WO2004083435A1 - Nab proteins and their use in diagnostic and therapeutic applications in heart disease - Google Patents

Abstract

The present invention relates to the use of NAB (NGFI-A-binding protein) nucleic acid molecules and proteins encoded by said nucleic acid molecules for the preparation of a pharmaceutical composition for the treatment of a heart disease. Furthermore, the invention relates to the use of NAB nucleic acid molecules and proteins for the preparation of a diagnostic composition for detecting a heart disease. Moreover, the invention relates to methods for diagnosing a heart disease or a susceptibility to a heart disease in a subject. The invention further relates to methods for identifying compounds which are able to modulate NAB protein interaction or biological activity.

Description

NAB PROTEINS AND THEIR USE IN DIAGNOSTIC AND THERAPEUTIC APPLICATIONS IN HEART DISEASE

The present invention relates to the use of NAB (NGFI-A-binding protein) nucleic acid molecules and proteins encoded by said nucleic acid molecules for the preparation of a pharmaceutical composition for the treatment of a heart disease. Furthermore, the invention relates to the use of NAB nucleic acid molecules and proteins for the preparation of a diagnostic composition for detecting a heart disease. Moreover, the invention relates to methods for diagnosing a heart disease or a susceptibility to a heart disease in a subject. The invention further relates to methods for identifying compounds which are able to modulate NAB protein interaction or biological activity.

NAB proteins constitute an evolutionary conserved family of co-repressors that specifically interact with and repress transcription mediated by the members of the NGFI-A (Egr-1 , Krox24, zif/268) family of transcription factors including NGFI-C, Krox20 and Egr-3. NGFI-A, in the following also referred to as Egr-1 , is a zinc-finger transcription factor encoded by an immediate-early gene which is induced by a wide variety of extra-cellular stimuli. Egr-1 has been implicated in cell differentiation, macrophage differentiation, and long term potentiation and activates numerous target genes including growth factors such as, inter alia, TNF-α, HGF, VEGF, TGF-β or PDGF A/B (Russo MW, Sevetson BR, Milbrandt J. Identification of NAB1 , a repressor of NGFI-A- and Krox20- mediated transcription. Proc. Natl. Acad. Sci. USA 1995; 92: 6873-6877; Gashler A, Sukhatme VP. Early Growth Response Protein 1 (Egr-1 ): Prototype of a Zinc-finger Family of Transcription Factors. Prog. Nucleic Res. Mol. Biol. 1995, 50: 191-224; Silverman ES, Collins T. Pathways of Egr-1 Mediated Gene Transcription in Vascular Biology, Am. J. Pathol. 1999, 154: 665-70; Silverman ES et al. The Transcription Factor Early-Growth-Response Factor 1 Modulates Tumor Necrosis Factor α, Immunoglobulin E, and Airway Responsiveness in Mice, Am. J. Respir. Crit. Care. Med. 2001 , 163: 778-785). A structure/function analysis of Egr-1 revealed a 34-amino acid inhibitory domain which was reported to be a target of the repressor protein NAB1 (NGFI-A-binding protein) (Russo MW, Sevetson Ϊ3R, Milbrandt J. Identification of NAB1 , a repressor of NGFI-A- and Krox20- mediated transcription. Proc. Natl. Acad. Sci. USA 1995; 92: 6873-6877). Through protein- protein interaction, this inhibitory domain of Egr-1 brings the transcriptional repressor NAB1 in close proximity to the transcription unit. Said repression function has been localized to the NAB conserved domain (NCD2), a region found in the carboxy- terminal half of all NAB proteins. Overexpression studies revealed that NAB1 is a.ble to completely block transcription mediated by Egr-1 (Thiel et al. Biochim. Biophys. Acta 2000, 1493 : 289-301).

Egr-1 is expressed, inter alia, in the heart and has been found to be increased in hypertrophied hearts of a transgenic mouse line that produces the polyomavirus large T-antigen gene in cardiomyocytes suggesting a role for Egr-1 in heart failure. However, a recent study demonstrated that the lack of Egr-1 expression does not preclude a hypertrophic response in the heart (Saadane, N. et al. Altered molecular response to adrenoreceptor induced cardiac hypertrophy in Egr-1 -deficient mice. Am. J. Physiol. Heart Circ. Physiol. 2000; 278: H796-H805).

Heart failure has become one of the leading causes of morbidity and mortality in the Western world (American Heart Association. Heart Disease and Stroke Statistics - 2003 Update. 2003). Current therapy for heart failure is primarily directed to using angiotensin-converting-enzyme (ACE) inhibitors, β-adrenergic receptor antagonists, diuretics and aldosterone antagonists. Of these only ACE-inhibitors, β-adrenergic receptor antagonists and aldosterone antagonists have been demonstrated to reduce mortality and prolong life in heart failure patients. Overall the prognosis of heart failure patients is still grim, with an average life expectancy after the first diagnosis of heart failure in the range of most malignant tumors (American Heart Association. Heart Disease and Stroke Statistics - 2003 Update. 2003). Heart transplantation is limited by the availability of donor hearts. These deficiencies in the current therapeutic options suggest an urgent need for novel therapeutic principles for the treatment of heart failure. Cardiac muscle hypertrophy is one of the most important adaptive physiological responses of the myocardium. In response to increased demands for cardiac work or following a variety of pathological stimuli which lead to cardiac injury, the heart adapts through the activation of a hypertrophic response in individual cardiac muscle cells, which is characterized by an increase in myocϋyte size, the accumulation of contractile proteins within individual cardiac cells, the activation of an embryonic gene expression pattern and the lack of a concomitant effect on muscle cell proliferation. Although the hypertrophic process can initially be compensatory, there can be a pathological transition in which the myocardi urn becomes dysfunctional (Hunter JJ, Chien KR. Signaling pathways for carcf iac hypertrophy and failure. N. Engl. J. Med. 1999; 341 : 1276-83; Chien KR. Meet ing Koch's postulates for calcium signaling in cardiac hypertrophy. J. Clin. Invest. 20 OO; 105: 1339-42; Braunwald E. Heart disease. Philadelphia: W. B. Saunders; 20O2). Numerous studies have been performed to uncover the signalling pathways underlying cardiac hypertrophy and have led to the identification of several signal transduction pathways involved in cardiomyocyte hypertrophy (Molkentin JD, Dorn IG, 2nd. Cytoplasmic signaling pathways that regulate cardiac hypertrophy. Annu Rev Physiol. 2001 ;63:391-426). The current concept comprising these findings suggests an intricate network of parallel and interconnected signalling pathways promoting to cardiomyocyte hypertrophy (Molkentin JD, Dorn IG, 2nd. Cytoplasmic signaling pathways that regulate cardiac hypertrophy. Annu. Rev. Physiol. 2001; 63: 391-426). While much progress has been made in uncovering pathways leading to cardiomyocyte hypertrophy, very little information is known about mechanisms inhibiting or reversing cardiomyocyte hypertrophy. These mechanisms could serve ideally as therapeutic targets for heart failure therapy. Therefore, new methods and means for diagnosing and treating heart diseases including cardiomyocyte hypertrophy and heart failure are highly desirable but not yet available.

Thus, the technical problem of the present invention is to comply with the needs described above. The solution to this technical problem is achieved by providing the embodiments characterized in the claims.

The present invention relates to the use of a nucleic acid molecule selected from the group consisting of:

(a) a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 1 or 3;

(b) a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4; (c) a nucleic acid encoding a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO: 2 or 4, wherein the fragment has repression of Egr-1 activity; and

(d) a nucleic acid of at least 70%, 80%, 90% or 95% identity to the nucleic acid of (a), (b) or (c), for the preparation of a pharmaceutical composition for the treatment of a heart disease.

The present invention is based on the surprising finding that NAB1 suppresses cardiac hypertrophy in a mouse model of cardiac hypertrophy. NAB1 mRNA and protein expression was found to be enhanced in murine heart failure in left-ventricular myocardium of a βradrenergic receptor transgenic mouse model (Engelhardt et al. Progressive hypertrophy and heart failure in betal -adrenergic receptor transgenic mice. Proc. Natl. Acad. Sci. U S A. 1999; 96:7059-64). In order to further analyze the biological role of NAB1 in heart diseases, transgenic mice overexpressing murine NAB1 under the control of the αMHC promoter (Engelhardt et al., loc. cit.; Subramanian et al. Transgenic analysis of the thyroid-responsive elements in the alpha-cardiac myosin heavy chain gene promoter. J Biol Chem. 1993; 268:4331-6) were generated. To assess the ability of overexpressed NAB1 to suppress Egr-1 - mediated transcriptional activity in the hearts of NAB1 -transgenic mice, direct gene transfer of Egr-1 -reporter plasmids carrying a luciferase gene into the hearts of NAB1 -transgenic and wild-type mice was performed. A portion of the animals was then given a dose of the hypertrophy inducing agents isoproterenol and phenylephrine intraperitoneally before determination of Egr-1 -dependent luciferase activity in lysates from left ventricular myocardium. Determination of luciferase activity in the hearts from wild-type and NAB-transgenic mice revealed a marked increase of Egr-1 -dependent luciferase activity in wild-type mice treated with the hypertrophic agents isoproterenol and phenylephrine. In sharp contrast, NAB1 -transgenic mice did not show any increase in luciferase activity indicating complete suppression of Egr-1 transcriptional activity through overexpression of NAB1 in vivo. Thus, these data showed that NAB1 suppresses Egr1 -activation through hypertrophic stimuli in vivo. This was further confirmed by expression analysis of TNFα, a known target gene for Egr-1. TNFα-expression was found to be significantly suppressed in NAB1- transgenic mice in comparison to wildtype mice. To investigate the role for NAB1 in cardiac hypertrophy, wild-type and NAB1- transgenic mice were treated with isoprenaline and phenylephrine through subcutaneously implanted osmotic minipumps. After 6 days, the animals were killed and their hearts removed. The ventricular weight of wild-type animals had significantly increased under the treatment as compared to untreated wild-type animals. However, NAB1 -transgenic mice showed a blunted response to the hypertrophic stimulation. The heart weight to body weight ratio of NAB1 -transgenic mice increased only marginally as compared to untreated mice and was significantly lower compared to treated wild-type mice. These data clearly demonstrate that NAB1 suppresses cardiac hypertrophy in vivo. Furthermore, wild type and NAB1 -transgenic mice were subjected to transverse aortic constriction and heart weight to body weight ratio was analysed 5 weeks after the procedure. Overexpression of NAB1 led to a significant reduction of the pressure-induced hypertrophic response, demonstrating that pressure overload-induced cardiac hypertrophy is inhibited by NAB1. In another study, NAB1 expression was assessed by Western blotting in left ventricular myocardium from heart failure patients and compared to nonfailing donor hearts, wherein a significant upregulation of NAB1 protein in human heart failure was detected. The surprising finding of the present invention that NAB1 prevents cardiac hypertrophy in mouse models of cardiac hypertrophy opens up novel methods and means for diagnosing and treating heart diseases, including cardiomyocyte hypertrophy, remodeling post-myocardial infarction or heart failure. Furthermore, the invention provides new screening methods for the identification of modulators of NAB1 interaction and biological activity as defined below.

For the use in the screening, diagnostic or therapeutic aspects of the invention, NAB1 polypeptides and nucleic acid sequences encoding NAB1 polypeptides from any origin or species can be utilized. The mouse and human NAB1 DNA sequences are listed, for example, on GenBank under accession numbers NM_008667 (SEQ ID NO: 1) and NM_005966 (SEQ ID NO: 3), respectively. The mouse and human NAB1 amino acid sequences are identified, for example, under accession numbers NP_032693 (SEQ ID NO: 2) and NP_005957 (SEQ ID NO: 4), respectively. Rat NAB1 is a 570 amino acid nuclear protein and is described in Proc. Nat. Acad. Sci. USA 1995, 92, p. 6873-6877. The rat DNA sequence is listed on GenBank under accession number U 17253. Moreover, mouse and human NAB2 protein sequences are indicated in Molecular and Cellular Biology, 1996, 16, 3545-3553. The mouse and human NAB2 nucleotide sequences can be found, e.g., under GenBank Accession numbers NM_008668 and NM_005967, respectively. The mouse and human Egr-1 nucleotide sequences are shown, for instance, in GenBank Accession numbers NM_007913 and NM_001964, respectively, see also references cited therein. The mouse and human Egr-2 nucleotide sequences have Genbank Accession numbers NM_010118 and J04076, respectively. Moreover, the mouse and human nucleotide sequences for Egr-3 can be found, e.g., under GenBank accession numbers NM_018781 and NM_004430, respectively. The mouse and human NGFI-C nucleotide sequences have GenBank Accession numbers NM_020596 and NM_001965, respectively. The references listed within the GenBank Accession Numbers provide further information with respect to the cloning and biochemical characterization of the mentioned genes and their gene products.

References to NAB1 polypeptides and nucleic acids described hereinafter are generally applicable to the sequences of any origin, particularly the mouse and human sequences described above. Preferably, said nucleic acid comprises the nucleic acid sequence of SEQ ID NO: 1 or 3 or encodes an amino acid sequence shown in SEQ ID NO: 2 or 4 as further described below.

The nucleic acids for the use in the screening, diagnostic or therapeutic aspects of the invention also comprise variants of the herein above described nucleic acids. Said variants, for instance, encode fragments, analogs and derivatives of the NAB1 polypeptide. Preferably, said nucleic acids encode fragments, analogs and derivatives having "repression of Egr-1 activity" as set forth below. A variant of the polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. Such non- naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms.

Among variants in this regard are variants that differ from the aforementioned nucleic acids by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.

It is apparent to the person skilled in the art that the nucleic acids for the use in the screening, diagnostic or therapeutic aspects of the invention are also directed to nucleic acids encoding fragments of the NAB1 polypeptide. Preferably, said nucleic acids encode fragments of the amino acid sequence of SEQ ID NO: 2 or 4, wherein the fragment has repression of Egr-1 activity. The term "repression of Egr-1 activity" refers to the fact that NAB proteins constitute an evolutionary conserved family of co- repressors that specifically interact with and repress transcription mediated by the members of the NGFI-A (Egr-1, Krox24, zif/268) family of immediate early gene transcription factors including NGFI-C, Krox20 and Egr-3 (Russo et al., 1995; loc. cit). Thus, "repression of Egr-1 activity" (sometimes hereinafter also termed biological activity) as used herein refers to repression by NAB1 of transcription mediated by the members of the NGFI-A (Egr-1, Krox24, zif/268) family of immediate early gene transcription factors comprising, inter alia, Egr-1 , Egr-2, Egr-3, NGFI-C, and Krox20. The present inventors found that repression of Egr-1 mediated transcription by NAB1 is particularly useful in the therapeutic and diagnostic means and methods according to the invention. Said Egr-1 repression function has been localized to the NAB conserved domain to (NCD2), a region found in the carboxy-terminal half of all NAB proteins (Swirnoff AH, Apel ED, Svaren J, Sevetson BR, Zimonjie DB, Popesen NC, Milbrandt J. NAB1 , a Corepressor of NGF1-A (Egr-1), Contains an Active Transcriptional Repression Domain. Mol. Cell. Biol. 1998, 18: 512-24). Assays for testing transcriptional repressor activity such as conventional reporter gene assays have been described in the art (Swirnoff AH, Apel ED, Svaren J, Sevetson BR, Zimonjie DB, Popesen NC, Milbrandt J. NAB1 , a Corepressor of NGF1-A (Egr-1), Contains an Active Transcriptional Repression Domain Mol. Cell. Biol. 1998, 18: 512- 24; Bahouth SW, Beauchamp MJ, Vu KM. Reciprocal Regulation of β-| -Adrenergic Receptor Gene Transcription by Sp1 and Early Growth Response Gene 1: Induction of EGR-1 Inhibits the Expression of the β-| -Adrenergic Receptor Gene. Mol. Pharmacol. 2002, 61 : 379-390; von der Kammer H, Mayhaus M, Albrecht C, Enderich J, Wegner M, Nitsch CM. Muscarinic Acethylcholine Receptors Activate Expression of the Egr Gene Family of Transcription Factors. J. Biol. Chem. 273: 14538-14544). For example, a reporter gene plasmid carrying Egr-1 -binding sites (Thiel G, Schoch S, Peterson D. Regulation of Synapsin I Gene Expression by the Zinc Finger Transcription Factor zif268/egr-1, J. Biol. Chem. 1994, 169: 15294-15301) can be transiently transfected into host cells (such as, e.g., CHO-K1 or HEK 293) in combination with a suitable control construct or in combination with an NAB1 gene- expression construct. Reporter genes are well known to the person skilled in the art and include, for instance, luciferase, GFP, CAT, lacZ, or the like. After transfection, the reporter gene activity is determined as described, for instance, in Thiel et al. (loc. cit.). In comparison to the reporter gene activity in the cells transfected with the control construct, a decrease in the reporter gene activity in the cells transfected with the NAB1 gene-expression construct is indicative for a repression of Egr-1 mediated transcription activity by NAB1. A decrease in the reporter gene activity as referred to herein is preferably a decrease of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to the reporter gene activity obtained with the control construct. It is apparent to a person skilled in the art that such a system can also be used to identify modulators of the repression of Egr-1 activity of NAB1 as described in more detail below. For example, the addition of a compound which is an agonist/activator of the NAB1 protein will cause an additional decrease in the reporter gene activity. Said reporter gene assays can also be performed in vivo as shown below.

Fragments as referred to herein comprise portions of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or 150 amino acid residues of the NAB1 protein. Preferably said fragments are biologically active fragments, i.e. fragments which have repression of Egr-1 activity. Said fragments, for example, may interact with the inhibitory domain of a transcription factor (TF), e.g., Egr-1, thereby inhibiting the ability of the TF to activate transcription of target genes. Known target genes for Egr- 1 are, e.g., TNF-α, TGF-β or PDGF A/B (Silverman ES, Collins T. Pathways of Egr-1 Mediated Gene Transcription in Vascular Biology, Am. J. Pathol., 1999, 154: 665-70).

The nucleic acids for the use in the screening, diagnostic or therapeutic aspects of the invention also comprise nucleic acids at least 70%, 75%, 80%, 85%, 90% or 95% identitical to the nucleic acids in SEQ ID NO: 1 or 3, to the nucleic acids encoding NAB1 polypeptides comprising the amino acid sequence of SEQ ID NO: 2 or 4 or to nucleic acids encoding NAB1 polypeptide fragments, wherein the fragment has repression of Egr-1 activity. Preferably, said nucleic acids which are at least 70%, 80%, 90% or 95% identical to the above-defined nucleic acids encode a polypeptide having repression of Egr-1 activity. By a nucleic acid having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the nucleic acid is identical to the reference sequence except that the nucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a nucleic acid having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be a. nucleic acid sequence as indicated above. As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 70%, 75%, 80%, 85%, 90% or 95% identical to a nucleotide sequence as indicated above can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence as referred to above) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. Appl. Biosci. 6:237-245 (1990)). In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity.

Nucleic acids generally refer to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA or cDNA. Thus, for instance, nucleic acids as used herein refer to, among others, single-and double- stranded DNA, DNA that is a mixture of single-and double stranded regions or single- or double- and triple-stranded regions, single-and double-stranded RNA, and RNA that is mixture of single-and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded, or a mixture of single- and double-stranded regions. In addition, nucleic acids as used herein refer to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term nucleic acids include DNAs and RNAs as described above that contain one or more modified bases. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "nucleic acids" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are nucleic acids as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term nucleic acids as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. Polynucleotides embraces short polynucleotides often referred to as oligonucleotide(s), which can be used, for instance, as probes or primers for detecting or amplifying the NAB1 nucleic acids. Preferably, said oligonucleotides are 10, 15, 20, 25 or 30 nucleotides in length and hybridize with the above identified NAB1 nucleic acids under stringent hybridization conditions according to Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994).

Nucleic acids encoding NAB1 transcriptional repressor proteins may be in the form of DNA, cDNA or RNA such as mRNA obtained by cloning or produced by chemical synthetic techniques. Thus, nucleic acids may be naturally occurring, e. g. DNA or RNA, or may be synthetic analogs, as known in the art. Such analogs may be preferred for use as probes or therapeutic agents because of superior stability under assay and cellular conditions, respectively. Modifications in the native structure, including alterations in the backbone, sugars or heterocyclic bases, have been shown to increase intracellular stability and binding affinity. Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3'-0'- 5'-S-phosphorothioate, 3'-S-5'-0-phosphorothioate, 3'-CH₂-5'-0-phosphonate and 3'- NH-5'-0-phosphoroamidate. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage. Sugar modifications are also used to enhance stability and affinity. The α-anomer of deoxyribose may be used, where the base is inverted with respect to the natural β-anomer. The 2'-OH of the ribose sugar may be altered to form 2'-0-methyl or 2'-0-allyl sugars, which provides resistance to degradation without comprising affinity. Modification of the heterocyclic bases must maintain proper base pairing. Some useful substitutions include deoxyuridine for deoxythymidine; 5-methyl-2'-deoxycytidine and 5-bromo-2'- deoxycytidine for deoxycytidine. 5-propynyl-2'-deoxyuridine and 5-propynyl-2'- deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and deoxycytidine, respectively.

The DNA may be single or double stranded. Single stranded DNA may be the coding or sense strand or it may be the non-coding or anti-sense strand. For therapeutic use, the polynucleotide is in a form capable of being expressed to a functional NAB1 transcription repressor protein at the site in the subject to be treated, preferably the heart. The polynucleotides may also be used for in vitro production of an NAB1 polypeptide for administration in a further therapeutic aspect of the invention as described in detail below.

Nucleic acids which encode a polypeptide of NAB1 may include, but are not limited to the coding sequence for NAB1 polypeptide, or biologically active fragments thereof. Thus, the polynucleotide may be provided together with additional, non-coding sequences, including for example, but not limited to non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription (including termination signals, for example), ribosome binding, mRNA stability elements, and additional coding sequence which encode additional amino acids, such as those which provide additional functionalities. Nucleic acids also include, but are not limited to, polynucleotides comprising a structural gene for NAB1 and its naturally associated genetic elements.

The above definitions apply mutatis mutandis to all of the methods and uses described herein. In a preferred embodiment of the use of the invention, the nucleic acid as defined above is comprised by a vector.

Said vector may be a cloning vector or an expression vector, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host/cells. Nucleic acids for the use in the screening, diagnostic or therapeutic aspects of the invention may be joined to a vector containing selectable markers for propagation in a host. Generally, a plasmid vector is introduced in a precipitate such as a calcium phosphate precipitate or rubidium chloride precipitate, or in a complex with a charged lipid or in carbon-based clusters, such as fullerens. Should the vector be a virus, it may be packaged in vitro using an appropriate packaging cell line prior to application to host cells.

Preferably, the nucleic acid is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells or isolated fractions thereof. Expression of said polynucleotide comprises transcription of the nucleic acid, preferably into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the lac, trp or tac promoter in E. coli, and examples for regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40- , RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNAI, pcDNA3 (Invitrogen), pSPORTI (GIBCO BRL). Preferably, said vector is an expression vector and/or a gene transfer vector. Expression vectors derived fr-om viruses such as retroviruses, adenoviruses, vaccinia virus, adeno-associated vir-us, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vector of the invention into targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombin ant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994). Alternatively, the polynucleotides and vectors of "the invention can be reconstituted into liposomes for delivery to target cells. The term "isolated fractions thereof" refers to fractions of eukaryotic or prokaryotic cells or tissues which are capable of transcribing or transcribing and translating RNA from the vector of the invention. Said fractions comprise proteins which are required for transcription of RNA or transcription of RNA and translation of said RNA into a polypeptide. Said isolated fractions may be, e.g., nuclear and cytoplasmic fractions of eukaryotic cells such as of reticulocytes. Kits for transcribing and translating RNA which encompass the said isolated fractions of cells or tissues are commercially available, e.g., as TNT reticulolysate (Promega).

Preferably, the vector is a viral vector such as adenovirus, adeno-associated virus, retrovirus (e.g. lentivirus) or herpesvirus.

Said viral vectors are particularly suitable for gene therapy. Gene therapy, which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors, methods or gene-delivering systems for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911 -919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Onodua, Blood 91 (1998), 30-36; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-2251 ; Verma, Nature 389 (1997), 239-242; Anderson, Nature 392 (Supp. 1998), 25-30; Wang, Gene Therapy 4 (1997), 393-400; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957; US 5,580,859; US 5,589,466; US 4,394,448 or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, and references cited therein. Further suitable gene therapy constructs for use especially in heart cells/tissues are known in the art (for example, Hajjar RJ, del Monte F, Matsui T, Rosenzweig A. Prospects for Gene Therapy for Heart Failure. Circ. Res., 2000, 86: 616-21). The nucleic acid molecules and vectors may be designed for direct introduction or for introduction via liposomes, viral vectors (e.g. adenoviral, retroviral), electroporation, ballistic (e.g. gene gun) or other delivery systems into the cell. Additionally, a baculoviral system can be used as eukaryotic expression system for the above-defined nucleic acid molecules. The introduction and gene therapeutic approach should, preferably, lead to the expression of a functional NAB1 polypeptide in the heart, whereby said expressed polypeptide is particularly useful in the treatment, amelioration and/or prevention of heart diseases, including, inter alia, cardiac hypertrophy, heart failure, or remodeling post-myocardial infarction (Braunwald E., Heart Disease, 1997; 2002; Saunders, Philadelphia).

Furthermore, nucleic acids for use in the diagnostic, screening or therapeutic aspects of the invention or a vector comprising said nucleic acids can be used for genetically engineering host cells, e.g., in order to express and isolate the NAB1 polypeptide as exemplified below.

Said host cell may be a prokaryotic or eukaryotic cell; see supra. The polynucleotide or vector which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally. In this respect, it is also to be understood that the recombinant DNA molecule of the invention can be used for "gene targeting" and/or "gene replacement", for restoring a mutant gene or for creating a mutant gene via homologous recombination; see for example Le Mouellic et al., Proc. Natl. Acad. Sci. USA, 87 (1990), 4712-4716; Joyner, Gene Targeting, A Practical Approach, Oxford University Press.

The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal, mammalian or, preferably, human cell. Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae. The term "prokaryotic" is meant to include all bacteria which can be transformed or transfected with a polynucleotide for the expression of a NAB1 polypeptide. Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. A polynucleotide coding for a NAB1 polypeptide can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art. Methods for preparing fused, operably linked genes and expressing them in bacteria or animal cells are well-known in the art (Sambrook, supra). The genetic constructs and methods described therein can be utilized for expression of NAB1 polypeptides in, e.g., prokaryotic hosts. In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted polynucleotide are used in connection with the host. The expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells. The transformed prokaryotic hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth. The NAB1 proteins can then be isolated from the grown medium, cellular lysates, or cellular membrane fractions. The isolation and purification of the microbially or otherwise expressed polypeptides may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies (Ausubel, loc. cit.)

Nucleic acids for use in the diagnostic, screening or therapeutic aspects of the invention can be utilized for the generation of transgenic non-human animals. Said animals preferably display an increased level of NAB1 expression and/or biological activity in the heart and comprise at least one allele of the NAB1 encoding gene. Promoters which drive heart specific expression of a transgene are described in the art. Particularly preferred as a transgenic non-human animal is mouse. A method for the production of a transgenic non-human animal, for example transgenic mouse, comprises introduction of a NAB1 polynucleotide or targeting vector into a germ cell, an embryonic cell, stem cell or an egg or a cell derived therefrom. The non-human animal can be used in accordance with a screening method of the invention described herein. Production of transgenic embryos and screening of those can be performed, e.g., as descπbed by A. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press. The DNA of the embryonal membranes of embryos can be analyzed using, e.g., Southern blots with an appropriate probe; see supra. Methods for producing transgenic flies, such as Drosophila melanogaster are also described in the art, see for example US-A-4,670,388, Brand & Perrimon, Development (1993) 118: 401-415; and Phelps & Brand, Methods (April 1998) 14: 367-379. Transgenic worms such as C. elegans can be generated as described in Mello, et al., (1991) Efficient gene transfer in C. elegans: extrachromosomal maintenance and integration of transforming sequences. Embo J 10, 3959-70, Plasterk, (1995) Reverse genetics: from gene sequence to mutant worm. Methods Cell Biol 48, 59-80.The invention also relates to transgenic non-human animals such as transgenic mice, rats, hamsters, dogs, monkeys, rabbits, pigs, C. elegans and fish such as Torpedo fish comprising a NAB1 gene. Preferably, said transgenic non- human animal is mouse.

Preferably, the modification is activation or overexpression of said gene or leads to the enhancement of the synthesis/biological activity of the corresponding NAB1 protein, particularly in the heart. This allows for example the study of the interaction of the aforementioned polypeptides on the onset of the clinical symptoms of a disease related to disorders in the heart. All the applications that have been herein before discussed with regard to a transgenic animal also apply to animals carrying two, three or more transgenes for example encoding different of the aforementioned nucleic acid molecules. It might be also desirable to inactivate or, more preferably, to enhance protein expression or function at a certain stage of development and/or life of the transgenic animal. This can be achieved by using, for example, tissue specific, developmental and/or cell regulated and/or inducible promoters which drive the expression of the transgene. A suitable inducible system is for example tetracycline- regulated gene expression as described, e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. 89 USA (1992), 5547-5551) and Gossen et al. (Trends Biotech. 12 (1994), 58-62). Similar, the expression of the mutant protein(s) may be controlled by such regulatory elements. As mentioned, the transgenic non-human animal, preferably mammal and cells of such animals contain (preferably stably integrated into their genome) at least one of the aforementioned nucleic acid molecule(s) or part thereof, wherein the transcription and/or expression of the nucleic acid molecule or part thereof leads to induction of the synthesis of (a) corresponding protein(s), preferably in the heart. Techniques how to achieve this are well known to the person skilled in the art. In cases where the aforementioned gene/nucleic acid is enhanced, optionally in combination with a modification of the function and/or expression of one or more further gene products such as other NAB family members, e.g. NAB2, interrelationships of gene products in the onset or progression of the diseases of the heart may be assessed. In this regard, it is also of interest to cross transgenic non- human animals having different transgenes for assessing further interrelationships of gene products in the onset or progression of said disease. Consequently, the offspring of such crosses is also comprised by the scope of the present invention.

The invention further relates to the use of a polypeptide selected from the group consisting of:

(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4;

(b) a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:2 or 4, wherein the fragment has repression of Egr-1 activity; and

(c) a polypeptide comprising an amino acid sequence at least 70%, 80%, 90% or 95% identical to the polypeptide of (a) or (b), for the preparation of a pharmaceutical composition for the treatment of a heart disease.

Polypeptide(s) as used herein, include but are not limited to polypeptides selected from the group consisting of: polypeptides comprising the amino acid sequence of SEQ ID NO: 2 or 4; a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:2 or 4, wherein the fragment has repression of Egr-1 activity; and a polypeptide comprising an amino acid sequence at least 70%, 80%, 90% or 95% identical to the polypeptide as described above. "Fragments" as used herein refer preferably to oligopeptides comprising 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or 150 amino acid residues of the mentioned polypeptide sequences. Furthermore, said polypeptide comprising an amino acid sequence at least 70%, 80%, 90% or 95% identical to the above-mentioned polypeptide has preferably repression of Egr-1 activity. The above definitions of "sequence identity" and "repression of Egr-1 activity" apply mutatis mutandis to all of the methods and uses described herein.

The basic structure of polypeptides and the recombinant or synthetic production as well as isolation methods of polypeptides are well known and have been described in innumberable textbooks and other publications in the art (see, e.g., Sambrook; Ausubel; loc. cit.). In this context, the term polypeptide is used herein to refer to any peptide or protein comprising two or more amino acid joined to each other in a linear chain by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are may types. It will be appreciated that polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids, and that many amino acids, including the terminal amino acids, may be modified in a given polypeptide, either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques which are well known to the art. Even the common modification that occur naturally in polypeptides are too numerous to list exhaustively here, but they are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art.

Among the known modifications which may be present in polypeptides for use in the present invention are, to name an illustrative few, acetylatioin, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-linkings, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sjflation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Such modifications are well known to those of skill and have been described in great detail in the scientific literature. Several particularly common modifications, glycosylation, lipid attachment, sufation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, for instance, are described in most basic texts, such as, for instance PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES, 2^nd ed., T. E. Creighton, W. H. Freeman and Company, New York (1993). Many detailed reviews are available on this subject, such as, for example, those provided by Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1 to 12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, ed., Academic Press, New York (1983); Seifert et al., Meth. Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62 (1992). It will be appreciated, as is well known and as noted above, that polypeptides are not always entirely linear. For instance, polypeptides may be generally as a result of posttranslational events, including natural processing event and events brought about by human manipulation which do not occur naturally. Circular, branched and branched circular polypeptides may be synthesized by non-translational natural process and by entirely synthetic methods, as well. Modifications can occur anywhere in polypeptides, including the peptide backbone, the amino acid side- chains and the amino or carboxyl termini. In fact, blockage of the amino or carboxyl group in a polypeptide, or both, by a covalent modification, is common in naturally occurring and synthetic polypeptides and such modifications may be present in polypeptides of the present invention, as well. For instance, the amino terminal residue of polypeptides made in E. coli or other cells as defined above, prior to proteolytic processing, almost invariably will be N-formylmethionine. During posttranslational modification of the peptide, a methionine residue at the NH2-terminus may be deleted. Accordingly, this invention contemplates the use of both the methionine-containing and the methionineless amino terminal variants of the protein of the invention. The modifications that occur in a polypeptide often will be a function of how it is made. For polypeptides made by expressing a cloned gene in a host, for instance, the nature and extend of the modifications in large part will be determined by the host cell posttranslational modification capacity and the modification signals present in the polypeptide amino acid sequence. For instance, as is well known, glycosylation often does not occur in bacterial hosts such as, for example, E. coli. Accordingly, when glycosylation is desired, a polypeptide should be expressed in a glycosylating host, generally a eukaryotic cell. Insect cell often carry out the same posttranslational glycosyltions as mammalian cells and, for this reason, insect cell expression systems have been developed to express efficiently mammalian proteins having native patters of glycosylation, inter alia. Similar considerations apply to ot ner modifications. It will be appreciated that the same type of modification may be present in the same or varying degree at several sites in a given polypeptide. Also , a given polypeptide may contain many types of modifications. In general, as us-ed herein, the term polypeptide encompasses all such modifications, particularly those that are present in polypeptides synthesized recombinantly by expressing a polynucleotide in a host cell.

Variant(s) of nucleic acids or polypeptides, as the term is used herein, are polynucleotides or polypeptides that differ from a reference polynucleotide or polypeptide, respectively. Variants in this sense are described below and elsewhere in the present disclosure in greater detail. (1) a polynucleotide that differs in nucleotide sequence from another, reference polynucleotide. Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical. As noted below, changes in the nucleotide sequence of the variant may be silent. That is, they may not alter the amino acids encoded by the polynucleotide. Where alterations are limited to silent changes of this type a variant will encode a polypeptides with the same amino acid sequence as the reference. Also as noted below, changes in the nucleotide sequence of the variant may alter the amino acid sequence of a polypeptides encoded by the reference polynucleotide. Such nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. (2) A polypeptide that differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference and the variant are closely similar overall and, in may regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, which may be present in any combination. The invention relates to therapeutic uses of a nucleic acid molecule comprising a sequence which encodes an NAB1 polypeptide. The invention relates also to therapeutic uses of fragments of said polynucleotide sequence which encode biologically active fragments of an NAB1 polypeptide, or variants of the polynucleotide sequence which, by virtue of the degeneracy of the genetic code, encode functional, i.e. biologically active fragments of NAB1, and to functionally equivalent allelic variants and related sequences modified by single or multiple baise substitution, addition and/or deletion which encode polypeptides having biologically activity as described above. These may be obtained by standard cloning procedures known to the persons skilled in the art.

The term "composition" as employed herein comprises at least one compound selected from the group consisting of: a nucleic acid molecule comprising a sequence encoding an NAB1 protein as defined above; an NAB1 protein as set forth above; ^n antibody directed against the NAB1 protein; an agonist or antagonist of the NAE31 polypeptide; or a host cell, a virus or a vector comprising said nucleic acid molecule. Preferably, such a composition is a pharmaceutical or a diagnostic composition.

The composition may be in solid, liquid or gaseous form and may be, inter alia, in a form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s). Said composition may comprise at least one, two, preferably three, more preferably four, most preferably five of the mentioned compounds, either alone or in combination. As mentioned, said composition may also comprise antibodies directed against NAB1 obtainable by methods described in the art (Ausubel, Sambrook, loc. cit.). Preferably, said antibodies bind specifically to the NAB1 polypeptides as defined above and comprise polyclonal as well as monoclonal antibodies. It is apparent to those skilled in the art that antibodies which bind to and enhance or increase NAB1 biological activity are particularly preferred.

It is preferred that said pharmaceutical composition, optionally comprises a pharmaceutically acceptable carrier and/or diluent. The herein disclosed pharmaceutical composition may be partially useful for the treatment of heart diseases. Said disorders comprise, but are not limited to cardiac hypertrophy, heart failure, or remodeling post-myocardial infarction (Braunwald, loc. cit.). Cardiac muscle hypertrophy is one of the most important adaptive physiological responses of the myocardium. In response to increased demands for cardiac work or following a variety of pathological stimuli which lead to cardiac injury, the heart adapts through the activation of a hypertrophic response in individual cardiac muscle cells, which is characterized by an increase in myocyte size, the accumulation of contractile proteins within individual cardiac cells, the activation of an embryonic gene expression pattern and the lack of a concomitant effect on muscle cell proliferation. Although t he hypertrophic process can initially be compensatory, there can be a pathologioal transition in which the myocardium becomes dysfunctional (Hunter JJ, Chien KIR. Signaling pathways for cardiac hypertrophy and failure. N Engl J M&d. 1999;341 :1276-83; Chien KR. Meeting Koch's postulates for calcium signaling in cardiac hypertrophy. J Clin Invest. 2000;105:1339-42; Braunwald E. Heart disease. Philadelphia: W. B. Saunders; 2002). While much progress has been made in uncovering pathways leading to cardiomyocyte hypertrophy, very little information is known about mechanisms inhibiting or reversing cardiomyocyte hypertrophy. Λs described above and further illustrated in the Examples, the invention is based on the surprising finding that NAB1 suppresses cardiac hypertrophy in vivo thereby providing novel methods and means for treating heart diseases/disorders such as heart failure, cardiac hypertrophy, or remodeling-post-myocardial infarction (Braunwald E. Heart disease. Philadelphia: W. B. Saunders; 2002).

Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. It is particularly preferred that said administration is carried out by injection and/or delivery, e.g., to a site in the heart, etc. The compositions of the invention may also be administered directly to the target site, preferably the heart, e.g., by biolistic delivery to an external or internal target site. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Proteinaceous pharmaceutically active matter may be present in amounts between 1 ng and 10 mg/kg body weight per dose; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is a continuous infusion, it should also be in the range of 1 ng to 10 mg units per kilogram of body weight per minute. Progress can be monitored by periodic assessment. The compositions of the invention may be administered locally or systemically. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents depending on the intended use of the pharmaceutical composition. Said agents may be drugs acting on the heart.

The terms "treatment" , 'treating" and like are used herein to generally mean obtaining a desired pharmaceutical and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a heart disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a heart disease and/or adverse effect attributed to the disease. The term "treatment" as used herein covers any treatment of a heart disease in a mammal, particularly a human, and includes: (a) preventing the heart disease from occurring in a subject which may be predisposed to said disease but has not yet been diagnosed as having it; (b) inhibiting the heart disease, i.e. arresting its development ; or (c) relieving the heart disease, i.e. causing regression of said disease. Preferably, the heart disease is cardiac hypertrophy, heart failure or remodeling post-myocardial infarction.

The invention also relates to the use of a nucleic acid molecule or polypeptide as defined above, for the preparation of a diagnostic composition for detecting a heart disease. Diagnostic compositions as used herein refer to at least one of the above-listed compounds comprising: a nucleic acid molecule comprising a sequence encoding an NAB1 protein as defined above; an NAB1 protein as set forth above; an antibody directed against the NAB1 protein; an agonist or antagonist of the NAB1 polypeptide; or a host cell, a virus or a vector comprising said nucleic acid molecule. For instance, oligonucleotides of the above defined nucleic acid molecules can be used, e.g., as probes or primers for the detection or amplification of NAB1 encoding nucleic acids, preferably in heart cells or in the heart. Preferably, said oligonucleotides comprise at least 10, 15, 20, 25 or 30 contiguous nucleotides of the NAB1 nucleic acids for the uses and methods of the invention. Diagnostic compositions also comprise antibodies which specifically bind to NAB1 proteins as already set forth above. The diagnostic compositions preferably further comprise means for detection, for instance, the mentioned compounds may be labeled. Methods for labeling of compounds are well described in the art (Ausubel, Sambrook, loc. cit). The diagnostic composition is preferably in soluble form/liquid phase but it is also envisaged that said compounds are bound to/attached to and/or linked to a solid support. Solid supports may be used in combination with the diagnostic composition as defined herein or the mentioned compounds may be directly bound to said solid supports. Such supports are well known in the art and comprise, inter alia, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes, wells and walls of reaction trays, plastic tubes etc. The compound(s) may be bound to many different carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetide. The nature of the carrier can be either soluble or insoluble for the purposes of the invention. Appropriate methods for labeling have been identified above. Suitable methods for fixing/immobilizing said compound(s) of the invention are well known and include, but are not limited to ionic, hydrophobic, covalent interactions and the like.

In addition, the invention relates to a method for diagnosing a heart disease or a susceptibility to a heart disease in a subject, comprising: (a) determining the presence or amount of expression of a nucleic acid molecule encoding NAB1 as defined above in a biological sample of said subject; and

(b) diagnosing a heart disease or a susceptibility to a heart disease, wherein the presence or an increased amount of expression of said nucleic acid molecule is indicative for said heart disease.

Moreover, the invention relates to a method for diagnosing a heart disease or a susceptibility to a heart disease in a subject, comprising:

(a) determining the amount of expression or activity of the polypeptide as defined above in a biological sample of said subject; and

(b) diagnosing a heart disease or a susceptibility to a heart disease wherein an increased amount of expression or activity for said polypeptide is indicative for said heart disease.

The invention provides diagnostic methods useful during diagnosis of a heart disorder in a subject, involving measuring the expression level of the above defined NAB1 proteins or nucleic acids encoding them in cells or body fluid from an individual and comparing the measured expression level with a standard level of protein or nucleic acid expression level, whereby an increase in the gene expression level compared to the standard is indicative of a heart disorder. Preferably, the subject is human.

By "determining the presence or amount of expression of a nucleic acid molecule" or "determining the amount of expression or activity of the polypeptide" is intended qualitatively or quantitatively measuring or estimating the level of the NAB1 polypeptide or the level of the mRNA encoding the NAB1 polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample). Preferably, the NAB1 polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard NAB1 polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having a disorder. As will be appreciated in the art, once a standard NAB1 polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison. Methods for determining the presence or amount of expression of a nucleic acid are well described in the art and comprise, for instance, Northern blot, RT-PCR, TaqMan or RNase protection. Methods for determining the amount of the expression of NAB1 protein, such as antibody-based techniques, are also well known to the person skilled in the art. It is apparent for the person skilled in the art that such diagnostic methods also include microarray-based techniques such as gene or protein arrays. The biological activity of NAB1 can be determined, e.g., by reporter gene assays which have been descπbed above. An increased amount of expression or activity for the above-mentioned nucleic acid or the encoded NAB1 polypeptide as used herein is indicative for a heart disease or a susceptibility to a heart disease.

The term "susceptibility to a heart disease" as used herein means that a person has an increased chance of developing a clinical significant symptomatic heart disease.

By "biological sample" is intended any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains the NAB1 polypeptide of the present invention or mRNA. Preferably, said biological sample is derived from the heart. As indicated, biological samples include body fluids which contain the NAB1 polypeptide of the present invention, and other tissue sources found to express the NAB1 polypeptide. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.

In a preferred embodiment of the uses or methods according to the invention, the heart disease is cardiac hypertrophy, heart failure, or remodeling post-myocardial infarction (Braunwald , loc. cit).

The invention furthermore relates to a method for identifying an agonist or antagonist of the NAB1 polypeptide as defined above, comprising:

(a) determining the interaction of the polypeptide as defined above and

Egr-1 in the presence of a compound suspected to be an agonist or antagonist; (b) determining, if said interaction has been altered in the presence of said compound in comparison to interaction observed in the absence of said compound.

In a preferred embodiment of the screening method according to the invention, an increase in the interaction between NAB1 and Egr-1 is indicative for an agonist of the NAB1 polypeptide and a decrease in the interaction is indicative for an antagonist of said polypeptide.

An NAB1 polypeptide may be used to screen for molecules that bind to the polypeptide or for molecules to which the polypeptide binds. The binding of the polypeptide and the molecule may activate (agonist), increase, inhibit (antagonist), or decrease activity of the polypeptide or the molecule bound. Examples of such molecules include antibodies, oligonucleotides, proteins, or small organic molecules.

Preferably, the screening for these molecules involves producing appropriate cells which express the NAB1 polypeptide, for example as a secreted protein or on the cell membrane. Methods for NAB1 isolation from nuclear extracts, e.g. by immunoprecipitation, are also well known to the person skilled in the art. Preferred cells include cells from mammals, such as heart cells, yeast, Drosophila, or E. coli. Cells expressing the NAB1 polypeptide (or cell membrane containing the expressed polypeptide) are then preferably contacted with a test compound potentially containing the molecule to observe binding, stimulation, or inhibition of activity of either the NAB1 polypeptide or the molecule. The assay may simply test binding of a candidate compound to the polypeptide, wherein binding is detected by a label, or in an assay involving competition with a labeled competitor. Further, the assay may test whether the candidate compound results in a signal generated by binding to the polypeptide. Preferably, said assay is a reporter-gene assay as described above. Alternatively, the assay can be carried out using cell-free preparations, polypeptide/molecule affixed to a solid support as described above, chemical libraries, or natural product mixtures. The assay may also simply comprise the steps of mixing a candidate compound with a solution containing a polypeptide, measuring polypeptide/molecule activity or binding, and comparing the polypeptide/molecule activity or binding to a standard. In another aspect, the invention relates to a method for identifying compounds which are capable of activating or inhibiting the repression of Egr-1 activity of the polypeptide as defined above, comprising:

(a) introducing of an Egr-1 reporter construct into the heart of a non-human animal or heart cells which express the NAB1 polypeptide as defined above;

(b) determining Egr-1 dependent reporter gene activity in the presence of a compound suspected to be capable of activating or inhibiting the repression of Egr-1 activity of said polypeptide; and

(c) determining if said reporter gene activity has been altered in the presence of said compound in comparison to the reporter gene activity observed in the absence of said compound.

In a preferred embodiment of this method of the invention, an increase in the reporter gene activity is indicative for an antagonist of the above-defined polypeptide and a decrease in the reporter gene activity is indicative for an agonist of said polypeptide.

To assess whether a compound is capable of modulating the repression of Egr-1 activity, an Egr-1 reporter construct as described above can be introduced into heart cells or into the heart of a non-human animal which express the NAB1 polypeptide. In the next step, Egr-1 dependent reporter gene activity is determined in the presence of a compound suspected to be capable of activating or inhibiting the repression of Egr-1 activity of said NAB1 polypeptide followed by determining if said reporter gene activity has been altered in the presence of said compound in comparison to the reporter gene activity observed in the absence of said compound. To assess the ability of a compound to modulate the repression of EGR-1 activity of NAB1 in heart cells or in the hearts of NAB1 -transgenic mice, direct gene transfer of Egr1 -reporter plasmids into the cells or into hearts of NAB1 -transgenic and wild-type mice can be performed as already set forth above. An increase in the reporter gene activity in the presence of said compound is indicative for an antagonist of the above-defined polypeptide and a decrease in the reporter gene activity is indicative for an agonist of said polypeptide. Examples of such compounds which are capable of activating or inhibiting the repression of Egr-1 activity of the NAB1 polypeptide include antibodies, oligonucleotides, antisense molecules, morpholino oligomers, proteins, or small organic molecules.

The terms "antagonist/inhibitor and agonist/activator" in accordance with the present invention are chemical agents that modulate the action of NAB1 , either through altering its biological activity, as defined above, or through modulation of expression, e.g., by affecting transcription or translation, for example, by an antisense molecule or morpholino oligomers specifically hybridizing to the above mentioned nucleic acids encoding NAB1 which can be produced by methods known in the art. "Specifically hybridizing" refers to stringent hybridization conditions as, for example, indicated in Sambrook (loc. cit) In some cases the antagonist/inhibitor or agonist/activator may also be a substrate or binding molecule.

The term "activator," as used herein, includes both substances necessary for the NAB1 to become active in the first place, and substances which merely accentuate its activity. For example, the addition of a compound which is an agonist/activator of the NAB1 protein causes an additional decrease in the reporter gene activity tested in a reporter gene assay described above. An additional decrease in the reporter gene activity as referred to herein which is caused by the presence of the agonist is preferably a decrease of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% as compared to the reporter gene activity obtained in the absence of the agonist. The term "inhibitor" includes both substances which reduce the activity of the NAB1 and these which nullify it altogether. An "antagonist" or "agonist" that modulates the repression of Egr1 activity of NAB1 and causes for example a differential response in a reporter gene assay described above to a compound that alters directly or indirectly the activity NAB1 or the amount of active NAB1. Preferably, the antagonist/inhibitor and agonist/activator of NAB1 are small chemical agents which directly interact with NAB1. Therefore, there will preferably be a direct relationship between the molar amount of compound required to inhibit or stimulate NAB1 activity and the molar amount of NAB1 present in the cell.

These and other embodiments are disclosed or are obvious from and encompassed by the description and the example of the present invention. Further literature concerning any one of the methods, uses and compounds to be employed in accordance with the present invention may be retrieved from public libraries, using for example electronic devices. For example the public database "Medline" may be utilized which is available on Internet, e.g. under http://www.ncbi.nlm.nih.gov/PubMed/medline.html. Further databases and addresses, such as http://www.ncbi.nlm.nih.gov/. http://www.infobioqen.fr/, http://www.fmi.ch/bioloqy/research tools.html. http://www.tiqr.org/. are known to the person skilled in the art and can also be obtained using, e.g., http://www.lycos.com. An overview of patent information in biotechnology and a survey of relevant sources of patent information useful for retrospective searching and for current awareness is given in Berks, TIBTECH 12 (1994), 352-364.

THE FIGURES SHOW:

Figure 1 : NAB1-mRNA expression is enhanced in heart failure

A. NAB1-mRNA expression was determined in left-ventricular myocardium of β adrenergic receptor transgenic mice by semi-quantitative RT-PCR. RNA was isolated from the left ventricles of transgenic and wild-type mice , transcribed into cDNA with oligodt-primers and NAB1-mRNA expression was determined with gene-specific primers for murine NAB1 by RT-PCR. The expression level in the transgenic animals at each age group was related to the respective wild- type expression levels.

B. Determination of NAB1 mRNA expression in human left ventricular myocardium from non-failing donor hearts as compared to failing hearts.

Figure 2: Enhanced expression of NAB1 -protein in heart failure

A. A polyclonal antibody directed against NAB1 was generated through immunizing rabbits with purified GST-tagged murine NAB1 and affinity- purification of the antiserum. Western blotting of lysates from left-ventricular myocardium demonstrate enhanced expression of NAB1 in the hearts of βr adrenergic receptor transgenic mice.

B. Enhanced expression of NAB1 -protein in hearts from mice subjected to aortic banding. Wild-type mice were subjected to transverse aortic constriction and NAB1 -expression was assessed in lysates from left ventricular myocardium.

Figure 3: Generation of NAB1 -transgenic mice

A. Transgenic vector for the generation of NAB1 -transgenic mice. The coding sequence of murine NAB1 was fused to the murine αMHC-promotor and injected into the pronucleus of fertilized oocytes from FVB/N mice.

B. Genotyping of NAB1 -founder mice by PCR. Specific primers located in the 5^' region of the αMHC-promotor (forward primer) and the coding sequence of NAB1 (reverse primer) were used to screen the FO-generation for transgenic founder mice.

C. Determination of NAB1 -overexpression by Western blotting. Figure 4: Normal cardiac morphology of NAB1 -transgenic mice

A. Macroscopic morphology of NAB1 -transgenic mice at 12 months of age. Whole hearts were fixed by immersion into 4% paraformaldehyde, embedded into paraffin and coronal sections were obtained at a thickness of 5 μm. Hearts from NAB1 -transgenic mice did not display any overt cardiac pathology.

B. Normal heart weight-to-body weight ratio in NAB1 -transgenic mice. Hearts were excised, blotted dry onto filter paper and the atria removed before weight determination.

C. Cardiomyocyte size in NAB1 -transgenic and wild-type mice. Sections were photographed at a magnification of 200x with a cooled high-resolution CCD- camera (Visitron, Puchheim, Germany). For the determination of myocyte cross-sectional area, 50 individual cells per genotype from at least three different animals were analyzed morphometrically. Only nucleated cardiomyocytes from areas of transversely cut muscle fibres were included in the analysis. Quantification of left ventricular fibrosis was achieved by sirius red staining followed by semi-automated image analysis as described.

D. Histological analysis of wild-type and NAB1 -transgenic hearts. Midventricular sections were fixed by immersion in 4% paraformaldehyde in PBS for 24 hours and subsequently embedded in paraffin. Sections (4 μm) were stained with hematoxylin/eosin and picric acid/sirius red.

Figure 5: Determination of cardiac transcriptional activity in vivo.

A. Measuring egr-activity through direct gene transfer of Egr1 -reporter constructs into the hearts of anaesthetized mice. 20 μg of plasmid DNA were injected into the apex of wild-type and NAB1 -transgenic mice.

B. NAB1 suppresses Egr1 -activation through hypertrophic stimuli in vivo. Gene transfer of egr-1 reporter constructs was carried out for 52 wild-type and 25 NAB1 -transgenic animals. A portion of the animals was then given a dose of the hypertrophic agents isoproterenol and phenylephrine intraperitoneally 4 hours before determination of Egrl -dependent luciferase activity in lysates from left ventricular myocardium. Figure 6: NAB1 suppresses TNFα expression in vivo.

RNA was isolated from wild-type and NAB1 -transgenic mice, reverse transcribed into cDNA and TNFα-expression determined with gene-specific primers by RT-PCR. TNFα-expression was found to be significantly suppressed in NAB1 -transgenic mice.

Figure 7: NAB1 suppresses cardiac hypertrophy in vivo.

Wild-type and NAB1 -transgenic mice were treated with isoprenaline and phenylephrine (30 mg each/kg/day) for 6 days through subcutaneously implanted osmotic minipumps. The ventricular weights were determined after trimming of the atria and blotting of the ventricles on blotting paper.

Figure 8: Inhibition of pressure overload-induced cardiac hypertrophy through NAB1

Wild-type and NAB1 -transgenic mice were subjected to transverse aortic constriction and heart weight to body weight ratio was analysed 5 weeks after the procedure. Overexpression of NAB1 led to a significant reduction of the pressure-induced hypertrophic response.

Figure 9: Enhanced expression of NAB1 in human heart failure

NAB 1 expression was assessed by Western blotting in left ventricular myocardium from heart failure patients and compared to nonfailing donor hearts. A significant upregulation of NAB1 protein in human heart failure was detected. The invention will now be described by reference to the following examples which are merely illustrated and are not to be considered as a limitation of the scope of the present invention.

EXAMPLES:

Example 1: Determination of NABImRNA in murine and human cardiac failure

To assess whether NAB1-mRNA expression was enhanced in murine heart failure, NAB mRNA-expression was determined in left-ventricular myocardium of βr adrenergic receptor transgenic mice by semi-quantitative RT-PCR. RNA was isolated from the left ventricles of transgenic and wild-type mice, transcribed into cDNA with oligo dT-primers and NAB1-mRNA expression was determined with gene-specific primers for murine NAB1 by RT-PCR. It was found that NAB1 mRNA was significantly upregulated at various stages of heart failure in these animals. Already young mice clearly before the onset of manifest heart failure showed upregulation of NAB1 mRNA which further increased with the age of the animals. Then NAB1 mRNA expression was determined in human left ventricular myocardium from non-failing donor hearts as compared to failing hearts. In failing human myocardium upregulation of NAB1 was observed (Figure 1).

Example 2: Determination of NAB1-protein expression in heart failure

A polyclonal antibody directed against NAB1 was generated through immunizing rabbits with purified GST-tagged murine NAB1 and affinity-purification of the antiserum. Western blotting of lysates from left-ventricular myocardium demonstrate enhanced expression of NAB1 in the hearts of βi -adrenergic receptor transgenic mice. Enhanced expression of NAB1 -protein in hearts from mice subjected to aortic banding. Wild-type mice were subjected to transverse aortic constriction and NAB1- expression was assessed in lysates from left ventricular myocardium (Figure 2).

Example 3: Generation of NAB1 -transgenic mice

Transgenic mice overexpressing murine NAB1 under the control of the αMHC promoter were generated. The coding sequence of murine NAB1 was fused to the murine αMHC-promotor and injected into the pronucleus of fertilized oocytes from FVB/N mice. Genotyping of NAB1 -founder mice was carried out by PCR. Specific primers located in the 5^' region of the αMHC-promotor (forward primer) and the coding sequence of NAB1 (reverse primer) were used to screen the FO-generation for transgenic founder mice. Three transgenic founder mice were identified in the FO- generation and were used to establish independent transgenic lines. All animals were housed under SPF-conditions with free access to food and water (Figure 3).

Example 4: Morphological analysis

After cervical dislocation, hearts were excised, rinsed briefly in phosphate-buffered saline (PBS) and the atria were dissected before determination of ventricular weight. Midventricular sections were fixed by immersion in 4% paraformaldehyde in PBS for 24 hours and subsequently embedded in paraffin. Sections (4 μm) were stained with hematoxylin/eosin and picric acid/sirius red. The sections were photographed at a magnification of 200x with a cooled high-resolution CCD-camera (Visitron, Puchheim, Germany). For the determination of myocyte cross-sectional area, 50 individual cells per genotype from at least three different animals were analyzed morphometrically. Only nucleated cardiomyocytes from areas of transversely cut muscle fibres were included in the analysis. Quantification of left ventricular fibrosis was achieved by sirius red staining followed by semi-automated image analysis (Figure 4).

Example 5: Determination of Egr-1 transcriptional activity in vivo

To assess the ability of overexpressed NAB1 to suppress Egrl -mediated transcriptional activity in the hearts of NAB1 -transgenic mice, direct gene transfer of Egrl -reporter plasmids into the hearts of NAB1 -transgenic and wild-type mice was performed. Mice were anaesthetized with tribromoethanol and their thorax was opened at the 3^rd intercostal space. The heart was exteriorized and 20 μg of plasmid DNA encoding a Egrl -reporter construct were injected directly into the apex of wild- type and NAB1 -transgenic mice. Gene transfer of egr-1 reporter constructs was carried out for 52 wild-type and 25 NAB1 -transgenic animals. A portion of the animals was then given a dose of the hypertrophic agents isoproterenol and phenylephrine intraperitoneally 4 hours before determination of Egrl -dependent luciferase activity in lysates from left ventricular myocardium. Determination of luciferase activity in the hearts from wild-type and NAB-transgenic mice revealed a marked increase of Egr1- dependent luciferase activity in wild-type mice treated with the hypertrophic agents isoproterenol and phenylephrine. In sharp contrast, NAB1 -transgenic mice did not show any increase in luciferase activity indicating complete suppression of Egr-1 transcriptional activity through overexpression of NAB1 in vivo (Figure 5).

Example 6: NAB1 suppresses Egrl -dependent gene expression in vivo

Total RNA was prepared according to the method of Chomczynski well described in the art. After preparation of the RNA, the concentration was determined by UV- absorbance and denaturing agarose gel electrophoresis was performed. The RNA was visualized using ethidium bromide staining followed by digital image aquisition with a CCD-camera. The samples studied were free of degradation as assessed by the comparison of the band intensities of the 28S and the 18S bands. The 18S band intensities of wild-type and transgenic animals were essentially identical, and were used to normalise the specific RNA levels (Figure 6).

For RT-PCR analysis of TNFα-expression, 1 μg of total RNA was reverse transcribed with reverse transcriptase (Superscript, Lifem Sciences). PCR-products were amplified using gene specific primers and electrophoresed on 5% polyacrylamide/8M urea gels. The radioactivity incorporated into the respective bands was analyzed by phosphoimager analysis (Fuji BAS 3000, Osaka, Japan).

Example 7: NAB1 suppresses cardiac hypertrophy in vivo

Wild-type and NAB1 -transgenic mice were treated with isoprenaline and phenylephrine (30 mg each/kg/day) for 6 days through subcutaneously implanted osmotic minipumps. The minipumps were implanted subcutaneously at the back of the animals under anaesthesia with tribromoethanol. Before and after filling with the pharmacological agents, the pumps were weighed to ensure proper loading of the devices. After 6 days, the animals were killed and there hearts removed. The ventricular weights were determined after trimming of the atria and blotting of the ventricles on blotting paper.

The ventricular weight of wild-type animals had significantly increased under the treatment as compared to wild-type animals. However, NAB1 -transgenic mice showed a blunted response to the hypertrophic stimulation. The heart weight to body weight ratio of NAB1 -transgenic mice increased only marginally as compared to untreated mice and was significantly lower compared to treated wild-type mice (Figure 7).

Example 8: Inhibition of pressure overload-induced cardiac hypertrophy through NAB1

Wild-type and NAB1 -transgenic mice were subjected to transverse aortic constriction and heart weight to body weight ratio was analysed 5 weeks after the procedure. Overexpression of NAB1 led to a significant reduction of the pressure-induced hypertrophic response (Figure 8).

Example 9: Enhanced expression of NAB1 in human heart failure

NAB 1 expression was assessed by Western blotting in left ventricular myocardium from heart failure patients and compared to nonfailing donor hearts. A significant upregulation of NAB1 protein in human heart failure was detected (Figure 9).

Claims

1. Use of a nucleic acid molecule selected from the group consisting of:

(a) a nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 1 or 3;

(b) a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4;

(c) a nucleic acid encoding a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO: 2 or 4, wherein the fragment has repression of Egr-1 activity; and

(d) a nucleic acid of at least 70%, 80%, 90% or 95% identity to the nucleic acid of (a), (b) or (c), for the preparation of a pharmaceutical composition for the treatment of a heart disease.

2. The use of claim 1 , wherein the nucleic acid is comprised by a vector.

3. The use of claim 2, wherein the vector is a viral vector.

4. Use of a polypeptide selected from the group consisting of:

(a) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4;

(b) a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:2 or 4, wherein the fragment has repression of Egr-1 activity; and

(c) a polypeptide comprising an amino acid sequence at least 70%, 80%, 90% or 95% identical to the polypeptide of (a) or (b), for the preparation of a pharmaceutical composition for the treatment of a heart disease.

5. Use of a nucleic acid molecule as defined in claim 1 or a polypeptide as defined in claim 4, for the preparation of a diagnostic composition for detecting a heart disease.

6. A method for diagnosing a heart disease or a susceptibility to a heart disease in a subject, comprising:

(a) determining the presence or amount of expression of a nucleic acid molecule as defined in claim 1 in a biological sample of said subject; and

(b) diagnosing a heart disease or a susceptibility to a heart disease, wherein the presence or an increased amount of expression of said nucleic acid molecule is indicative for said heart disease.

7. A method for diagnosing a heart disease or a susceptibility to a heart disease in a subject, comprising:

(a) determining the amount of expression or activity of the polypeptide as defined in claim 4 in a biological sample of said subject; and

(b) diagnosing a heart disease or a susceptibility to a heart disease wherein an increased amount of expression or activity for said polypeptide is indicative for said heart disease.

8. The use of any one of claims 1 to 5 or the method of claim 6 or 7, wherein the heart disease is selected from the group consisting of:

(a) cardiac hypertrophy;

(b) heart failure; and

(c) remodeling post-myocardial infarction.

9. A method for identifying an agonist or antagonist of the polypeptide as defined in claim 4, comprising:

(a) determining the interaction of the polypeptide as defined in claim 4 and Egr-1 in the presence of a compound suspected to be an agonist or antagonist; (b) determining, if said interaction has been altered in the presence of said compound in comparison to interaction observed in the absence of said compound.

10. The method of claim 9, wherein an increase in the interaction is indicative for an agonist of the polypeptide of claim 4 and a decrease in the interaction is indicative for an antagonist of the polypeptide of claim 4.

11. A method for identifying compounds which are capable of activating or inhibiting the repression of Egr-1 activity of the polypeptide as defined in claim 4, comprising:

(a) introducing of an Egr-1 reporter construct into the heart of a non-human animal or heart cells which express the polypeptide as defined in claim 4;

(b) determining Egr-1 dependent reporter gene activity in the presence of a compound suspected to be capable of activating or inhibiting the biological activity of said polypeptide; and

(c) determining if said reporter gene activity has been altered in the presence of said compound in comparison to the reporter gene activity observed in the absence of said compound.

12. The method of claim 11 , wherein an increase in the reporter gene activity is indicative for an antagonist of the polypeptide of claim 4 and a decrease in the reporter gene activity is indicative for an agonist of the polypeptide of claim 4.