variants, derivatives, and the like thereof. Thus, the term "molecule" includes both the nucleic acid and amino acid sequences of BAG3.

[0035] "Isolated nucleic acid or cDNA" refers to a nucleic acid segment or fragment separated from sequences adjacent to it in the naturally occurring state, e.g., a DNA fragment removed from sequences normally adjacent to the fragment (e.g., sequences adjacent to the fragment in the genome in which it naturally occurs), and refers to a nucleic acid sequence from which one or more introns have been removed. The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany the nucleic acid, e.g., RNA or DNA, or proteins that naturally accompany it in a cell. Thus, the term includes recombinant DNA that is incorporated, e.g., in a vector, an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or recombinant DNA that exists as a separate molecule, independent of other sequences (e.g., as a cDNA, or a genomic or cDNA fragment produced by PCR or restriction enzyme cleavage). It also includes recombinant DNA, e.g., DNA that is part of a hybrid gene that encodes additional polypeptide sequences.

[0036] "Polynucleotide" means a nucleic acid that is single-stranded or parallel and anti-parallel. Thus, a polynucleotide can be either a single-stranded or double-stranded nucleic acid.

[0037] The term "variant" when used in the context of a polynucleotide sequence may include a polynucleotide sequence related to a wild-type gene. This definition may also include, for example, "allelic," "splice," "species," or "polymorphic" variants. A splice variant may have significant identity to a reference molecule, but will generally have more or fewer polynucleotides due to alternative splicing of exons during mRNA processing. The corresponding polypeptide may have additional functional domains or the absence of domains. Species variants are polynucleotide sequences that differ from one species to another. Of particular utility for the present invention are variants of wild-type gene products. Variants may result from at least one mutation in the nucleic acid sequence, resulting in an altered mRNA, or resulting in a polypeptide whose structure or function may or may not be altered. Any given natural or recombinant gene may have no alleles, one or many alleles. Common mutational changes that give rise to variants are generally due to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone or in combination with the others one or more times in a given sequence.

[0038] Unless otherwise indicated, the terms "peptide," "polypeptide," or "protein" are used interchangeably herein, typically referring to peptide sequences of various sizes.

[0039] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to act as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene codes for a protein when transcription and translation of the mRNA corresponding to the gene produces the protein in a cell or other biological system. The coding strand, which is the nucleotide sequence identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as "encoding" a protein or other product of the gene or cDNA.

[0040] Unless otherwise specified, the term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNAs can contain introns.

[0041] "Non-naturally occurring amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. Other terms that may be used synonymously with the term "non-naturally occurring amino acid" are "non-naturally encoded amino acid", "non-naturally occurring amino acid", "non-naturally occurring amino acid", and various hyphenated and non-hyphenated versions thereof. The term "non-naturally occurring amino acid" includes, but is not limited to, amino acids that occur naturally by modification of a naturally encoded amino acid (including, but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine), and includes amino acids that are not themselves incorporated into a growing polypeptide chain by the translation complex without user manipulation. Examples of non-naturally encoded naturally occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine. In addition, the term "non-naturally occurring amino acid" includes, but is not limited to, amino acids that do not occur in nature and may be obtained synthetically or by modification of non-naturally occurring amino acids.

[0042] As used herein, the term "ectopic expression" refers to a non-wild type pattern of gene expression. It includes expression at a non-wild type level, i.e., overexpression or underexpression; an expression pattern that differs from the wild type with respect to the time or stage at which the gene is expressed, such as increased or decreased expression (compared to the wild type) at a predefined developmental period or stage; an expression pattern that differs from the wild type with respect to decreased expression (compared to the wild type) in a predefined cell or tissue type; an expression pattern that differs from the wild type with respect to the splicing size, amino acid sequence, post-translational modification, or biological activity of the expressed polypeptide; an expression pattern that differs from the wild type with respect to the effect of environmental or extracellular stimuli on the expression of the gene, such as increased or decreased expression (compared to the wild type) in the presence of increased or decreased strength of the stimulus.

[0043] The term "modulate" means that any of the described activities of the compounds specifically described herein are, for example, increased, enhanced, increased, agonized (acting as an agonist), promoted, decreased, reduced, inhibited, blocked, or antagonized (acting as an antagonist). Modulation can increase the activity by more than 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, etc., relative to the baseline value. Modulation can also decrease the activity below the baseline value.

[0044] As used herein, the term "agent" is meant to include any molecule, chemical, composition, drug, therapeutic, chemotherapeutic, or biological agent that can prevent, ameliorate, or treat a disease or other medical condition. This term includes small molecule compounds, antisense reagents, siRNA reagents, antibodies, enzymes, peptides, organic or inorganic molecules, natural or synthetic compounds, and the like. Agents can be assayed according to the methods of the present invention at any stage during clinical trials, pretrial testing, or subsequent FDA approval.

[0045] As defined herein, a "therapeutically effective" amount (i.e., effective dose) of a compound or agent means an amount sufficient to produce a therapeutically (e.g., clinically) desired result. The composition can be administered from one or more times per day, including once every other day, to one or more times per week. Those skilled in the art will understand that certain factors, including but not limited to, the severity of the disease or disorder, previous treatments, the overall health and / or age of the subject, and the presence of other diseases, can affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a compound of the present invention can include a single treatment or a series of treatments.

[0046] The terms "determining," "measuring," "assessing," "detecting," "assessing," and "assaying" are used interchangeably herein to refer to any form of measurement, including determining whether an element is present or not. These terms include both quantitative and / or qualitative determinations. Evaluation can be relative or absolute. "Assaying for the presence of" includes determining the amount of something present, as well as determining whether it is present or absent.

[0047] The term "assay", as used herein, whether in the singular or plural, is not to be construed or limited as referring to only one assay having a particular step, but is intended to include, but is not limited to, any additional steps, materials, various iterations, permutations, etc. that may be used. Thus, when the term "assay" is used in the singular, it is for descriptive purposes only.

[0048] A "label" or a "detectable label" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radiolabeled molecules, fluorophores, luminescent compounds, electron-dense reagents, enzymes (e.g., commonly used in ELISA), biotin, digoxigenin, or haptens; proteins can be detected, for example, by incorporating a label into a peptide that is used to detect an antibody specifically reactive with the peptide.

[0049] The term "high throughput screening" or "HTS" refers to methods that utilize different technologies and disciplines, such as optical, chemical, biological, or image analysis, to enable rapid and highly parallel biological research and drug discovery. HTS methods are known in the art and are generally performed in multi-well plates using automated liquid handling and detection equipment, although it is envisioned that the methods of the present invention may be performed in microarrays or microfluidic systems.

[0050] As used herein, the term "library" or "drug library" refers to a plurality of chemical molecules (test compounds), a plurality of nucleic acids, a plurality of peptides, or a plurality of proteins, organic or inorganic compounds, synthetic molecules, natural molecules, or a combination thereof.

[0051] As used herein, the term "target" or "target molecule" refers to any type of molecule or structure to be detected, manipulated, or characterized. The molecule can be an intracellular molecule, e.g., a nucleic acid sequence, a peptide, a structure (e.g., intracellular membrane, ribosome, etc.), a surface molecule (e.g., receptor), an extracellular molecule (e.g., cytokine, enzyme, viral particle, organism, biological sample, etc.

[0052] As used herein, "biological sample" includes solid samples and body fluid samples. Biological samples used in the present invention may include biological fluids such as cells, protein, or membrane extracts of cells, blood, or ascites or brain fluid (e.g., cerebrospinal fluid). Examples of solid biological samples include, but are not limited to, samples taken from tissues of the central nervous system, bone, breast, kidney, cervix, endometrium, head and neck, gallbladder, parotid gland, prostate, pituitary gland, muscle, esophagus, stomach, small intestine, colon, liver, spleen, pancreas, thyroid, heart, lung, bladder, fat, lymph nodes, uterus, adrenal gland, testis, tonsils, thymus, and skin, or samples taken from tumors. Examples of "body fluid samples" include, but are not limited to, blood, serum, semen, prostatic fluid, semen, urine, feces, saliva, sputum, mucus, bone marrow, lymph, and tears.

[0053] As used herein, "cardiac disease" refers to any type of cardiac disease, including heart failure, myocardial disease, cardiomyopathy, hypertrophic cardiomyopathy, dilated cardiomyopathy, atherosclerosis, coronary artery disease, ischemic heart disease, myocarditis, viral infection, wound, hypertensive heart disease, valvular disease, congenital heart disease, myocardial infarction, congestive heart failure, arrhythmias, diseases resulting in cardiac remodeling, etc. Cardiac disease can be due to any cause, such as, for example, damage to cardiac tissue, such as loss of contractility (as may be demonstrated, for example, by a reduced ejection fraction).

[0054] Cardiac damage or injury characterized by inadequate cardiac function includes any defect or lack of normal cardiac function or the presence of abnormal cardiac function. Abnormal cardiac function can be the result of disease, injury, and / or aging. As used herein, abnormal cardiac function includes morphological and / or functional abnormalities of cardiomyocytes, cardiomyocyte populations, or the heart itself. Non-limiting examples of morphological and functional abnormalities include physical deterioration and / or death of cardiomyocytes, abnormal proliferation patterns of cardiomyocytes, abnormalities in the physical association between cardiomyocytes, under- or overproduction of one or more substances by cardiomyocytes, failure of cardiomyocytes to produce one or more substances that they normally produce, and transmission of electrical impulses in abnormal patterns or at abnormal times. Abnormalities at a larger level include motility abnormalities, reduced ejection fraction, changes observed by echocardiography (e.g., dilation), changes in EKG, changes in exercise tolerance, reduced capillary perfusion, and changes observed by angiography. Abnormal cardiac function is associated with many disorders, including, for example, ischemic heart disease, such as angina pectoris, myocardial infarction, chronic ischemic heart disease, hypertensive heart disease, pulmonary heart disease (cor pulmonale), valvular heart disease, such as rheumatic fever, mitral valve prolapse, calcification of the mitral annulus, carcinoid heart disease, infective endocarditis, congenital heart disease, myocardial disease, such as myocarditis, dilated cardiomyopathy, hypertensive cardiomyopathy, cardiac disorders resulting in congestive heart failure, and cardiac tumors, such as primary sarcomas and secondary tumors.Heart injury also includes wounds, such as, for example, knife wounds; biological (e.g., viruses; autoimmune diseases) or chemical (e.g., chemotherapy, drugs); surgery; transplantation, etc.

[0055] The term "diagnosis" as used herein means to identify the presence or nature of a pathological condition. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives"). Diseased individuals not detected by the assay are "false negatives". Subjects who are not diseased and test negative in the assay are called "true negatives". The "specificity" of a diagnostic assay is "1 minus the false positive rate", where the "false positive" rate is defined as the proportion of non-diseased subjects who test positive. A particular diagnostic method may not provide a definitive diagnosis of a condition, but is sufficient if it provides a positive indication that aids in diagnosis.

[0056] The term "diagnosing" as used herein refers to classifying a disease or condition, determining the severity of a disease, monitoring the progression of a disease, predicting the prognosis and / or chance of recovery of a disease. The term "detecting" may also optionally include any of the above. Diagnosis of a disease according to the present invention may be achieved by determining the level of a polynucleotide or polypeptide of the present invention in a biological sample obtained from a subject, and correlating the determined level with a tendency for disease or the presence or absence of disease. It should be noted that a "biological sample obtained from a subject" may optionally include a sample that has not been physically removed from the subject, as described in more detail below.

[0057] "Treatment" is an intervention performed with the intent to prevent the onset or to modify the pathology or symptoms of the disorder. Thus, "treatment" refers to both therapeutic treatment and prophylactic or preventative treatment. "Treatment" may also be specified as palliative care. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. Thus, "treating" a condition, disorder, or condition or "treatment" thereof includes (1) preventing or delaying the appearance of clinical symptoms of the condition, disorder, or condition in a human or other mammal that may be affected by or susceptible to the condition, disorder, or condition, but that has not yet experienced or exhibited clinical or subclinical symptoms of the condition, disorder, or condition; (2) inhibiting the condition, disorder, or condition, i.e., arresting, reducing, or delaying the onset of the disease or its recurrence, or at least one of its clinical or subclinical symptoms (in the case of maintenance therapy); or (3) relieving the disease, i.e., causing regression of the condition, disorder, or condition, or at least one of its clinical or subclinical symptoms. The benefit to the individual being treated is either statistically significant or at least perceptible to the patient or physician.

[0058] The terms "patient," "individual," or "subject" are used interchangeably herein and refer to the mammalian subject being treated, preferably including a human patient. In some cases, the methods of the present invention find use in laboratory animals, including, but not limited to, rodents, including mice, rats, and hamsters, and primates, in veterinary applications, and in the development of animal models for disease.

[0059] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of a reaction assay, such delivery systems include systems that allow for the storage, transportation, or delivery of reaction reagents (e.g., oligonucleotides in appropriate containers, enzymes, etc.) and / or supporting materials (e.g., buffers, instructions for performing the assay, etc.) from one location to another. For example, a kit includes one or more enclosures (e.g., boxes) that contain the relevant reaction reagents and / or supporting materials. As used herein, the term "fragmented kit" refers to a delivery system that includes two or more separately separated containers, each containing a subportion of the total kit components. The containers can be delivered to the intended recipient together or separately separated. For example, a first container may contain an enzyme for use in the assay and a second container contains an oligonucleotide. The term "fragmented kit" is intended to include, but is not limited to, kits that include analyte-specific reagents (ASRs) regulated by section 520(e) of the Federal Food, Drug, and Cosmetic Act. Indeed, any delivery system that includes two or more separately separated containers, each containing a subportion of the total kit components, is included in the term "fractionalized kit." In contrast, a "combination kit" refers to a delivery system that includes all of the components of a reaction assay in a single container (e.g., in a single box that houses each of the desired components). The term "kit" includes both fractionalized kits and combination kits.

[0060] composition The most common cause of dilated cardiomyopathy and heart failure (HF) is ischemic heart disease, but in one-third of all patients, the cause remains undefined and patients are diagnosed with idiopathic dilated cardiomyopathy (IDC). The study conducted herein employed whole-exome sequencing to identify causative variants in a large family with autosomal dominant transmission of dilated cardiomyopathy. Sequencing and subsequent informatics revealed a novel 10-nucleotide deletion in the BCL2-associated aganogen-3 (BAG3) gene (Ch10:del 121436332_12143641:del.1266_1275 [NM 004281]), which segregated in all affected individuals. This deletion predicted a shift in the reading frame and a resulting deletion of 135 amino acids from the C-terminus of the protein. Consistent with genetic variants in genes encoding other sarcomeric proteins, there was a large amount of genetic heterogeneity in affected family members. Interestingly, it was also found that BAG3 protein levels were significantly reduced in hearts derived from unrelated patients with end-stage HF who underwent heart transplantation when compared with non-failing controls. Reduced levels of BAG3 protein may be associated with both familial and non-familial forms of dilated cardiomyopathy. Therefore, modulation of BAG3 expression or amount of BAG3 in patients would be of great benefit.

[0061] In embodiments, therapeutic agents for the treatment of diseases associated with BAG3, related molecules, and the pathway modulate the expression of BAG3 or the amount of BAG3 in a cell.

[0062] In some embodiments, the compositions comprise nucleic acid sequences of BCL2-associated aganocyte gene 3 (BAG3), including but not limited to cDNA, sense, and / or antisense sequences of BAG3.

[0063] In some embodiments, the composition comprises an expression vector having an isolated nucleic acid or cDNA sequence or a synthetic nucleic acid sequence encoding a BCL2-associated aganogen 3 (BAG3) molecule. The term "nucleic acid sequence" is used for brevity and includes, but is not limited to, an isolated nucleic acid or cDNA sequence, a synthetic or synthetic nucleic acid sequence, a chimeric nucleic acid sequence, a homolog, an ortholog, a variant, a mutant, or a combination thereof.

[0064] In some embodiments, the nucleic acid sequence of BAG3 comprises at least about 50% sequence identity to wild-type BAG3 or its cDNA sequence. In other embodiments, the nucleic acid sequence of BAG3 comprises at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to wild-type BAG3 or its cDNA sequence.

[0065] In some embodiments, the nucleic acid sequence of BAG3 further comprises one or more mutations, substitutions, deletions, variants, or a combination thereof.

[0066] In some embodiments, the homology, sequence identity, or complementarity of the BAG3 nucleic acid sequence containing one or more mutations, substitutions, deletions, variants, or combinations thereof to the native or wild-type or cDNA sequence of BAG3 is about 50% to about 60%. In some embodiments, the homology, sequence identity, or complementarity is about 60% to about 70%. In some embodiments, the homology, sequence identity, or complementarity is about 70% to about 80%. In some embodiments, the homology, sequence identity, or complementarity is about 80% to 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%.

[0067] In one embodiment, the expression vector encodes the BCL2-associated asanogene 3 (BAG3) gene or its cDNA sequence, or a modified sequence thereof. In one embodiment, the expression vector encodes a nucleic acid sequence that comprises at least about 50% sequence identity with wild-type BCL2-associated asanogene 3 (BAG3) or its cDNA sequence. In other embodiments, the nucleic acid sequence comprises at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with wild-type BCL2-associated asanogene 3 (BAG3) or its cDNA sequence.

[0068] A wide variety of host / expression vector combinations can be used in expressing the BAG3 DNA sequence of the present invention. For example, useful expression vectors can consist of segments of chromosomal, non-chromosomal, and synthetic DNA sequences. Suitable vectors include derivatives of SV40, and known bacterial plasmids, such as E. coli plasmids, col E1, pCR1, pBR322, pMal-C2, pET, pGEX (Smith et al., Gene 67:31-40, 1988), pMB9 and their derivatives, plasmids such as RP4; phage DNA, such as many derivatives of phage 1, such as NM989, and other phage DNA, such as M13 and filamentous single-stranded phage DNA; vectors useful in eukaryotic cells, such as yeast plasmids or their derivatives, such as 2μ plasmids, vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNA, such as plasmids modified to use phage DNA or other expression control sequences, and the like.

[0069] Numerous vectors are known to be capable of mediating the transfer of gene products into mammalian cells, as known in the art and described herein. A "vector" (sometimes described as a gene delivery or gene transfer "vehicle") refers to a macromolecule or complex of molecules, including a polynucleotide, that is delivered to a host cell, either in vitro or in vivo. The polynucleotide delivered may include a coding sequence of interest in gene therapy. For example, vectors include viral vectors (adenoviruses ("Ad"), adeno-associated viruses (AAV), and vesicular stomatitis viruses (VSV), and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes that can mediate the delivery of polynucleotides to host cells. Vectors may also include other components or functionalities that further regulate gene delivery and / or gene expression, or that otherwise provide advantageous properties to the targeted cells. As described and explained in more detail below, such other components include, for example, components that affect binding or targeting to cells (including components that mediate cell type or tissue specific binding); components that affect uptake of vector nucleic acid by cells; components that affect localization of polynucleotides within cells after uptake (such as agents that mediate nuclear localization); and components that affect expression of polynucleotides. Such components may also include markers, such as detectable and / or selectable markers, that can be used to detect or select for cells that have taken up and expressed the nucleic acid delivered by the vector. Such components may be provided as natural features of the vector (such as the use of certain viral vectors with components or functionality that mediate binding and uptake), or the vector may be modified to provide such functionality. Other vectors include those described by Chen et al; BioTechniques, 34:167-171 (2003). A wide variety of such vectors are known in the art and are generally available.

[0070] Suitable nucleic acid delivery systems include sequences derived from at least one of a viral vector, typically an adenovirus, an adeno-associated virus (AAV), a helper-dependent adenovirus, a retrovirus, or a hemagglutinating virus of Japan-liposome (HVJ) complex. Preferably, the viral vector includes a strong eukaryotic promoter operably linked to a polynucleotide, such as, for example, a cytomegalovirus (CMV) promoter.

[0071] More preferred vectors include viral vectors, fusion proteins, and chemical complexes.Retroviral vectors include Moloney murine leukemia virus and HIV-based viruses.One HIV-based viral vector contains at least two vectors, where gag and pol genes are derived from the HIV genome, and env genes are derived from another virus.DNA viral vectors are preferred. These vectors include pox vectors, such as orthopox or avipox vectors, herpes virus vectors, such as herpes simplex virus type I (HSV) vectors [Geller, AI et al., J. Neurochem, 64:487 (1995); Lim, F., et al., DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al., Proc Natl. Acad. Sci.: USA:90 7603 (1993); Geller, AI, et al., Proc Natl. Acad. Sci USA: 87:1149 (1990)], adenovirus vectors [LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet. 3: 219 (1993); Yang, et al., J. Virol. 69:2004 (1995)], and adeno-associated virus vectors [Kaplitt, MG, et al., Nat. Genet. 8:148 (1994)].

[0072] Poxvirus vectors introduce genes into the cytoplasm of cells. Avian poxvirus vectors result in only short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors can be indications for some inventive embodiments. Adenovirus vectors result in shorter-term expression (e.g., less than about one month) than adeno-associated virus (AAV), and in some embodiments, may show longer expression. In some embodiments, the expression vector is an AAV9 vector. The particular vector selected will depend on the target cell and the condition being treated. Selection of an appropriate promoter can be easily accomplished. Preferably, a high expression promoter will be used. An example of a suitable promoter is the 763 base pair cytomegalovirus (CMV) promoter. Rous sarcoma virus (RSV) (Davis, et al., Hum Gene Ther 4:151 (1993)) and MMT promoters can also be used. Certain proteins can be expressed using their native promoters. Other elements that can enhance expression can also be included, such as enhancers or systems that result in high levels of expression, such as the tat gene and tar element. The cassette can then be inserted into a vector, such as a plasmid vector, such as pUC19, pUC118, pBR322, or other known plasmid vectors that contain an E. coli origin of replication. See Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory press, (1989). The plasmid vector can also include a selectable marker, such as the β-lactamase gene for ampicillin resistance, provided that the marker polypeptide does not adversely affect the metabolism of the treated organism. The cassette can also be linked to a nucleic acid binding moiety in a synthetic delivery system, such as the system disclosed in WO 95 / 22618.

[0073] If desired, the polynucleotide of the present invention can also be used with micro-delivery vehicles such as cationic liposomes and adenovirus vectors.For a review of procedures for liposome preparation, targeting and delivery of 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, RA, Bethesda Res. Lab. Focus, 11(2):25 (1989).

[0074] Replication-defective recombinant adenovirus vectors can be produced according to known techniques. 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).

[0075] Another delivery method is to use a single-stranded DNA producing vector that can produce BAG3 intracellularly, for example in cardiac tissue. See, e.g., Chen et al., BioTechniques, 34:167-171 (2003), which is incorporated herein by reference in its entirety.

[0076] Expression of BAG3 may 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, 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), the troponin 1 promoter (Bhavsar et al. (1996) Genomics, Vol. 35(1):11-23); Na + / Ca 2+exchanger 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), α7 integrin promoter (Ziober and Kramer (1996) J. Bio. Chem., Vol. 271(37):22915-22), brain natriuretic peptide promoter (LaPointe et al. (1996) Hypertension, Vol. 27(3 Pt2):715-22) and αB-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).

[0077] Other promoters which may be used to control BAG3 gene expression include the cytomegalovirus (CMV) promoter (US Pat. Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' terminal repeat of Rous sarcoma virus (Yamamoto, et al., Cell 22:787-797, 1980), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78: 1441-1445, 1981), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39-42, 1982); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Kamaroff, et al., Proc. Natl. Acad. Sci. USA 75:3727-3731, 1978), or the tac promoter (DeBoer, et al., Proc. Natl. Acad. Sci. USA 80:21-25, 1983); see also "Useful proteins from recombinant bacteria" in Scientific American, 242:74-94, 1980; promoter elements derived from yeast or other fungi, such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, the alkaline phosphatase promoter; and transcriptional control regions of animals that exhibit tissue specificity and are used in transgenic animals: the elastase I gene control region, which is active in pancreatic acinar cells (Swift et al., Cell 38:639-646, 1984; Ornitz et al., Cold Spring Harbor Symp. Quant. Biol.50:399-409, 1986; MacDonald, Hepatology 7:425-515, 1987); the insulin gene control region, which is active in pancreatic beta cells (Hanahan, Nature 315:115-122, 1985); the immunoglobulin gene control region, which is active in lymphoid cells (Grosschedl et al., Cell 38:647-658, 1984; Adames et al., Nature 318:533-538, 1985; Alexander et al., Mol. Cell. Biol. 7:1436-1444, 1987); the mouse mammary tumor virus control region, which is active in testis, breast, lymphocytes, and mast cells (Leder et al., Cell 45:485-495, 1986); the albumin gene control region, which is active in the liver (Pinkert et al., al., Genes and Devel. 1:268-276, 1987), the α-fetoprotein gene control region that is active in the liver (Krumlauf et al., Mol. Cell. Biol. 5:1639-1648, 1985; Hammer et al., Science 235:53-58, 1987), the α1-antitrypsin gene control region that is active in the liver (Kelsey et al., Genes and Devel. 1:161-171, 1987), the β-globin gene control region that is active in myeloid cells (Mogram et al., Nature 315:338-340, 1985; Kollias et al., Cell 46:89-94, 1986), and the myelin basic protein gene control region that is active in oligodendrocyte cells in the brain (Readhead et al., Cell 48:703-712, 1987), the myosin light chain-2 gene control region which is active in skeletal muscle (Sani, Nature 314:283-286, 1985), and the gonadotropin releasing hormone gene control region which is active in the hypothalamus (Mason et al., Science 234:1372-1378, 1986).

[0078] Yeast expression systems may also be used in accordance with the present invention to express BAG3. For example, the non-fusion pYES2 vector (XbaI, SphI, ShoI, NotI, GstXI, EcoRI, BstXI, BamH1, SacI, Kpn1, and HindIII cloning sites; Invitrogen) or the fusion pYESHisA, B, C (XbaI, SphI, ShoI, NotI, BstXI, EcoRI, BamH1, SacI, KpnI, and HindIII cloning sites, ProBond resin purified, enterokinase cleaved N-terminal peptide; Invitrogen), to name just two, may be used in accordance with the present invention. A yeast two-hybrid expression system may be prepared in accordance with the present invention.

[0079] One preferred delivery system is a recombinant viral vector incorporating one or more polynucleotides therein, preferably about one polynucleotide. Preferably, the viral vector used in the methods of the present invention is a vector that is capable of carrying about 10 8 ~Approx. 5×10 10 In embodiments in which the polynucleotide is administered by a non-viral vector, use of about 0.1 nanograms to about 4000 micrograms, such as about 1 nanogram to about 100 micrograms, is often useful.

[0080] In some embodiments, the vector is an adenovirus-associated virus vector (AAV), such as AAV9. The term "AAV vector" refers to a vector derived from an adeno-associated virus serotype, including but not limited to AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, and AAV-8. An AAV vector may have one or more AAV wild-type genes, preferably the rep and / or cap genes, deleted in whole or in part, but may retain functional adjacent ITR sequences. Despite the high degree of homology, different serotypes have tropism for different tissues. The receptor for AAV1 is not known, but AAV1 is known to transduce skeletal and cardiac muscles more effectively than AAV2. Most of the studies are done with pseudotyped vectors in which vector DNA flanked by AAV2 ITRs is packaged into the capsid of other serotypes, so it is clear that the biological differences are related to the capsid rather than the genome. Recent evidence shows that the DNA expression cassette packaged in AAV1 capsid is at least 1 log 10 more effective in transducing cardiomyocytes than that packaged in AAV2 capsid. In one embodiment, the viral delivery system is an adeno-associated viral delivery system. The adeno-associated virus can be of serotype I (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).

[0081] Some of those skilled in the art have circumvented some of the limitations of adenovirus-based vectors by using adenovirus "hybrid" viruses that incorporate the desired properties from adenovirus and other types of viruses as a means to create unique vectors with highly specialized properties.For example, viral vector chimeras have been created with adenovirus and adeno-associated virus (AAV).These aspects of the invention do not depart from the scope of the invention described herein.

[0082] The nucleic acid encoding the BAG3 protein of the present invention can be delivered to the cardiac muscle by methods known in the art (see, for example, US Patent Appln. Publication No. US 2009 / 0209631). For example, cardiac cells of a large mammal can be transfected by a method comprising dilating blood vessels of the coronary circulation by administering a vasodilator prior to and / or simultaneously with administration of the nucleic acid. In some embodiments, the method comprises administering the nucleic acid into blood vessels of the coronary circulation in vivo, the nucleic acid being injected into the blood vessels for at least about 3 minutes or more, the coronary circulation being not or substantially not isolated from the systemic circulation of the mammal, and the nucleic acid transfecting the cardiac cells of the mammal.

[0083] In some embodiments, the subject 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 subject may suffer from a cardiac disorder, such as heart failure, ischemia, myocardial infarction, congestive heart failure, arrhythmia, transplant rejection. In a preferred embodiment, the subject suffers from heart failure. In other specific embodiments, the subject suffers from arrhythmia. In one embodiment, the subject is a human. For example, the subject is between 18 and 65 years old. In other embodiments, the subject is a non-human animal.

[0084] In one embodiment, the subject has or is at risk for heart failure, eg, non-ischemic cardiomyopathy, mitral regurgitation, ischemic cardiomyopathy, or aortic stenosis or regurgitation.

[0085] In some embodiments, transfection of cardiac cells with a nucleic acid molecule encoding a BAG3 protein or a nucleic acid molecule encoding 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 cardiac cells increases the fractional shortening of the lateral ventricle by at least 25% when measured about 4 months after injection, compared to the fractional shortening of the lateral ventricle before injection of the polypeptide. In some embodiments, transfection of cardiac cells results in an improvement in a measure of cardiac function selected from the group consisting of expression of BAG3 protein, fractional shortening, ejection fraction, cardiac output, time constant of ventricular relaxation, and regurgitant volume.

[0086] Treatment can be judged by assessing the effect of treatment on parameters related to contractility. For example, SR Ca 2+ ATPase activity or intracellular Ca 2+ Additionally, 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.

[0087] Modified Nucleic Acid Sequences: The present invention is not intended to be limited by the nature of the nucleic acid used, so long as it modulates the expression or amount of BAG3 in a cell or in a patient to which the nucleic acid composition is administered as a therapeutic agent. The nucleic acid may be DNA or RNA and may exist in double-stranded, single-stranded, or particularly double-stranded form.

[0088] Nucleic acids useful in the present invention include, but are not limited to, oligonucleotides and polynucleotides such as antisense DNA and / or RNA; ribozymes; DNA for gene therapy; viral fragments including viral DNA and / or RNA; DNA and / or RNA chimeras; mRNA; plasmids; cosmids; genomic DNA; cDNA; gene fragments; single-stranded DNA, double-stranded DNA, supercoiled DNA and / or triple helix DNA; DNA in various structural forms including Z-DNA, and the like. Nucleic acids can be prepared by any conventional means typically used to prepare nucleic acids in large quantities. For example, DNA and RNA can be chemically synthesized 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 yields via in vitro transcription using plasmids such as pGEM®T vectors or SP65 (Promega Corporation, Madison, WI).

[0089] Thus, certain preferred nucleic acid sequences of the present invention are chimeric nucleic acid sequences. In the context of the present invention, a "chimeric nucleic acid sequence" or "chimera" comprises two or more chemically distinct regions, each made of at least one nucleotide. These sequences typically comprise at least one region of modified nucleotides that confer one or more advantageous properties (e.g., increased nuclease resistance, increased cellular uptake, increased binding affinity to target, etc.).

[0090] The chimeric nucleic acid sequence of the present invention can be formed as a complex structure 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.Representative United States patents that disclose the preparation of such hybrid structures include, but are not limited to, US Pat. 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.

[0091] Specific examples of some modified nucleic acid sequences envisioned for the present invention include those that contain modified backbones, such as phosphorothioate, phosphotriesterase, methylphosphonate, short chain alkyl or cycloalkyl intersugar linkages, or short chain heteroatom or heterocyclic intersugar linkages. Examples of oligonucleotides with phosphorothioate backbones and oligonucleotides with heteroatom backbones include, but are not limited to, CH2-NH--O--CH2, CH, --N(CH3)-0--CH2 [known as methylene (methylimino) or MMI backbones], CH2-0--N(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2, and 0--N(CH3)-CH2-CH2 backbones, with the natural phosphodiester backbone represented as O--P--O--CH). The amide backbone disclosed by De Mesmaeker et al. (1995) Acc. Chem. Res. 28:366-374 is also an example. In other embodiments, the nucleic acid sequence comprises a morpholino backbone structure (Summerton and Weller, US Pat. No. 5,034,506). In other embodiments, the phosphodiester backbone of the nucleic acid sequence is replaced with a polyamide backbone, such as a peptide nucleic acid (PNA) backbone, in which the nucleotides are linked directly or indirectly to the aza nitrogen atoms of the polyamide backbone (Nielsen et al. (1991) Science 254, 1497). The nucleic acid sequence may also have one or more substituted sugar moieties. Examples include OH, SH, SCH3, F, OCN, OCHOCH3, OCH3O(CH2). n CH3, O(CH2) n NH2, or O(CH2) n CH3, where n is 1 to about 10; C1 to C 10Examples of substituents include lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl, or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; reporter groups; intercalating agents; groups for improving the pharmacokinetic properties of oligonucleotides; or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Other modifications include, for example, 2'-methoxyethoxy [2'-O--CH2CHOCH3, also known as 2'-O-(2-methoxyethyl)] (Martin et al., (1995) Hely. Chim. Acta, 78, 486), 2'-methoxy (2'-O--CH3), 2'-propoxy (2'-OCH2CH2CH3) and 2'-fluoro (2'-F). Similar modifications can also be made at any position in the oligonucleotide, at the 2' or 3' position of the sugar of the 3' terminal nucleotide and at the 5' position of the 5' terminal nucleotide. Nucleic acid sequences can also have sugar mimetics, such as cyclobutyls in place of the pentofuranosyl group.

[0092] Preferred modified oligonucleotide backbones include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phototriesters, methyl and other alkyl phosphonates, including 3' alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phototriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reverse polarity, where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.Various salts, mixed salts, and free acid forms are also included.

[0093] Preferred modified oligonucleotide backbones that do not contain phosphorus atoms have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkane internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages.These include morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; backbones that contain alkenes; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; those with amide backbones; and others with mixed N, O, S and CH2 component parts.

[0094] Nucleic acid sequences may also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base") modifications or substitutions. 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 found rarely or occasionally in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 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-(aminoalkylamino)adenine, or other hetero-substituted alkyl adenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine, and 2,6-diaminopurine (Kornberg, A., DNA Replication, WH Freeman & Co., San Francisco, 1980, pp 75-77; Gebeyehu, G., (1987) et al. Nucl. Acids Res. 15:4513). "Universal" bases known in the art may be included, such as inosine.

[0095] Another modification includes chemically linking to the oligonucleotide, one or more moieties, or complexes that enhance the activity of the nucleotide or enhance the intracellular uptake of the nucleotide. Such moieties include, but are not limited to, fatty moieties, such as cholesterol moieties, cholesteryl moieties, cholic acid, thioethers, such as hexyl-5-tritylthiol, aliphatic chains, such as dodecanediol or undecyl residues, phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamines or polyethylene glycol chains, or adamantane acetic acid. Nucleic acid sequences that include lipophilic moieties and methods for preparing such oligonucleotides are known in the art, for example, in US Pat. Nos. 5,138,045, 5,218,105, and 5,459,255.

[0096] It is not necessary for all portions of a given nucleic acid sequence to be uniformly modified, and indeed more than one of the above-mentioned modifications may be incorporated within a single nucleic acid sequence, or within a single nucleoside within such a sequence. The present invention also includes oligonucleotides that are chimeric oligonucleotides, as defined below.

[0097] In another embodiment, the BAG3 nucleic acid molecules of the present invention are conjugated to other moieties, including but not limited to abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, lipids, or polyhydrocarbon compounds. One of skill in the art will recognize that these molecules can be linked to any nucleotide or nucleotides comprising the nucleic acid molecule at any site in the sugar, base, or phosphate group.

[0098] In another embodiment, the BAG3 nucleic acid sequence comprises one or more nucleotides substituted with locked nucleic acid (LNA). The LNA modified nucleic acid sequence may have a size similar to the parent or native sequence, or may be larger, or preferably smaller. Such LNA modified oligonucleotides preferably contain less than about 70%, more preferably less than about 60%, and most preferably less than about 50% LNA monomers, and their size is about 1-25 nucleotides.

[0099] Antisense BAG3-oligonucleotides: In another preferred embodiment, expression of BAG3 in a cell or patient is regulated by one or more target nucleic acid sequences that regulate expression of BAG3, such as, for example, transcriptional regulatory elements.

[0100] In a preferred embodiment, the oligonucleotide comprises at least 5 consecutive bases that are complementary to a nucleic acid sequence, where the oligonucleotide specifically hybridizes to BAG3 in vivo or in vitro and regulates the expression of BAG3. In another preferred embodiment, the oligomeric compound of the present invention also comprises variants in which different bases are present at one or more nucleic acid positions in the compound. For example, if the first nucleotide is adenosine, variants containing thymidine, guanosine, or cytidine at this position can be produced. This can be done at any position of the oligonucleotide. These compounds are then tested using the methods described herein to determine their ability to inhibit the expression of target nucleic acid.

[0101] In some embodiments, the homology, sequence identity, or complementarity between the oligonucleotide and the target is about 50% to about 60%. In some embodiments, the homology, sequence identity, or complementarity is about 60% to about 70%. In some embodiments, the homology, sequence identity, or complementarity is about 70% to about 80%. In some embodiments, the homology, sequence identity, or complementarity is about 80% to 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%.

[0102] In another preferred embodiment, the oligonucleotide comprises a combination of phosphorothioate internucleotide linkages and a combination of at least one internucleotide linkage selected from the group consisting of alkyl phosphonates, phosphorodithioates, alkyl phosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters, and / or combinations thereof.

[0103] In another preferred embodiment, the oligonucleotide optionally comprises at least one modified nucleobase, including a peptide nucleic acid, a locked nucleic acid (LNA) molecule, an analog, a derivative, and / or a combination thereof.

[0104] An oligonucleotide is specifically hybridizable if there is a sufficient degree of complementarity such that binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid resulting in a loss of activity, and to avoid non-specific binding of the oligonucleotide to non-target nucleic acid sequences under conditions where specific binding is desired, i.e., physiological conditions, in the case of in vivo assays or therapeutic treatments, and conditions under which the assay is performed, in the case of in vitro assays.

[0105] Any oligonucleotide, whether DNA, RNA, chimeric, substituted, etc., can specifically hybridize if binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA molecule so as to result in loss of utility, and if there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions where specific binding is desired, i.e., physiological conditions in the case of in vivo assays or therapeutic treatments, and conditions under which the assay is performed in the case of in vitro assays.

[0106] The specificity and sensitivity of antisense are also utilized by those skilled in the art for therapeutic use.Antisense oligonucleotides are used as therapeutic moieties in the treatment of disease conditions in animals and humans.Antisense oligonucleotides have been safely and effectively administered to humans, and many clinical trials are currently being conducted.Therefore, it has been established that oligonucleotides can be useful therapeutic procedures that can be configured to be useful in treatment regimens for the treatment of cells, tissues, and animals, especially humans.

[0107] In the embodiment of the present invention, oligomeric oligonucleotides, particularly oligonucleotides, bind to target nucleic acid molecules and regulate the expression of molecules encoded by target genes.The functions of DNA that are disrupted include, for example, replication and transcription.The functions of RNA that are disrupted include, for example, translocation of RNA to the site of protein translation, translation of protein from RNA, splicing of RNA to generate one or more mRNA species, and all important functions such as catalytic activity that can be bound to or promoted by RNA.This function can be upregulated or inhibited depending on the desired function.

[0108] Oligonucleotides include antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternative splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of a target nucleic acid. Therefore, these compounds can be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds.

[0109] In the context of the present invention, targeting of oligonucleotide to a specific nucleic acid molecule can be a multi-step process.The process usually starts with the identification of the target nucleic acid whose function is to be regulated.This target nucleic acid can be, for example, a cellular gene (or the mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state.

[0110] The targeting process also usually includes determining at least one target region, segment, or site within the target nucleic acid for antisense interaction to occur, such that a desired effect, such as, for example, regulating expression, occurs. In the context of the present invention, the term "region" is defined as a portion of the target nucleic acid that has at least one identifiable structure, function, or characteristic. A region within a target nucleic acid is a segment. A "segment" is defined as a smaller portion or sub-portion of a region within a target nucleic acid. As used in the present invention, a "site" is defined as a position within a target nucleic acid.

[0111] In another preferred embodiment, the 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.

[0112] In another preferred embodiment, the antisense oligonucleotide binds to a natural antisense polynucleotide and modulates the expression and / or function of the target molecule. An example of a "function" may be to inhibit a negative regulator of transcription, thus allowing increased expression of a desired molecule, such as BAG3.

[0113] In another preferred embodiment, antisense oligonucleotides bind to sense polynucleotides and modulate the expression and / or function of a target molecule.

[0114] In an embodiment of the present invention, oligonucleotide binds to the antisense strand of specific target.Oligonucleotide has a length of at least 5 nucleotides, and each oligonucleotide can be synthesized to target overlapping sequences, so that oligonucleotide is synthesized to cover the full length of target polynucleotide.Target also includes coding region and non-coding region.

[0115] According to the present invention, antisense compounds include single-stranded or double-stranded RNA interference (RNAi) compounds, such as antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, siRNA compounds, and other oligomeric compounds that hybridize to at least a portion of a target nucleic acid to regulate its function. As such, they can be DNA, RNA, DNA-like, RNA-like, or mixtures thereof, or one or more mimetics thereof. These compounds can be single-stranded, double-stranded, circular, or hairpin oligomeric compounds, and can include structural elements such as internal or terminal bulges, mismatches, or loops. Antisense compounds are usually prepared in a linear form, but can be combined or prepared to be circular and / or branched. Antisense compounds can include constructs, such as, for example, double strands hybridized to form a fully or partially double-stranded compound, or single strands with sufficient self-complementarity to allow hybridization and formation of a fully or partially double-stranded compound. The two strands can be linked to leave the 3' or 5' ends free internally, or to form a continuous hairpin structure or loop. The hairpin structure can include an overhang at either the 5' or 3' end, which results in an extension of the single stranded character. The double stranded compound can optionally include an overhang at the end. Further modifications can include a conjugate group attached to one of the ends, selected nucleobase position, sugar position, or one of the internucleoside linkages. Alternatively, the two strands can be linked via a non-nucleic acid moiety or linker group. When formed from only one strand, the dsRNA can form a self-complementary hairpin type molecule that forms a duplex that doubles back on itself. Thus, the dsRNA can be fully or partially double stranded. Specific regulation of gene expression can be achieved by stable expression of dsRNA hairpin in transgenic cell lines, but in a preferred embodiment, gene expression is up-regulated.When formed from a single strand that forms a duplex, or a self-complementary hairpin-type molecule that forms a duplex back on itself, the duplex (or the duplex-forming region of the single strand) is a complementary RNA strand that base pairs in a Watson-Crick fashion.

[0116] In another preferred embodiment, the desired oligonucleotide or antisense compound comprises at least one of an antisense RNA, an antisense DNA, a chimeric antisense oligonucleotide, an antisense oligonucleotide containing a modified linkage, an interfering RNA (RNAi), a short interfering RNA (siRNA); a micro interfering RNA (miRNA); a small temporal RNA (stRNA); or a short hairpin RNA (shRNA); a small RNA-induced gene activation (RNAa); a small activating RNA (saRNA), or a combination thereof.

[0117] dsRNA can also activate gene expression, a mechanism called "small RNA-induced gene activation" or RNAa. dsRNA targeting gene promoters induces strong transcriptional activation of associated genes. RNAa has been demonstrated in human cells using synthetic dsRNAs called "small activating RNAs" (saRNAs).

[0118] Small double-stranded RNA (dsRNA) can also act as a small activating RNA (saRNA). Without wishing to be bound by theory, by targeting sequences in gene promoters, the saRNA will induce target gene expression in a phenomenon called dsRNA-induced transcriptional activation (RNAa).

[0119] In some embodiments, the ribonucleic acid sequence is specific to the regulatory segment of genome that controls the transcription of BAG3. Thus, the candidate therapeutic agent can be a dsRNA that activates the expression of BAG3 in cells and is administered to a patient in need of treatment.

[0120] Peptide: In another embodiment, the BAG3 peptide is encoded by the nucleic acid comprising the wild type, chimeric or cDNA sequence of BCL2-associated aganase 3 (BAG3).The peptide can also be a synthetic peptide of BCL2-associated aganase 3 (BAG3).

[0121] It should be understood that the peptide sequence is not limited to the native form of the BCL2-associated asanogene 3 (BAG3) molecule or its cDNA sequence. Those skilled in the art will recognize that conservative amino acid changes can be made that do not usually change the function of a protein or peptide, even though they change the original sequence of the protein or peptide. Conservative amino acid substitutions typically include substitutions within glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, phenylalanine, and tyrosine.

[0122] Conservative substitutions can also be made based on the type of amino acid: aliphatic (valine, isoleucine, leucine, and alanine); charged (aspartic acid, glutamic acid, lysine, arginine, and histidine); aromatic residues (phenylalanine, tyrosine, and tryptophan); and sulfur-containing (methionine and cysteine). The polypeptide sequence has at least about 68% identity, at least about 70% identity, or at least about 71% identity to the BCL2-associated asanogene 3 (BAG3) nucleic acid sequence or its cDNA sequence.

[0123] The determination of percent identity between two nucleotide or nucleic acid sequences can be achieved using mathematical algorithms.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 by Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877).This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990, J. Mol. Biol. 215:403-410), and can be evaluated, for example, at the National Center for Biotechnology Information (NCBI) World Wide Web Site, which has the universal resource locator http: / / blast(dot)ncbi(dot)nlm(dot)nih(dot)gov / blast.cgi / . BLAST nucleotide searches can be performed with the NBLAST program (referred to on the NCBI website as "blastn") using parameters of gap penalty=5; gap extension penalty=2; mismatch penalty=3; match reward=1; expectation value 10.0; and word size=11 to obtain nucleotide sequences having homology to the nucleic acids described herein. BLAST protein searches can be performed with the XBLAST program or the NCBI "blastp" program using parameters of expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences having homology to the protein molecules described herein. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402).Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterative search that detects distance relationships between molecules and relationships between molecules that share common patterns. When using BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. In calculating percent identity, typically exact matches are counted.

[0124] The present invention also includes polynucleotides encoding hybrid proteins comprising a BCL2-associated athanogene 3 (BAG3) polypeptide functionally fused directly or indirectly to a second polypeptide sequence via a peptide linker. Linker sequences are well known in the art. In one embodiment, the hybrid protein comprises a BAG3 polypeptide or a BAG3 polypeptide functionally fused to a detectable moiety, such as a reporter polypeptide, which is directly or indirectly fused to the N-terminus or C-terminus of the BAG3 polypeptide. Exemplary reporter polypeptides include luciferase (LUC), green fluorescent protein (GFP), and GFP derivatives.

[0125] The hybrid protein comprising BAG3 polypeptide or its fragment can be linked to other types of polypeptides in addition to or instead of reporter polypeptide.These additional polypeptides can be any amino acid sequence that is useful for peptide purification, identification, and / or therapeutic or prophylactic application.In addition, the additional polypeptide can be a signal peptide, or a targeting peptide, etc.

[0126] In some cases, other additions, substitutions, or deletions may increase the stability of the polypeptide (including, but not limited to, resistance to proteolysis) or increase the affinity of the polypeptide for its suitable receptor, ligand, and / or binding protein. In some cases, other additions, substitutions, or deletions may increase the solubility of the polypeptide. In some embodiments, sites are selected for incorporation of non-naturally occurring amino acids, in addition to other sites for substitution with naturally encoded amino acids or non-naturally occurring amino acids, in order to increase the solubility of the polypeptide after expression in a recombinant host cell. In some embodiments, the polypeptide includes another addition, substitution, or deletion that modulates affinity for associated ligands, binding proteins, and / or receptors, modulates receptor dimerization (including, but not limited to, increasing or decreasing), stabilizes receptor dimers, modulates circulating half-life, modulates release or bioavailability, facilitates purification, or improves or changes a particular route of administration. Similarly, non-naturally occurring amino acid polypeptides may include chemical or enzymatic cleavage sequences, protease cleavage sequences, 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 conjugated molecules (including but not limited to biotin) that improve detection (including but not limited to GFP), purification, transport through tissue or cell membranes, prodrug release or activation, size reduction, or other properties of the polypeptide.

[0127] The methods and compositions described herein include the incorporation of one or more non-natural amino acids into polypeptides.One or more specific non-natural amino acids can be incorporated at one or more positions that do not interfere with the activity of polypeptides.This can be achieved by making "conservative" substitutions, including but not limited to, substituting hydrophobic amino acids with non-natural or natural hydrophobic amino acids, substituting large amino acids with non-natural or natural large amino acids, substituting hydrophilic amino acids with non-natural or natural hydrophilic amino acids, and / or inserting non-natural amino acids into positions that are not required for activity.

[0128] A variety of biochemical and structural approaches can be employed to select desirable sites for substitution with non-natural amino acids within a polypeptide. Any position of the polypeptide chain is suitable for selection for incorporating non-natural amino acids, and selection can be based on rational design for any or unspecified desired purpose, or by random selection. Selection of desirable sites can be based on producing non-natural amino acid polypeptides with desired properties or activities (which may be further modified or remain unmodified), including, but not limited to, agonists, superagonists, partial agonists, inverse agonists, antagonists, receptor binding molecules, receptor activity modulators, modulators of binding to binding partners, binding partner activity modulators, binding partner conformation modulators, dimer or multimer formation, no change in activity or properties compared to natural molecules, or manipulating any physical or chemical properties of the polypeptide, such as solubility, aggregation, or stability. For example, positions in a polypeptide that are required for the biological activity of the polypeptide can be identified using methods including, but not limited to, point mutation analysis, alanine scanning, or homolog scanning methods. Residues other than those identified as important for biological activity by methods including, but not limited to, alanine scanning or homolog scanning mutagenesis can be good candidates for substitution with non-natural amino acids, depending on the desired activity required for the polypeptide. Alternatively, sites identified as important for biological activity can also be good candidates for substitution with non-natural amino acids, depending further on the desired activity required for the polypeptide. Another option would be to make successive substitutions with non-natural amino acids at each position in the polypeptide chain and observe the effect on the activity of the polypeptide. Any means, technique, or method for selecting positions for substitution with non-natural amino acids into any polypeptide is adapted for use in the methods, techniques, and compositions described herein.

[0129] Candidate Agents and Screening Assays The compositions embodied herein may also be applied in the field of drug development and target validation.The present invention includes the use of the nucleic acid sequences and peptides embodied herein in drug development efforts to elucidate the relationship that exists between Bcl-2-associated anthanogene-3 (BAG3) polynucleotides and disease states, phenotypes, or conditions.These methods include contacting a sample, tissue, cell, or organism with a compound, measuring the nucleic acid or protein levels of Bcl-2-associated anthanogene-3 (BAG3) polynucleotides and / or related phenotypes or chemical endpoints at a time point after treatment, and optionally detecting or modulating Bcl-2-associated anthanogene-3 (BAG3) polynucleotides, including comparing the measurements to untreated samples or samples treated with additional compounds of the present invention.

[0130] The screening assay of the present invention suitably includes and embodies animal models, cell-based systems, and non-cell-based systems.The nucleic acid sequences and peptides embodied herein are used to identify agents of therapeutic interest, for example, by screening libraries of compounds, or by identifying compounds of interest by any of a variety of drug screening or analysis techniques or synthesis of new compounds.The genes, their alleles, fragments, or oligopeptides used in such screening can be free in solution, attached to solid support, present on cell surface, or localized intracellularly.Measurements are carried out as described in detail in the Examples section below.In an embodiment, screening of candidate agents is carried out to identify those that regulate the translation of BAG3.

[0131] The assay can be in vitro or in vivo. In vitro formats of interest include cell-based formats, where contact occurs by, for example, introducing a substrate into a medium, such as an aqueous medium, in which the cells are present. In other embodiments, the assay can be in vivo, where a multicellular organism containing cells is employed. Contact between the targeting vector encoding the nucleic acid sequence specifically shown herein and the target cell can be achieved using any convenient protocol. In these embodiments, where the target cell is present as part of a multicellular organism, such as an animal, the vector is conveniently administered (e.g., injected, fed, etc.) to the multicellular organism, such as the whole animal, and administration can be systemic or local, for example, directly to a specific tissue and / or organ of the multicellular organism.

[0132] Multicellular organisms of interest include, but are not limited to, insects, vertebrates, e.g., birds, such as chickens; mammals, including rodents, e.g., mice, rats; ungulates, e.g., pigs, cows, horses; primates, e.g., dogs, cats, monkeys, apes, humans, etc. Thus, target cells of interest include, but are not limited to, insect cells, vertebrate cells, particularly bird cells, e.g., chicken cells; mammalian cells, including mouse, pig, ungulate, sheep, horse, rat, dog, cat, monkey, and human cells, etc.

[0133] Target cells containing a BAG3 polynucleotide or polypeptide are contacted with a test compound and translation of BAG3 is determined or assessed by detecting the presence or absence of a signal from a detectable moiety, e.g., a luciferase substrate, i.e., by screening the cells (either in vitro or in vivo) for the presence of a luciferase-mediated luminescent signal. The detected signal is then employed to assess transcriptional and / or translational activity of BAG3 in the presence of the test agent.

[0134] The luminescence signal can be detected using any convenient luminescence detection device. In certain embodiments, the detector of interest includes, but is not limited to, photomultiplier tube (PMT), avalanche photodiode (APD), charge-coupled device (CCD); complementary metal oxide semiconductor (CMOS detector), and the like. The detector can be present in a signal detection device, such as a luminometer, that can detect the signal once or multiple times over a predetermined period as desired. Data can be collected in this manner at frequent intervals over a given assay period, such as once every 10 ms.

[0135] In certain embodiments, the subject method is carried out in a high-throughput (HT) format. In the subject HT embodiments of the present invention, a plurality of different cells are assayed or tested simultaneously. By simultaneously tested, it is meant that each of the plurality of cells is tested substantially simultaneously. In general, the number of cells tested simultaneously in the subject HT methods ranges from about 10 to 10,000, usually about 100 to 10,000, and in certain embodiments, about 1000 to 5000. A variety of high-throughput screening assays for determining the activity of candidate agents are known in the art, including, for example, those described in Schultz (1998) BioorgMed Chem Lett 8:2409-2414; Fernandes (1998) Curr Opin Chem Biol 2:597-603, the disclosures of which are incorporated herein by reference; and those described in US Pat. No. 6,127,133, and are readily adapted to the present invention.

[0136] In some embodiments, a method for screening for an agent that modulates the transcription and / or translation of Bcl-2 associated anthanogene-3 (BAG3) comprises the steps of contacting a BAG3 molecule with an agent, wherein the BAG3 molecule comprises an isolated nucleic acid or cDNA sequence of Bcl-2 associated anthanogene-3 (BAG3) operably linked to a detectable moiety, and at least one stop codon between the BAG3 and the detectable moiety; evaluating the transcription level of BAG3 in the absence of the candidate agent to obtain a reference level of transcription and / or translation, and evaluating the transcription and / or translation of BAG3 in the presence of the candidate agent to obtain a test level of transcription and / or translation, wherein the candidate agent is identified as an agent that increases transcription and / or translation when the test level of transcription and / or translation is higher than the reference level of transcription and / or translation.

[0137] In embodiments, the detectable moiety comprises a luminescent moiety, a chemiluminescent moiety, a fluorescent moiety, a bioluminescent moiety, an enzyme, a natural or synthetic moiety.

[0138] Any method known in the art can be used to evaluate translation.In a preferred embodiment, translation is evaluated using mammalian cells transfected with an expression vector comprising the nucleic acid of the present invention.Transfection can be transient, or cells can be stably transformed with the expression vector.Cell-based assays such as those described in Butcher et al., 2007, J Biol Chem. 282:2853-28539 can be used.Alternatively, in vitro translation assays can be used.

[0139] In the context of an expression vector, the vector can be readily introduced into a host cell, such as, for example, a mammalian, bacterial, yeast, or insect cell, by any method in the art. For example, the expression vector can be transferred into the host cell by physical, chemical, or biological means.

[0140] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, photoporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0141] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells.Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus type I, adenovirus, and adeno-associated virus, etc.See, for example, US Pat. No. 5,350,674 and 5,585,362.

[0142] Chemical means for introducing polynucleotide into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.The preferred colloidal system for use as a delivery vehicle in vitro and in vivo is liposome (i.e., artificial membrane vesicle).The preparation and use of such systems are well known in the art.

[0143] When non-viral delivery system is used, preferred delivery vehicle is liposome.The above delivery system and protocol can be found in "Gene Targeting Protocols, 2ed.", Kmiec ed., Humana Press, Totowa, NJ, pp 1-35 (2002) and "Gene Transfer and Expression Protocols, Vol. 7, (Methods in Molecular Biology)", Murray ed., Humana Press, Totowa, NJ, pp 81-89 (1991).

[0144] Candidate agent: The method can be carried out using any test compound as a candidate agent. Test compounds useful in carrying out the method of the present invention can be obtained using any of the many approaches in combinatorial library methods known in the art, including biological libraries, spatially-addressable parallel solid phase or solution phase libraries, synthetic library methods that require deconvolution, "one-bead one-compound" library methods, and synthetic library methods that use affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer, or small molecule libraries of compounds (Lam, 1997, Anticancer Drug Des. 12:145).

[0145] Examples of methods for the synthesis of 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.

[0146] Libraries of compounds can be stored 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), bacteria (US Pat. No. 5,223,409), spores (US Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), 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).

[0147] 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 that contain compounds that mimic polyamines (i.e., polyamine analogs) can also be screened.

[0148] In certain embodiments, the candidate agents are small molecule or large molecule ligands. By small molecule ligand is meant a ligand that ranges in size from about 50 to about 10,000 daltons, usually from about 50 to about 5,000 daltons, more usually from about 100 to about 1000 daltons. By large molecule is meant a ligand that ranges in size from about 10,000 daltons or greater in molecular weight.

[0149] The method may be performed repeatedly using different test conditions, such as different concentrations of the test candidate and / or duration of reaction time. Test candidates identified by the method may be further tested by methods convenient in the art to confirm specificity, dose dependency, in vivo efficacy, etc. Test candidates may be used as lead compounds for developing additional test candidates.

[0150] As indicated above, the present invention finds use in monitoring the transcriptional and / or translational activity of BAG3 in assays in which testing is performed with cells. In these embodiments, the cells are cultured under specific user-defined conditions (e.g., in the presence or absence of cytokines, nutrients, and / or candidate therapeutic agents) and monitored for emitted light.

[0151] A prototype compound or agent may be believed to have therapeutic activity based on any information available to one of skill in the art. For example, a prototype agent may be believed to have therapeutic activity based on information contained in the Physician's Desk Reference. In addition, by way of non-limiting example, a compound may be believed to have therapeutic activity based on the experience of a clinician, the structure of the compound, structure-activity relationship data, EC 50 , Assay Data, IC 50 It may be believed to have therapeutic activity based on assay data, evidence from animal or clinical studies, or other evidence, or a combination of such evidence.

[0152] A therapeutically active compound or agent is an agent that has therapeutic activity, including, for example, the ability of the agent to induce a particular response when administered to a subject or tested in vitro. Therapeutic activity includes treatment of a disease or condition, including both preventive and ameliorative treatment. Treatment of a disease or condition can include improvement of the disease or condition in any amount, including prevention, amelioration, and elimination of the disease or condition. Therapeutic activity can be performed against any disease or condition, including, in a preferred embodiment, a disease or disorder associated with injury by reactive oxygen intermediates. To determine therapeutic activity, any method that can evaluate the therapeutic activity of a compound can be used. For example, clinical evaluation, EC 50 , and I.C. 50 Both in vivo and in vitro methods, including assays and dose-response curves, can be used.

[0153] Candidate compounds for use with the assay of the present invention, or identified as useful pharmacological agents by the assay of the present invention, may be pharmacological agents known in the art or variants thereof, or may be compounds not previously known to have any pharmacological activity. Candidate compounds may be naturally occurring or designed in the laboratory. Candidate compounds may include a single diastereomer, multiple diastereomers, a single enantiomer, or multiple enantiomers.

[0154] Candidate compounds may be isolated, for example, from microorganisms, animals, or plants, and may be recombinantly produced, or may be synthesized by chemical methods known in the art. If desired, the candidate compounds of the present invention may be obtained using any of a number of combinatorial library methods known in the art, including, but not limited to, biological libraries, spatially addressable parallel solid-phase or liquid-phase libraries, synthetic library methods that require deconvolution, "one bead one compound" library methods, and synthetic library methods that use affinity chromatography selection. The biological library approach is limited to polypeptide libraries. The other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds, and are the preferred approaches in the present invention. See Lam, Anticancer Drug Des. 12:145-167 (1997).

[0155] In one aspect, the present invention provides a method for identifying a candidate compound as a suitable prodrug. Suitable prodrugs include any prodrug that can be identified by the methods of the present invention. Any method apparent to one of skill in the art can be used to identify a candidate compound as a suitable prodrug.

[0156] In another aspect, the present invention provides a method for screening a candidate compound for suitability as a therapeutic agent. Screening for suitability as a therapeutic agent can include evaluation of one, several, or a number of criteria for a compound that can affect the ability of the compound as a therapeutic agent. For example, factors such as efficacy, safety, efficiency, retention, localization, tissue selectivity, degradability, or intracellular persistence can be considered. In one embodiment, a method for screening a candidate compound for suitability as a therapeutic agent is provided, where the method includes providing the candidate compound identified as a suitable prodrug, determining the therapeutic activity of the candidate compound, and determining the intracellular persistence of the candidate compound. Intracellular persistence can be measured by any technique obvious to those skilled in the art, such as, for example, radioactive tracer, heavy isotope labeling, or LCMS.

[0157] In screening compounds for suitability as therapeutic agents, the intracellular persistence of the candidate compounds is evaluated. In a preferred embodiment, the agents are evaluated for their ability to modulate the translation of the compositions embodied herein over the course of the candidate therapeutic agent's response.

[0158] In another preferred embodiment, soluble and / or membrane-bound forms of the compositions embodied herein, such as, for example, proteins, variants, or biologically active portions thereof, can be used in assays to screen candidate agents. When membrane-bound forms of the proteins are used, it may be desirable to utilize solubilizing agents. Examples of such solubilizing agents include non-ionic surfactants such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, TRITON™ X-100, TRITON™ X-114, THESIT™, isotridecypoly(ethylene glycol ether), 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamino]-2-hydroxy-1-propanesulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propanesulfonate.

[0159] Cell-free assays may also be used and involve preparing a reaction mixture containing a BAG3 molecule (nucleic acid or peptide) containing a bioluminescent moiety and a test compound under conditions and for periods of time that allow for measurement of transcriptional and / or translational activity over time, as well as measurement of the concentration of the test agent.

[0160] In other embodiments, the candidate agent is an antisense oligonucleotide. In embodiments, BAG3 expression (e.g., protein) in a sample (e.g., in vivo or in vitro cells or tissues) treated with the antisense oligonucleotide of the present invention is evaluated by comparison with BAG3 expression in a control sample. For example, the translation of BAG3 is monitored by the signal emitted by the detectable moiety and compared with mock-treated or untreated samples. Alternatively, a comparison with a sample treated with a control antisense oligonucleotide (e.g., one with an altered or different sequence) can be made according to the desired information. In another embodiment, the difference in transcription and / or translation activity in treated versus untreated samples can be compared by the difference in expression of different nucleic acids (including any standard, such as housekeeping genes, deemed suitable by the researcher) in treated versus untreated samples.

[0161] The observed difference can be presented as desired, such as in the form of a ratio or percentage, for use in comparison with a control. In some embodiments, the level of BAG3 protein in a sample treated with an antisense oligonucleotide is increased or decreased by about 1.25-fold to about 10-fold, or more, compared to an untreated sample or a sample treated with a control nucleic acid. Preferably, the level of BAG3 protein is increased. In embodiments, the level of BAG3 protein is increased or decreased by at least about 1.25-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, or at least about 10-fold or more. In embodiments, the level or amount of BAG3 is preferably increased.

[0162] Microarray: Identification of nucleic acid sequences capable of binding to target molecules can be achieved by immobilizing a nucleic acid library on a substrate surface such that each unique nucleic acid is located at a defined position to form an array. In general, the immobilized library of nucleic acids is exposed to a biomolecule or candidate agent under conditions that favor binding of the biomolecule to the nucleic acid. The nucleic acid array will then be analyzed by the methods embodied herein to determine which nucleic acid sequences have been bound to the biomolecule. Preferably, the biomolecule will have a predetermined label for use in detecting the position of the bound nucleic acid.

[0163] Assays using fixed arrays of BAG3 nucleic acid sequences can be used for determining the sequence of unknown nucleic acids; single nucleotide polymorphism (SNP) analysis; analysis of BAG3 gene expression patterns from particular species, tissues, cell types, etc.; gene identification, etc.

[0164] In further embodiments, oligonucleotides or longer fragments derived from any BAG3 polynucleotide sequence can be used as targets in microarrays. Microarrays can be used to monitor the identification and / or expression levels of multiple genes or gene transcripts, to simultaneously identify the genes with which the target gene or its product interacts, and / or to evaluate the effect of candidate therapeutic agents in regulating the expression products of genes, such as, for example, in mediating neurological disorders. This information can be used to determine gene function and develop and monitor the activity of therapeutic agents.

[0165] Microarrays can be prepared, used, and analyzed using methods known in the art (see, e.g., Brennan et al., 1995, US Pat. No. 5,474,796; Schena et al., 1996, Proc. Natl. Acad. Sci. USA 93:10614-10619; Baldeschweiler et al., 1995, PCT application WO95 / 251116; Shalon, et al., 1995, PCT application WO95 / 35505; Heller et al., 1997, Proc. Natl. Acad. Sci. USA 94:2150-2155; and Heller et al., 1997, US Pat. No. 5,605,662). In other embodiments, the microarray contains BAG3 peptides or other desired molecules that can be assayed to identify candidate agents.

[0166] In another preferred embodiment, the method for screening candidate agents for treating or preventing cardiac disease or disorders comprises contacting sample with candidate agents and measuring the effect that the agent has on target.For example, the agent may regulate BAG3 expression, and then the agent may be further studied for any possible therapeutic effect (e.g., increase or decrease of the monitored parameter such as expression).Abnormal expression state may be caused by pathology such as heart failure, disease, cancer, genetic defect, and / or toxin.

[0167] Antibodies: Useful diagnostic assays may include one or more antibodies that specifically bind to BAG3. In some embodiments, the antibodies specifically bind to mutant BAG3, such as the BAG3 polypeptide disclosed herein having a 10 amino acid deletion as shown in FIG. 2. We use the term antibody to broadly refer to immunoglobulin-based binding molecules, including conventional antibodies (e.g., G-class tetrameric antibodies (e.g., IgG1)), fragments thereof that retain the ability to bind to their intended target (e.g., Fab' fragments), and single-chain antibodies (scFvs). Antibodies may be polyclonal or monoclonal, and may be produced by human, mouse, rabbit, sheep, or goat cells, or by hybridomas derived from these cells. Antibodies may be humanized, chimeric, or bispecific.

[0168] Antibodies may take on a variety of configurations and may comprise proteins consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Any of a variety of antibody structures may be used, including intact antibodies, antibody multimers, or antibody fragments, or other variants thereof that contain functional antigen-binding regions of an antibody. The term "immunoglobulin" may be used synonymously with "antibody." Antibodies may be monoclonal or polyclonal in origin. Regardless of the origin of the antibody, suitable antibodies include intact antibodies, e.g., IgG tetramers having two heavy (H) and two light (L) chains, single-chain antibodies, chimeric antibodies, humanized antibodies, complementarity determining region (CDR)-grafted antibodies, and antibody fragments, e.g., Fab, Fab', F(ab')2, scFv, Fv, and recombinant antibodies derived from such fragments, e.g., camelid antibodies, microantibodies, diabodies, and bispecific antibodies.

[0169] Intact antibodies consist of antigen-binding variable regions (V H and V L ), and the light chain constant domain (C L ) and heavy chain constant domain C H1 , C H2and C H3 The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. As is well known in the art, H and V L The region is further divided into regions of hypervariability called "complementarity determining regions" (CDRs), which are interspersed with more conserved framework regions (FRs).

[0170] Anti-BAG3 antibodies may be derived from any class of immunoglobulin, such as, for example, IgA, IgG, IgE, IgD, IgM (and subtypes thereof, such as IgG1, IgG2, IgG3, and IgG4), and the immunoglobulin light chains may be of the kappa or lambda type. Recognized human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes, as well as a number of immunoglobulin variable region genes.

[0171] The term "antigen-binding portion" of an immunoglobulin or antibody generally refers to the portion of an immunoglobulin that specifically binds to a target, in this case, the epitope that includes amino acid residues in the BAG3 polypeptide. Thus, an antigen-binding portion of an immunoglobulin is a molecule in which one or more immunoglobulin chains are not full-length, but do not specifically bind to an intracellular target. Examples of antigen-binding portions or fragments include (i) Fab fragments, a monovalent fragment consisting of VLC, VHC, CL, and CH1 domains; (ii) F(ab')2 fragments, a bivalent fragment that includes two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fv fragments consisting of the VLC and VHC domains of a single arm of an antibody; and (v) isolated CDRs that have sufficient framework to specifically bind, for example, to the antigen-binding portion of the variable region. The antigen-binding portion of the light chain variable region and the antigen-binding portion of the heavy chain variable region, e.g., the two domains of the Fv fragment, VLC and VHC, can be joined using recombinant methods by a synthetic linker that allows the VLC and VHC regions to be produced as a single protein chain that pairs to form a monovalent molecule, known as a single chain Fv (scFv). Such scFvs can be targeting agents of the present invention and are included in the term "antigen-binding portion" of an antibody.

[0172] An "Fv" fragment is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in tight con-covalent association. In this conformation, the three hypervariable regions of each variable domain interact to form the V H -V LThe six hypervariable regions define an antigen-binding site on the surface of the dimer. The six hypervariable regions confer antigen-binding specificity, but 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 improve stability, the VH-VL domains may be linked by a flexible peptide linker, such as (Gly4Ser)3 (SEQ ID NO:7), to form single-chain Fv or scFV antibody fragments, or may be engineered to form disulfide bonds by introducing two cysteine ​​residues into the framework regions to produce disulfide-stabilized Fvs (dsFvs). Fragments of antibodies are suitable for use in the provided methods, so long as they retain the desired specificity of the full-length antibody and / or sufficient specificity to specifically bind to a BAG3 polypeptide.

[0173] The compositions of the 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 determined, for example, by Scatchard analysis (Scatchard, Ann. NY Acad, Sci. 51 :660-672 (1949)) can be readily determined by one of ordinary skill in the art.

[0174] In some embodiments, the anti-BAG3 antibody is at least 1.5-fold, 2-fold, 5-fold, 10 ... 3 Double, 10 4 Double, 10 5 Double, 10 6 These may bind to their target epitopes or mimicking decoys at 100-fold or more.

[0175] In some embodiments, the anti-BAG3 antibody is -4 M or less, 10 -7 M or less, 10 -9In some embodiments, the binding affinity of the anti-BAG3 antibody for its respective target is at least 1×10 6 In some embodiments, the binding affinity of the anti-BAG3 antibody to BAG3 is at least 5×10 6 Ka, at least 1 × 10 7 Ka, at least 2 × 10 7 Ka, at least 1 × 10 8 Ka or greater. Antibodies may also be described or specified in terms of their binding affinity to BAG3. In some embodiments, the binding affinity is greater than or equal to 5×10 -2 Less than M, 10 -2 Less than M, 5×10 -3 Less than M, 10 -3 Less than M, 5×1O -3 Less than M, 10 -4 Less than M, 5×10 -5 Less than M, 10 -5 Less than M, 5×10 -6 Less than M, 10 -6 Less than M, 5×10 -7 Less than M, 10 -7 Less than M, 5×10 -8 Less than M, 10 -8 Less than M, 5×10 -9 Less than M, 5×10 -10 Less than M, 10 -10 Less than M, 5×10 -11 Less than M, 10 -11 Less than M, 5×1O -12 Less than M, 10 -12 Less than M, 5×10 -13 Less than M, 10 -13 Less than M, 5×10 -14 Less than M, 10 -14 Less than M, 5×10 -15 Less than M or 10 -15 These include those with a Kd less than M or lower.

[0176] In some embodiments, the antibodies do not bind to known related polypeptide molecules; for example, they bind to BAG3 but not to known related polypeptides. In some embodiments, the antibodies specifically bind to mutant BAG3 polypeptides, such as, for example, a BAG3 polypeptide having a 10 base pair deletion as shown in Figure 2, but which is not a wild-type BAG3 polypeptide. Antibodies can be screened against known related polypeptides to isolate an antibody population that specifically binds to BAG3.

[0177] The diagnostic assays of the present invention may include concurrent immunoelectrophoresis, radioimmunoassay (RIA), radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. The anti-BAG3 antibody may include a tag, which may also be indicated as a reporter or marker (e.g., a detectable marker). The detectable marker may be any molecule that is covalently attached to the anti-BAG3 antibody, or a biologically active fragment thereof, that allows for qualitative and / or quantitative evaluation of the expression or activity of the tagged peptide. The activity may include biological activity, physicochemical activity, or a combination thereof. Both the formation and location of the detectable marker may vary, so long as the labeled antibody retains biological activity. Many different markers may be used, and the selection of a particular marker will depend on the application desired. For example, a labeled anti-BAG3 antibody can be used to assess levels of BAG3 or mutant BAG3 in biological samples such as urine, saliva, cerebrospinal fluid, blood, or biopsy samples, or to assess clinical response to cardiovascular therapy, such as the BAG3 constructs described above.

[0178] Exemplary detectable labels include barium, diatrizoate, iodized poppy seed oil ethyl ester, gallium citrate, iocarmic acid, iosetamic acid, iodamide, iodipamide, iodoxamic acid, iogulamide, iohexol, iopamidol, iopanoic acid, ioprocemic acid, iosefamic acid, ioseric acid, iosulamide meglumine, iosemetic acid, iotasul, iotetric acid, iothalamic acid, iotroxic acid, ioxaglic acid, ioxotrizoic acid, radiopaque or contrast agents, such as fluorescein isothiocyanate, ipodate, meglumine, metrizamide, metrizoate, propriodone, and thallium chloride. Alternatively or additionally, the detectable label can be, for example, a fluorescent label, such as fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, 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 aequorin.

[0179] Suitable markers include, for example, enzymes, photoaffinity ligands, radioisotopes, and fluorescent or chemiluminescent compounds. Methods for introducing detectable markers into peptides are well known in the art. Markers can be added during or after synthesis. Recombinant anti-BAG3 antibodies or their biologically active variants can also be labeled by adding a labeled precursor (e.g., a radiolabeled amino acid) to the culture medium in which 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 that can be easily labeled with 125I, such as tyrosine. In some embodiments, additional functional groups that support effective labeling can be added to fragments of anti-BAG3 antibodies or their biologically active variants. For example, a 3-tributyltin benzoyl group can be added to the N-terminus of the native structure; subsequent replacement of the tributyltin group with 125I will create a radiolabeled iodobenzoyl group.

[0180] Any method known in the art can be used to detect such labels, such as, for example, positron emission tomography (PET), SPECT imaging, magnetic resonance imaging, x-ray, or detectable by ultrasound.

[0181] In another preferred embodiment, a method of treating a patient having a cardiac disease or disorder, wherein the patient has reduced BAG3 levels compared to baseline levels, comprising administering a pharmaceutical composition comprising a therapeutically effective amount of at least one BAG3 inducer that increases expression of a BAG3 molecule.

[0182] In another embodiment, a method for preventing or treating a subject at risk of or suffering from a cardiac disease or disorder, comprising administering to the subject a pharmaceutical composition comprising at least one agent that regulates the expression of BAG3 or a therapeutically effective amount of a BAG3 polynucleotide or polypeptide. In a preferred embodiment, the cardiac disease and / or disorder is heart failure.

[0183] In another embodiment, a method of treating heart failure in a patient, comprising administering a pharmaceutical composition comprising a therapeutically effective amount of at least one agent that regulates expression of BAG3 or a BAG3 polynucleotide or polypeptide.

[0184] In another embodiment, a method for preventing or treating a cardiac disease or disorder in a subject, comprising administering a pharmaceutical composition comprising a therapeutically effective amount of at least one agent that regulates expression of BAG3 or a BAG3 polynucleotide or polypeptide.

[0185] In yet another embodiment, a method for preventing or treating a cardiac disease or disorder in a subject, comprising administering a pharmaceutical composition comprising a therapeutically effective amount of at least one agent that modulates the expression of BAG3 or a BAG3 polynucleotide or polypeptide, and one or more therapeutic agents prescribed by a medical caregiver. In an embodiment, the at least one agent that modulates the expression of BAG3 or a BAG3 polynucleotide or polypeptide, and one or more therapeutic agents prescribed by a medical caregiver are administered sequentially or simultaneously.

[0186] Diagnostics, Treatments, Kits The compositions and compounds herein of the present invention may be utilized for diagnostics, therapeutics, and prophylaxis, as well as research reagents and components of kits.

[0187] The compositions disclosed herein are generally and variously useful for treating subjects with cardiac diseases or disorders, such as, for example, heart failure or dilated cardiomyopathy. Subject, patient, or individual may be referred to interchangeably. A subject is effectively treated whenever it produces a clinically useful result. This may mean, for example, a complete remission of disease symptoms, a reduction in the severity of disease symptoms, or a delay in disease progression. These methods may further include a) identifying a subject (e.g., a patient, more specifically, a human patient) with cardiac disease or disorder; and b) providing the patient with a composition comprising a nucleic acid encoding a BAG3 polypeptide. The nucleic acid encoding a BAG3 polypeptide may be inserted into a vector, such as, for example, an AAV vector, that is administered to the patient. For example, the subject may be identified using standard clinical tests related to cardiac function. The amount of such a composition provided to the subject that results in a complete remission of infection symptoms, a reduction in the severity of infection symptoms, or a delay in infection progression is considered a therapeutically effective amount. The method may also include a monitoring step to aid in optimal dosing and scheduling, as well as to predict results. In some methods of the present invention, first, it can be determined whether the patient has a reduced level of BAG3, and then it can be determined whether the patient should be treated with one or more compositions described herein.BAG3 level can be assayed, for example, using anti-BAG3 antibody, and then compared with a reference level to determine whether the patient has an increased level of BAG3.Monitoring can also be used to quickly distinguish responsive patients from non-responsive patients.

[0188] Cardiovascular disorders applicable to the therapeutic and / or prognostic methods of the present invention include: BAG3 The disorder may be responsive to regulation.We believe that the specific events occurring in the course of treatment will be understood, but the composition of the present invention is not limited to those that act by affecting specific cellular mechanisms.Any form of cardiovascular disorder associated with misregulation of BAG3 is within the scope of the present invention.

[0189] The method of the present invention can be shown in relation to the preparation of medicaments.Thus, the present invention includes the use of the agent and composition described herein in the preparation of medicaments.The compounds described herein are useful in therapeutic compositions and regimens for use in the treatment of diseases or conditions described herein (e.g., cardiovascular disorders disclosed herein), or for the manufacture of medicaments for such use.

[0190] Any of the compositions described herein can be administered to any part of the host body for subsequent delivery to target cells. The compositions can be delivered to, but are not limited to, the brain, cerebrospinal fluid, joints, nasal mucosa, blood, lungs, intestines, muscle tissue, skin, or peritoneal cavity of a mammal. With respect to the route of delivery, the compositions can be administered by intravenous, intracranial, intraperitoneal, intramuscular, subcutaneous, intramuscular, rectal, vaginal, intrathecal, intratracheal, intradermal, or transdermal injection, by oral or intranasal administration, or by gradual perfusion over time. In a further example, an aerosol preparation of the composition can be given to the host by inhalation.

[0191] The dosage required will depend on the route of administration, the nature of the formulation, the nature of the patient's disease, the size, weight, surface area, age, and sex of the patient, other drugs being administered, and the judgment of the attending physician. Suitable dosages range from 0.01 to 1,000 mg / kg. Variations in the required dosage are expected given the diversity of intracellular targets and the differing efficiencies of various routes of administration. Variations in these dosage levels are adjusted using standard empirical routines for optimization, as is well understood in the art. Doses can be single or multiple (e.g., 2- or 3-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 50-fold, 100-fold, 150-fold, or more). Encapsulation of the compound in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) can increase the efficiency of delivery.

[0192] The duration of treatment with any of the compositions provided herein can be anything from as short as one day to as long as the lifespan of the host (e.g., many years). For example, the compound can be administered once a week (e.g., for 4 weeks to many months or years); once a month (e.g., for 3 to 12 months, or many years); or once a year for 5, 10, or more years. It is also understood that the frequency of treatment can vary. For example, the compound can be administered once (or twice, three times, etc.) daily, weekly, monthly, or yearly.

[0193] Any compound provided herein may be administered in an effective amount to an individual in need of treatment. As used herein, the term "effective" refers to any amount that induces a desired response in a patient but does not induce significant toxicity. Such an amount may be determined by evaluating the patient's response after administration of a known amount of a specific composition. In addition, the level of toxicity, if any, may be determined by evaluating the patient's clinical symptoms before and after administration of a known amount of a specific composition. It is known that the effective amount of a specific composition administered to a patient may be adjusted according to the desired outcome and the patient's response and level of toxicity. Significant toxicity may vary for each specific patient and depends on multiple factors, including but not limited to the patient's disease state, age, and tolerance to side effects.

[0194] Any method known in the art can be used to determine whether a particular reaction is induced.Clinical methods that can evaluate the disease extent of a particular condition can be used to determine whether a reaction is induced.The particular method used to evaluate the reaction will depend on the nature of the patient's disorder, the patient's age and sex, other drugs administered, and the judgment of the attending physician.

[0195] The simultaneous administration of two or more therapeutic agents does not require that the agents be administered at the same time or by the same route, as long as there is an overlap in the period during which the agents exert their therapeutic effect.Consequent or sequential administration, such as administration on different days or weeks, is considered.The composition can also be administered together with another standard therapeutic agent for the treatment of cardiovascular disease.

[0196] In another preferred embodiment, the agent regulates the expression of Bcl-2-associated anthanogene-3 (BAG3) in patients suffering from or at risk of developing a disease or disorder associated with a molecule or pathway associated with BAG3. Examples of such associated diseases or disorders include cardiac disease or disorder, skeletal muscle disease or disorder, multiple sclerosis, senile plaques, cerebral amyloid angiopathy, atherosclerosis, glioblastoma, amyloid deposits, neurodegenerative disease, neurofibrillary tangles, dementia, choriocarcinoma, astrocytoma, amyloidosis, hyperlipidemia, neurodegeneration, neoplastic transformation, prostate cancer, atherosclerotic plaque, obstructive disease, AIDS, metastasis, myocardial infarction, pulmonary fibrosis, necrosis, shock, melanoma, colorectal cancer, genetic susceptibility, psoriasis, cancer, inflammation, glioma, carcinoma, breast cancer, neuropathology, tumor, prostate cancer, vascular disease, cell injury, brain tumor, non-small cell lung cancer (NSCLC), hypercholesterolemia. Examples of skeletal muscle diseases include primary (genetic) diseases of muscle (e.g., muscular dystrophies and congenital myopathies, metabolic muscle diseases); acquired diseases (e.g., myositis, toxic myopathies); secondary diseases of muscle (e.g., neurogenic atrophy, atrophy resulting from chronic pulmonary embolism, heart disease, kidney disease, HIV / AIDs, cancer, sarcopenia, etc.).

[0197] Kits: The present invention further provides 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 carrying out any of the methods described herein. These systems and kits may include buffers, detection / imaging components, positive / negative control reagents, instructions, software, hardware, packaging, or other desirable components.

[0198] The kit provides a useful tool for screening test compounds capable of modulating the effect of a compound on a target molecule. The kit can be packaged in any suitable manner to aid in research, clinical, and laboratory testing, with the various parts typically in a container that includes instructions for use.

[0199] In certain embodiments, the kit may further comprise lipid and / or solvent.In certain embodiments, the kit may further comprise buffers and reagents required for the procedure, and instructions for carrying out the assay.In certain embodiments, the kit may further comprise, if necessary, agents for reducing background interference in the test, positive and negative control reagents, devices for carrying out the test, etc.

[0200] Also provided are kits for determining whether a subject has a mutation in the BAG3 polypeptide, such as the 10 base pair deletion disclosed herein, to diagnose a patient with or a predisposition to developing cardiovascular disease. The kits can also be utilized to monitor the efficiency of agents used to treat cardiovascular disease.

[0201] Administration of the Composition The agents identified by the methods embodied herein can be formulated and the compositions of the invention can be administered with one or more additional active ingredients, pharmaceutical compositions, or other compounds.The therapeutic agents of the invention can be administered to animals, preferably mammals, and most preferably humans.

[0202] In some embodiments, a pharmaceutical composition comprises a therapeutically effective amount of at least one agent that modulates expression of BAG3 or a BAG3 polynucleotide or polypeptide and is administered as part of a treatment.

[0203] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of at least one agent that modulates expression of BAG3 or a BAG3 polynucleotide or polypeptide, and one or more therapeutic agents prescribed by a medical caregiver.

[0204] In other embodiments, the pharmaceutical composition comprises at least one or more of the candidate therapeutic agents specifically described herein.

[0205] Pharmaceutical formulations may be for oral (solid or liquid), parenteral (intramuscular, intraperitoneal, intravenous (IV), or subcutaneous injection), intracardiac, transdermal (either passive or using iontophoresis or electroporation), transmucosal and systemic (intranasal, intravaginal, rectal, or sublingual) administration, or by inhalation route, or using biodegradable inserts, and may be formulated into dosage forms suitable for each route of administration.

[0206] The agent can be formulated in a pharma- ceutically acceptable carrier or diluent, such as saline or buffered salt solution. Suitable carriers and diluents can be selected based on the mode and route of administration and standard pharmaceutical practice. Descriptions of exemplary pharma-ceutically acceptable carriers and diluents and formulations can be found in Remington's Pharmaceutical Sciences and USP / NF, which are standard texts in the art. Other substances may be added to the composition to stabilize and / or preserve the composition.

[0207] The composition of the present invention can be administered to animals by any convenient technique. The composition can be directly administered to the target site, for example, by surgical delivery to the internal or external target site, or by catheter to a site accessible by blood vessel. Other methods of delivery, 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 composition is preferably formulated in a sterile, pyrogen-free form.

[0208] The compounds identified by the present invention may also be orally administered to a patient in such a manner that the concentration of the drug is sufficient to inhibit bone resorption or to achieve any other therapeutic objective as disclosed herein. Typically, a pharmaceutical composition containing the compound is administered at an oral dose of about 0.1 to about 50 mg / kg in a manner compatible with the patient's condition. Preferably, the oral dose will be about 0.5 to about 20 mg / kg.

[0209] 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 injections are also useful. Typically, parenteral doses will be from about 0.01 to about 100 mg / kg; preferably, 0.1 to 20 mg / kg, in a manner that maintains drug concentrations in the plasma at a concentration effective to increase expression of BAG3. The compound may be administered 1 to 4 times daily at a level that achieves a total daily dose of from about 0.4 to about 400 mg / kg / day. The precise amount of a compound of the present invention that is therapeutically effective and the route by which such a compound is optimally administered are readily determined by one of skill in the art by comparing the blood levels of the agent to the concentration required to have a therapeutic effect. Prodrugs of the compounds of the present invention may be prepared by any suitable method.

[0210] When the compounds, derivatives, salts, compositions, etc. of the present invention are administered in accordance with the present invention, it is expected that there will be no unacceptable toxicological effects. Compounds of the present invention that have the potential to have good bioavailability can be tested in one of several biological assays to determine the concentration of the compound required to have a given pharmacological effect.

[0211] In another preferred embodiment, a pharmaceutical or veterinary composition is provided that comprises one or more of the identified compounds and a pharma- ceutical or veterinary acceptable carrier. Other active substances may also be present as deemed appropriate or desirable for the disease or condition to be treated or prevented.

[0212] The carrier, or, if more than one is present, each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient.

[0213] The compounds identified by the methods herein will be suitable for use in the various drug delivery systems described above. Furthermore, to improve the in vivo serum half-life of the administered compounds, the compounds can be encapsulated, introduced into the lumen of liposomes, prepared as colloids, or other convenient techniques that provide an extended serum half-life of the compounds can be employed. For example, various methods are available for preparing liposomes, as described in Szoka, et al., US Pat. Nos. 4,235,871, 4,501,728, and 4,837,028, each of which is incorporated herein by reference. Furthermore, the drugs may be administered in targeted drug delivery systems, such as liposomes coated with tissue-specific antibodies. The liposomes will be targeted to and selectively taken up by organs.

[0214] Formulations include those suitable for rectal, nasal, topical (including buccal and sublingual), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) administration, but preferably, the formulation is an orally administered formulation. Formulations can be conveniently presented in unit dosage form, such as, for example, tablets and sustained release capsules, and can be prepared by any method well known in the art of pharmacy.

[0215] Such methods include the step of bringing into association the agent with the carriers as defined above. Generally, the formulations are prepared by uniformly and essentially bringing into association the active agent with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0216] Compounds identified using these methods can be formulated according to known methods by combining the compound in a mixture with a pharma- ceutically acceptable carrier vehicle to prepare a pharma- ceutically useful composition. Therapeutic agents are prepared for storage by mixing the active ingredient, having the desired degree of purity, in the form of a lyophilized formulation or aqueous solution, with any pharmacologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Suitable 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 (less than about 10 residues) polypeptides; 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 such as glucose, mannose, or dextrin; 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, NJ), PLURONICS™ (BASF Corporation, Mount Olive, NJ), or PEG.

[0217] Formulations to be used for in vivo administration must be sterile and pyrogen-free, which is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.

[0218] The dosage and desired drug concentration of the pharmaceutical composition of the present invention may vary depending on the specific use envisaged. The determination of suitable dosage and route of administration is within the ability of a physician. Animal experiments provide reliable guidance for determining effective dosage for human treatment. Interspecies scaling of effective dosage can be carried out according to the principle described by Mordenti, J. and Chappell, W. "The use of interspecies scaling in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, New York 1989, pp.42-96.

[0219] Formulations for oral administration 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 granules; as a solution or suspension of the active agent in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water or water-in-oil liquid emulsion.

[0220] The term "acceptable carrier" for compositions for oral administration (e.g., tablets and capsules) includes vehicles such as common excipients, such as binders, such as syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone (povidone), methylcellulose, ethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, sucrose, and starch; fillers and carriers, such as 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 fluids, talc wax, oils, and colloidal silica. Flavorings, such as peppermint, wintergreen oil, cherry flavoring, and the like, may also be used. It may be desirable to add coloring agents to make the dosage form easily identifiable. The tablets may be coated by methods well known in the art.

[0221] Tablets 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 powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active agent, 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 be optionally coated or scored and formulated to provide slow or controlled release of the active agent.

[0222] Other formulations suitable for oral administration include lozenges, which contain the active agent in a flavored base, usually sucrose and acacia or tragacanth; pastilles, which contain the active agent in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes, which contain the active agent in a suitable liquid carrier.

[0223] Parenteral formulations will generally be sterile.

[0224] Dose: An effective dose of the disclosed compositions of the present invention is administered to a subject 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 relevant response in the subject being treated). This response can be measured in many ways, such as cytokine profiles, cell types, cell surface molecules, etc., as described above. The actual dose level of the active ingredients in the disclosed compositions of the present invention can be modified to administer an amount of the active compound that is effective to achieve the desired therapeutic response for a particular subject. The dose level selected will depend on the activity of the therapeutic composition, the route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the condition and prior medical history of the subject being treated. However, it is within the skill of the art to start a dose of the compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. The potency of the composition may vary, and therefore the "effective amount of treatment" may vary. However, using the assay methods described herein, one of skill in the art can easily evaluate the potency and effectiveness of the disclosed candidate compounds of the present invention and adjust the treatment regimen accordingly.

[0225] The present invention has been described in detail with reference to preferred embodiments thereof, but it will be understood that those skilled in the art, upon consideration of this disclosure, may make modifications and improvements within the spirit and scope of the invention.

[0226] All documents described herein are incorporated herein by reference. All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each publication or patent document was individually indicated. By citation of various references in this document, applicants do not acknowledge that any particular reference is "prior art" to the present invention. EXAMPLES

[0227] While various aspects of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. The following non-limiting examples are illustrative of the present invention.

[0228] Example 1: Altered BAG3 protein levels are associated with both familial and non-familial dilated cardiomyopathy Mutations in Bcl-2-associated anthanogene-3 (BAG3), a 575 amino acid antiapoptotic protein that acts as a co-chaperone for heat shock proteins (HSPs), have been associated with FDC (Selcen D, et al., Annals of Neurology. 2009; 65:83-89; Odgerel Z, et al., Neuromuscular disorders :NMD. 2010; 20:438-442; Lee HC, et al., Clinical Genetics. 2012; 81:394-398). For example, Norton et al. recently identified a deletion of BAG3 exon 4 as a rare variant responsible for FDC in a family without neuropathy or peripheral muscle weakness (Norton N, et al., American Journal of Human Genetics. 2011; 88:273-282). Subsequent sequencing of BAG3 in subjects diagnosed with IDC identified four additional mutations that segregated in all relatives affected with the disease.A genome-wide association study performed in patients with HF secondary to IDC suggested that a nonsynonymous single nucleotide polymorphism (SNP) (c.757T>C, [p.Cys151Arg]) located within the BAG3 gene as contributing to sporadic dilated cardiomyopathy (Villard E, et al., European Heart Journal. 2011; 32:1065-1076).

[0229] In this study, novel BAG3 mutations were identified in a family with adult-onset FDC. Furthermore, we report here for the first time that BAG3 protein levels are significantly reduced in unrelated patients with non-familial IDC. This demonstrates that altered levels of BAG3 protein may be involved in the progression of HF.

[0230] material and method Materials: Families with adult-onset familial dilated cardiomyopathy were identified. After obtaining informed consent, participating family members underwent a physical examination by a heart failure cardiologist and blood was collected for subsequent DNA analysis. DNA was extracted using a DNA extraction kit (Qiagen, Valencia CA) and stored at -70°C. Whenever possible, electrocardiograms were obtained from affected family members who had not undergone heart transplantation. Family members without a recent electrocardiogram underwent transthoracic echocardiograms using a SonoHeart Elite (SonoSite Inc, Bothell, Washington, USA) portable echocardiographic system. Medical records were obtained from deceased individuals. Disease status was determined based on consensus guidelines (Mestroni L, et al., European Heart Journal. 1999; 20:93-102). Participating family members provided written informed consent prior to assessment, and the protocol was approved by the internal review boards of Thomas Jefferson University and the University of Colorado.

[0231] METHODS: Human cardiac tissue was obtained from nine subjects (six males, three females, mean age 47.6 ± 5.7 years) unrelated to the study family with end-stage heart failure who underwent heart transplantation at Temple University Hospital, from one affected family member at the time of heart transplantation at the University of Colorado, and from seven organ donors (one male, six females, mean age 59.3 ± 3.7 years) whose hearts were not suitable for donation due to incompatibility with blood type, age, or size. All patients who underwent transplantation had severe left ventricular dysfunction and cardiac dilatation with a mean left ventricular ejection fraction (LVEF) of 12.8 ± 1.4%. Two of the transplant recipients had HF secondary to ischemic cardiomyopathy, and the remaining had nonischemic IDC. At the time of transplantation, four of the transplant recipients received dobutamine only, five received milrinone only, and one received both milrinone and dobutamine. Echocardiograms were performed in all of the donors prior to donation, and all had normal left ventricular function by echocardiogram with a mean LVEF of 57.5±1.6%. Tissue aliquots were taken from the left ventricular free wall, rapidly frozen in liquid nitrogen, and stored at −70° C. as previously described (Bristow MR, et al., The Journal of Clinical Investigation. 1993; 92:2737-2745). The Institutional Review Boards at the University of Colorado and Temple University approved the tissue studies, and consent was obtained for all subjects.

[0232] Exome sequencing and bioinformatics: DNA from five affected and one unaffected family members was selected for exome sequencing with a target depth >100×. Exome enrichment was performed using the Agilent SureSelect Human Exon 51Mb kit (Agilent, Santa Clara, CA). Paired-end 100-nucleotide exome sequencing was performed using the Illumina HiSeq 2000 platform (San Diego, CA). Sequence reads that passed the Illumina chastity filter were subjected to a quality filtering step and refined, and refined reads for each pair were retained if they were greater than 50 nucleotides. The paired reads were then mapped to the reference human genome sequence (hgl9) with gSNAP (Wu TD, et al., Bioinformatics. 2010; 26:873-881). Sequence calling for variants (single nucleotide polymorphisms [SNPs] insertions and deletions [indels]) was performed using Broad's Genome Analysis Toolkit (McKenna A, et al., Genome Research. 2010; 20:1297-1303).

[0233] After variant detection, the program Annotate Variation (ANNOVAR) was used to classify variants (e.g., exonic, intronic, synonymous, nonsynonymous, splice variants, stop retention, stop loss, insertion, or deletion) and cross-reference all variants against various genetic diversity databases (e.g., dbSNP, 1000 Genomes Database, AVSIFT) to isolate rare variants (variants with an average allele frequency ≦1% not found in dbSNP, 1000 Genomes Database, aVSIFT) (Wang K, et al., Nucleic Acids Research. 2010; 38:e164). Only nonsynonymous changes (SNPs and indels) resulting in alternative splicing sites and / or aberrant stop codons were considered for further analysis. For nonsynonymous changes, all insertion and deletion variants were considered damaging, whereas SNP variants were cross-referenced against the dbNSFP database to determine whether the change to the protein structure was considered tolerated or damaging using four algorithms: Sorting Intolerant From Tolerant (SIFT), PolyPhen2, likelihood ratio test [LRT], or MutationTaster (Liu X, et al., Human Mutation. 2011; 32:894-899). Identified putative mutations were confirmed by conventional Sanger sequencing in both affected and unaffected family members (primers and conditions available as needed).

[0234] Western blot analysis of human cardiac tissue: Frozen tissue was homogenized in 40 mM Tris buffer, pH 7.5, containing 150 mM NaCl, 1% NP40, 1 mM DTT, and 1 mM EDTA. Samples were then centrifuged at 10,000 x g for 30 min at 4°C, and the supernatant was collected and resuspended in 350 μM Tris buffer, pH 6.8, containing 25% β-mercaptoethanol, 30% glycerol, 10% SDS, and 2% bromophenol blue. Protein concentration was determined using the method of Bradford, and samples were stored at -80°C. Equal amounts of protein (10 μg) were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were blocked for 1 h in 10% dry skim milk / Tris-buffered saline (pH 7.6) supplemented with 0.1% Tween-20 (TBS-T) and then incubated with polyclonal BAG3 antibody (Proteintech, Chicago, IL) in 5% dry skim milk with PBST for 2 h. Membranes were then incubated with goat anti-rabbit 800 and goat anti-mouse secondary antibodies for 1 h and scanned with a LI-COR Odyssey imaging system (Lincoln NE). All Western blot procedures were performed at room temperature. BAG3 signal intensity was normalized to GAPDH.

[0235] result Family History: The proband (Figure 1, III-5) was a 65-year-old woman of Eastern European descent who was referred to the Heart Failure Clinic at Thomas Jefferson University in June 2003 because of a family history of HF. She was first noted to have dilated cardiomyopathy at the age of 45 years. She was largely asymptomatic but was receiving diuretic beta-adrenergic receptor antagonists (β-blockers) and angiotensin-converting enzyme (ACE) inhibitors. Her vital signs were within normal limits, and her physical examination was notable only for a weak S3 heart sound. She had no peripheral muscle weakness, and her neurological examination was unremarkable. Her electrocardiogram showed normal sinus rhythm with mild LV hypertrophy and nonspecific ST-T wave changes. Her left ventricular ejection fraction was 20% on echocardiography. As seen in Figure 1 and Table 1, the proband had two sisters, one of whom (III-7) was asymptomatic with a normal physical examination, but her ejection fraction by echocardiography was 44%. The second sister (III-9) was phenotypically normal and had a normal echocardiogram.

[0236] The proband had three children. A son underwent heart transplantation at age 20 years after IDC (IV-5), and a second son was diagnosed with idiopathic dilated cardiomyopathy at age 20 years but remained asymptomatic at age 32 years, despite an ejection fraction of 33% (IV-4). A daughter had no cardiac symptoms, but her left ventricular ejection fraction by echocardiography was 48% and she had mild dilation of the left ventricle and aortic root without obvious aortic valve disease (IV-6). Her echocardiogram met the criteria for the diagnosis of dilated cardiomyopathy. Her electrocardiogram was normal. Neurological function was normal in all three children. The proband's affected sister (III-7) had one daughter who died at age 22 years from progressive heart failure secondary to IDC (IV-7): the other two children had normal echocardiograms. A cousin underwent a heart transplant for IDC at age 42 after diagnosis at age 40 (III-1), and one of his sons also underwent a heart transplant for IDC at the University of Colorado at age 30 (IV-1). Normal subjects were defined as "non-affected" if they had reached age 40 without symptoms and had normal echocardiograms that did not meet the criteria for the diagnosis of cardiomyopathy. A 10-year follow-up of all participants showed that functional capacity remained stable in all family members.

[0237] Genetic Analysis: As seen in Figure 1, the pedigree and clinical data are compatible with autosomal dominant adult-onset familial IDC. Exome sequencing of DNA from five affected family members (III-5, 7:IV-1, 4, 5) and one unaffected family member (III-9) had an average of 11.8 ± 0.96 Gb of pass-filter sequence read data per sample. After bioinformatics filtering, a 10-nucleotide deletion in the coding region of exon 4 of BAG3 (Ch10:del 121436332_12143641:del. 1266_1275 [NM 004281]) was shown to be present in all tested affected subjects and absent in the proband's healthy sister (III-9) (Figure 2). Additional family members were tested for the BAG3 deletion by Sanger sequencing, confirming the appropriate co-segregation of the deletion with the phenotype in affected (III-1, 5, 7 and IV-1, 4, 5, 6) and unaffected (III-9 and IV-8, 9, 10, 11, 12) individuals. This deletion is not found in existing databases and predicts a frameshift and introduction of a premature stop codon after 13 amino acids, truncating BAG3 by 140 amino acids at the carboxyl terminus. Thus, the abnormal BAG3 protein is predicted to have 435 amino acids instead of 575 amino acids. In addition, the amino acid sequence distal to the deletion: TIFF2024175055000002.tif5128 is predicted to be different from that found in the normal protein. Only one additional variant was found by exome sequencing and after bioinformatics filtering. The variant found in the IKZF5 gene (rs8192669) did not segregate with the IDC phenotype in other family members. Previous analysis of 52 genes associated with monogenic IDC for rare variants (≦1%) identified only nonsynonymous mutations in TTN, GATAD1, MYPN, ANKRD1, and RBM20: none of these variants segregated with the disease phenotype.

[0238] BAG3 expression in failing human hearts: To determine whether the BAG3 deletion (BAG3 del. NM_004281) found in this patient cohort results in reduced BAG3 protein levels, Western blot analysis was performed on myocardium obtained from an affected family member (IV-1) who underwent heart transplantation. The BAG3 protein levels in subject IV-1 were less than half of those found in heart tissue obtained from an organ donor whose heart could not be used for transplantation. As seen in Figures 3A and 3B, BAG3 levels in failing human hearts from patients with end-stage heart failure without known BAG3 mutations were significantly (p=0.0002) lower than those found in non-failing control hearts. Thus, reduced levels of BAG3 protein can be found in both individuals with BAG3 mutations and in end-stage failing human hearts.

[0239] Consideration There is growing recognition that genetic mutations may constitute a third cause of IDC. Indeed, researchers have begun to refer to these causes as familial dilated cardiomyopathy (FDC). Inheritance can occur in a variety of ways with the most common pattern of autosomal dominant inheritance. Mutations are most commonly found in genes encoding sarcomeres, leading to cardiac dysfunction, disruption of muscle fiber structure, and accumulation of degraded material in autophagic granules. Herein, we report that a 10 bp deletion in the gene encoding the sarcomere protein BAG3 segregates completely with affected individuals in families with an autosomal dominant pattern of FDC. We also report for the first time that BAG3 protein is substantially reduced in hearts of unrelated patients undergoing heart transplantation when compared to normal hearts derived from transplant recipients.

[0240] BAG3 is a 575 amino acid anti-apoptotic protein that is constitutively expressed in the heart and acts as a co-chaperone for heat shock proteins (HSPs). BAG3 binds to HSPs, regulates the ability of chaperone cytoskeletal proteins, including desmin, and is also involved in the degradation of cellular proteins via either the proteosome or autophagic pathways. BAG3 also protects cells from apoptotic death and inhibits myofibrillar degeneration in response to mechanical stress. Knockdown of BAG3 in zebrafish or neonatal cardiomyocytes, or homozygous disruption of BAG3 in mice, causes cardiac dysfunction, and BAG3 levels are reduced in skeletal muscle of spontaneously hypertensive rats.

[0241] The results of this study in a large family with FDC are consistent with earlier reports showing an association between mutations in BAG3 and the development of muscle pathology. Mutations in BAG3 were first shown to cause abnormal muscle function in two families with childhood-onset muscular dystrophy (Selcen D, et al., Annals of Neurology. 2009; 65:83-89; Odgerel Z, et al., Neuromuscular Disorders: NMD. 2010; 20:438-442), and IDC, a diffuse myocardial fibrosis phenotype, and sudden death were associated with markers in the chromosomal region 10q25-26 that contains the BAG3 locus. More recent studies have demonstrated a causal relationship between BAG3 mutations and the development of FDC in the absence of peripheral muscle weakness or neurological findings (Norton N, et al., American Journal of Human Genetics. 2011; 88:273-282; Villard E., et al., European Heart Journal. 2011; 32:1065-1076; Arimura T., et al., Human Mutation. 2011; 32:1481-1491).

[0242] As seen with genetic variants in other sarcomere genes, there was substantial genetic heterogeneity within this large family. For example, one of the proband's sons had early onset of severe disease requiring transplantation, while a sister with moderate disease and a middle-aged daughter with very mild disease have remained asymptomatic for over 10 years. In fact, the cardiac dysfunction of the proband's daughter was not recognized for careful phenotyping as part of this study. Identification of the causative mutation in this family provides an opportunity for guideline-style genetic testing, counseling of family members, and early identification of affected individuals. The finding that the use of angiotensin converting enzyme inhibitors improved survival in a small group of patients with Duchenne muscular dystrophy suggests that early treatment in families with mutations in sarcomere genes may be effective; however, further studies will be required to define the best treatment strategies.

[0243] It is reported here for the first time that BAG3 protein levels are significantly reduced in the hearts of unrelated patients with end-stage HF who have undergone heart transplantation and have no family history of myocardial disease. This finding is intriguing because it demonstrates that although mutations in BAG3 may be the cause of disease in FDC, changes in the levels of BAG3 protein may be involved in disease progression in patients with non-familial IDC. Nevertheless, these results demonstrate that BAG3 protein may be a novel target for therapeutic intervention in HF.

[0244] Example 2: Changes in BAG3 protein levels in failing mouse hearts Wild-type c57BL / 6 mice underwent trans-aortic banding (TAC) as described by Tilley et al. (Circulation 2014, Nov 11; 130(20): 1800-11). Eighteen weeks after TAC, left ventricular contractility was measured using a permeable catheter introduced into the left ventricle by a carotid approach as previously described. Contractility was measured during intravenous infusion of increasing doses of catecholamines (Figure 6B). Heart weight-to-body weight ratios were calculated after sacrifice (Figure 6A). Hearts were then frozen for later measurement of BAG3 levels. Myocardial proteins were extracted and resolved by gel electrophoresis and probed with mouse BAG3 antibody as described in Example 1. As shown in Figure 6C, there was a significant reduction in BAG3 levels by Western blotting in TAC mice compared to sham-operated controls. A representative Western blot is shown in Figure 6D.

[0245] Example 3: Changes in BAG3 protein levels in porcine hearts after balloon occlusion Hemodynamic index and BAG3 level were measured in non-infarcted left ventricular myocardium from pigs 4 weeks after left balloon occlusion of the left anterior descending artery. As shown in Figure 7A, Figure 7B, Figure 7C, and Figure 7D, ejection fraction, shortening fraction, end diastolic volume, and end systolic volume were significantly changed after balloon occlusion, respectively. As shown in Figure 7E by graph and in Figure 7F by Western blot, BAG3 level was decreased in pig hearts after balloon occlusion.

[0246] Table 1. Phenotypes of test subjects with and without a 10 nucleotide deletion in the BAG3 gene TIFF2024175055000003.tif146154

[0247] Sequence information SEQUENCE LISTING <110> TEMPLE UNIVERSITY OF THE COMMONWEALTH SYSTEM OF HIGHER EDUCATION <120> BAG3 AS A TARGET FOR THERAPY OF HEART FAILURE <150> US 61 / 934,483 <151> 2014-01-31 <160> 51 <170> PatentIn version 3.5 <210> 1 <211> 575 <212> PRT <213> Homo sapiens <400> 1 Met Ser Ala Ala Thr His Ser Pro Met 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 <210> 2 <211> 2608 <212> DNA <213> Homo sapiens <400> 2 gcggagctcc gcatccaacc ccgggccgcg gccaacttct ctggactgga ccagaagttt 60 ctagccggcc agttgctacc tccctttatc tcctccttcc cctctggcag cgaggaggct 120 atttccagac acttccaccc ctctctggcc acgtcacccc cgcctttaat tcataaaggt 180 gcccggcgcc ggcttcccgg acacgtcggc ggcggagagg ggcccacggc ggcggcccgg 240 ccagagactc ggcgcccgga gccagcgccc cgcacccgcg ccccagcggg cagaccccaa 300 cccagcatga gcgccgccac ccactcgccc atgatgcagg tggcgtccgg caacggtgac 360 cgcgaccctt tgccccccgg atgggagatc aagatcgacc cgcagaccgg ctggcccttc 420 ttcgtggacc acaacagccg caccactacg tggaacgacc cgcgcgtgcc ctctgagggc 480 cccaaggaga ctccatcctc tgccaatggc ccttcccggg agggctctag gctgccgcct 540 gctagggaag gccaccctgt gtacccccag ctccgaccag gctacattcc cattcctgtg 600 ctccatgaag gcgctgagaa ccggcaggtg caccctttcc atgtctatcc ccagcctggg 660 atgcagcgat tccgaactga ggcggcagca gcggctcctc agaggtccca gtcacctctg 720 cggggcatgc cagaaaccac tcagccagat aaacagtgtg gacaggtggc agcggcggcg 780 gcagcccagc ccccagcctc ccacggacct gagcggtccc agtctccagc tgcctctgac 840 tgctcatcct catcctcctc ggccagcctg ccttcctccg gcaggagcag cctgggcagt 900 caccagctcc cgcgggggta catctccatt ccggtgatac acgagcagaa cgttacccgg 960 ccagcagccc agccctcctt ccaccaagcc cagaagacgc actacccagc gcagcagggg 1020 gagtaccaga cccaccagcc tgtgtaccac aagatccagg gggatgactg ggagccccgg 1080 cccctgcggg cggcatcccc gttcaggtca tctgtccagg gtgcatcgag ccgggagggc 1140 tcaccagcca ggagcagcac gccactccac tccccctcgc ccatccgtgt gcacaccgtg 1200 gtcgacaggc ctcagcagcc catgacccat cgagaaactg cacctgtttc ccagcctgaa 1260 aacaaaccag aaagtaagcc aggcccagtt ggaccagaac tccctcctgg acacatccca 1320 attcaagtga tccgcaaaga ggtggattct aaacctgttt cccagaagcc cccacctccc 1380 tctgagaagg tagaggtgaa agttccccct gctccagttc cttgtcctcc tcccagccct 1440 ggcccttctg ctgtcccctc ttcccccaag agtgtggcta cagaagagag ggcagcccccc 1500 agcactgccc ctgcagaagc tacacctcca aaaccaggag aagccgaggc tcccccaaaa 1560 catccaggag tgctgaaagt ggaagccatc ctggagaagg tacaggggct ggagcaggct 1620 gtagacaact ttgaaggcaa gaagactgac aaaaagtacc tgatgatcga agagtatttg 1680 accaaagagc tgctggccct ggattcagtg gaccccgagg gacgagccga tgtgcgtcag 1740 gccaggagag acggtgtcag gaaggttcag accatcttgg aaaaacttga acagaaagcc 1800 attgatgtcc caggtcaagt ccaggtctat gaactccagc ccagcaacct tgaagcagat 1860 cagccactgc aggcaatcat ggagatgggt gccgtggcag cagacaaggg caagaaaaat 1920 gctggaaatg cagaagatcc ccacacagaa acccagcagc cagaagccac agcagcagcg 1980 acttcaaacc ccagcagcat gacagacacc cctggtaacc cagcagcacc gtagcctctg 2040 ccctgtaaaa atcagactcg gaaccgatgt gtgctttagg gaattttaag ttgcatgcat 2100 ttcagagact ttaagtcagt tggtttttat tagctgcttg gtatgcagta acttgggtgg 2160 aggcaaaaca ctaataaaag ggctaaaaag gaaaatgatg cttttcttct atattcttac 2220 tctgtacaaa taaagaagtt gcttgttgtt tgagaagtttt aaccccgttg cttgttgttc 2280 tgcagccctg tctacttggg cacccccacc acctgttagc tgtggttgtg cactgtcttt 2340 tgtagctctg gactggaggg gtagatgggg agtcaattac ccatcacata aatatgaaac 2400 attatcaga aatgttgcca ttttaatgag atgattttct tcatctcata attaaaatac 2460 ctgactttag agagagtaaa atgtgccagg agccatagga atatctgtat gttggatgac 2520 tttaatgcta cattttaaaa aaagaaaata aagtaataat ataactcaaa aaaaaaaaa 2580 aaaaaaaaaaaaaaaaaaaaaaaaaaa 2608 <210> 3 <211> 575 <212> PRT <213> Homo sapiens <400> 3 Master Sister Here Here Thr His Sister Pro Master Master Gln Val Here Sister 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 <210> 4 <211> 575 <212> PRT <213> Homo sapiens <400> 4 Met Ser Ala Ala Thr His Ser Pro Met 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 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> PRT <213> Homo sapiens <400> 5 Met Ser Ala Ala Thr His Ser Pro Met 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> PRT <213> Homo sapiens <400> 6 Met Ser Ala Ala Thr His Ser Pro Met 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 <210> 7 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 7 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 8 <211> 13 <212> PRT <213> Homo sapiens <400> 8 Lys Pro Ser Trp Arg Arg Tyr Arg Gly Trp Ser Arg Leu 1 5 10 <210> 9 <211> 85 <212> DNA <213> Homo sapiens <400> 9 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgctgaaag tggaagccat 60 cctggagaag gtacaggggc tggag 85 <210> 10 <211> 64 <212> DNA <213> Homo sapiens <400> 10 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgctgaaag tggaagccat 60 cctg 64 <210> 11 <211> 11 <212> DNA <213> Homo sapiens <400> 11 aggggctgga g 11 <210> 12 <211> 64 <212> DNA <213> Homo sapiens <400> 12 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgaagccat cctggagaag 60 gtac 64 <210> 13 <211> 64 <212> DNA <213> Homo sapiens <400> 13 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgaagccat cctggagaag 60 gtac 64 <210> 14 <211> 65 <212> DNA <213> Homo sapiens <400> 14 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgctgaaag tggaagccat 60 cctgg 65 <210> 15 <211> 66 <212> DNA <213> Homo sapiens <400> 15 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgctgaaag tggaagccat 60 cctgga 66 <210> 16 <211> 71 <212> DNA <213> Homo sapiens <400> 16 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgaagccat cctggagaag 60 gtacaggggc t 71 <210> 17 <211> 72 <212> DNA <213> Homo sapiens <400> 17 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgaagccat cctggagaag 60 gtacaggggc tg 72 <210> 18 <211> 68 <212> DNA <213> Homo sapiens <400> 18 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgctgaaag tggaagccat 60 cctggaga 68 <210> 19 <211> 73 <212> DNA <213> Homo sapiens <400> 19 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgctgaaag tggaagccat 60 cctggagaag gta 73 <210> 20 <211> 73 <212> DNA <213> Homo sapiens <400> 20 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgctgaaag tggaagccat 60 cctggacaag gta 73 <210> 21 <211> 70 <212> DNA <213> Homo sapiens <400> 21 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgctgaaag tggaagccat 60 cctggagaag 70 <210> 22 <211> 75 <212> DNA <213> Homo sapiens <400> 22 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgaagccat cctggagaag 60 gtacaggggc tggag 75 <210> 23 <211> 75 <212> DNA <213> Homo sapiens <400> 23 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgaagccat cctggagaag 60 gtacaggggc tggag 75 <210> 24 <211> 84 <212> DNA <213> Homo sapiens <400> 24 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgctgaaag tggaagccat 60 cctggagaag gtacaggggc tgga 84 <210> 25 <211> 75 <212> DNA <213> Homo sapiens <400> 25 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgaagccat cctggagaag 60 gtacaggggc tggag 75 <210> 26 <211> 82 <212> DNA <213> Homo sapiens <400> 26 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgctgaaag tggaagccat 60 cctggagaag gtacaggggc tg 82 <210> 27 <211> 75 <212> DNA <213> Homo sapiens <400> 27 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgaagccat cctggagaag 60 gtacaggggc tggag 75 <210> 28 <211> 85 <212> DNA <213> Homo sapiens <400> 28 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgctgaaag tggaagccat 60 cctggagaag gtacaggggc tggag 85 <210> 29 <211> 75 <212> DNA <213> Homo sapiens <400> 29 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgaagccat cctggagaag 60 gtacaggggc tggag 75 <210> 30 <211> 85 <212> DNA <213> Homo sapiens <400> 30 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgctgaaag tggaagccat 60 cctggagaag gtacaggggc tggag 85 <210> 31 <211> 74 <212> DNA <213> Homo sapiens <400> 31 aaaaccagga gaagccgagg ctccccgaaa acatccagga gtgctgaaag tggaagccat 60 cctggagaag gtac 74 <210> 32 <211> 85 <212> DNA <213> Homo sapiens <400> 32 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgctgaaag tggaagccat 60 cctggagaag gtacaggggc tggag 85 <210> 33 <211> 85 <212> DNA <213> Homo sapiens <400> 33 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgctgaaag tggaagccat 60 cctggagaag gtacaggggc tggag 85 <210> 34 <211> 75 <212> DNA <213> Homo sapiens <400> 34 aaaaccagga gaagccgagg ctcccccaaa acatccagga gtgaagccat cctggagaag 60 gtacaggggc tggag 75 <210> 35 <211> 80 <212> DNA <213> Homo sapiens <400> 35 caggagaagc cgaggctccc ccaaaacatc caggagtgct gaaagtggaa gccatcctgg 60 agaaggtaca ggggctggca 80 <210> 36 <211> 64 <212> DNA <213> Homo sapiens <400> 36 aagccgaggc tcccccaaaa catccaggag tgaagccatc ctggagaagg tacaggggct 60 ggag 64 <210> 37 <211> 63 <212> DNA <213> Homo sapiens <220> <221> modified_base <222> (4)..(4) <223> a, c, t, g, unknown or other <400> 37 agcngaggct cccccaaaac atccaggagt gaagccatcc tggacaaggt acaggggctg 60 gag 63 <210> 38 <211> 70 <212> DNA <213> Homo sapiens <400> 38 cgaggctccc ccaaaacatc caggagtgct gaaagtggaa gccatcctgg agaaggtaca 60 ggggctggag 70 <210> 39 <211> 58 <212> DNA <213> Homo sapiens <400> 39 aggctccccc aaaacatcca ggagtgaagc catcctggag aaggtacagg ggctggag 58 <210> 40 <211> 67 <212> DNA <213> Homo sapiens <400> 40 ggctccccca aaacatccag gagtgctgaa agtggaagcc atcctggaga aggtacaggg 60 gctggag 67 <210> 41 <211> 66 <212> DNA <213> Homo sapiens <400> 41 gctcccccaa aacatccagg agtgctgaaa gtggaagcca tcctggagaa ggtacagggg 60 ctggag 66 <210> 42 <211> 54 <212> DNA <213> Homo sapiens <400> 42 tcccccaaaa catccaggag tgaagccatc ctggagaagg tacaggggct ggag 54 <210> 43 <211> 62 <212> DNA <213> Homo sapiens <400> 43 ccccaaaaca tccaggagtg ctgaaagtgg aagccatcct ggagaaggta caggggctgg 60 of 62 <210> 44 <211> 47 <212> DNA <213> Homo sapiens <400> 44 gagtgctgaa agtggaagcc atcctggaga aggtacaggg gctggag 47 <210> 45 <211> 43 <212> DNA <213> Homo sapiens <220> <221> modified_base <222> (4)..(4) <223> a, c, t, g, unknown or other <400> 45 atcnaggagt gaagccatcc tggagaaggt acaggggctg gag 43 <210> 46 <211> 40 <212> DNA <213> Homo sapiens <400> 46 gaaagtggaa gccatcctgg agaaggtaca ggggctggag 40 <210> 47 <211> 39 <212> DNA <213> Homo sapiens <400> 47 aggagtgaag ccatcctgga gaaggtacag gggctggag 39 <210> 48 <211> 37 <212> DNA <213> Homo sapiens <400> 48 agtggaagcc atcctggaga aggtacaggg gctggag 37 <210> 49 <211> 34 <h2 style=";text-align:left;direction:ltr"><212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Homo sapiens<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 49<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgaagccatc ctggagaagg tacaggggct ggag 34<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 50<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 31<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Homo sapiens<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 50<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aaacatccag gagtgctgaa agtggaagcc a 31<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 51<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> DNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Homo sapiens<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 51<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aaacatccag gagtgmwgmm akyskrrrcc 30

Claims

[Claim 1] The invention described herein.

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