METHODS FOR TREATING AND DIAGNOSING HEART FAILURE

FIELD OF THE INVENTION:

The present invention relates to methods for treating and diagnosing heart failure.

BACKGROUND OF THE INVENTION:

Ventricular hypertrophy and HF are features of genetic cardiomyopathy and secondary to a number of pathologies (e.g. ischemia, pressure overload...). HF is the leading cause of hospitalisation in people older than 65 and has major clinical and economical consequences (Jessup M et al, N Engl J Med 2003 May 15;348(20):2007-18). The care costs have been estimated to amount to 2% of the total health care resources (Stewart S, et al Eur J Heart Fail 2002 June;4(3):361-71), a proportion that is expected to rise in the future. Furthermore, HF may be increasing in prevalence, perhaps related to better survival from myocardial infarction (MI) and alterations in demographics due to increased life expectancy (Felker GM, et al. Circ Heart Fail 2010 March;3(2):314-25). Despite improved treatments for HF, their impact on mortality has been relatively modest (Jhund PS, et al. Circulation 2009 February 3;119(4):515-23).

Therefore, a better understanding of the pathophysiology underlying the development and progression of HF, and how this can be influenced by potential new therapies, is highly relevant.

Remodeling of the heart elicits structural changes of the left ventricle (LV) wall which lead to contractile dysfunction and ultimately to HF. These changes can cause dilatation of the LV cavity which in turn increases wall stress and leads to further cavity dilatation (Yousef ZR et al. Heart 2000 January;83(l):76-80). Thus, the extent of LV dilatation, particularly at end- systole, is predictive of mortality and morbidity (Gaudron P, et al. Circulation 1993 March;87(3):755-63). The diagnosis of HF itself is often difficult and when is made, it often too late and it remains difficult to assess stability of the patient. Several diagnostic biomarkers, particularly natriuretic peptides, have been disappointing because of low predictive potential. In this context that interest in biomarkers of HF is increasing. Ahmad T, et al. Fiuzat. Nat Rev Cardiol 2012 June;9(6):347-59; van Kimmenade RR, et al. Clin Chem 2012 January;58(l): 127-38).

The fundamental mechanism of LV remodeling is multifactorial and a number of biological responses are required in the time course of remodeling. It involves alterations in specific signaling molecules and their respective downstream pathways. In more detail, a number of factors (including ischemia, pressure overload...) induce chronic exposure to ROS leading to oxidation and accumulation of DNA, proteins, lipids. Furthermore, ROS can directly contribute to the activation of several kinase pathways by inducing conformation changes (Burgoyne JR, et al. Science 2007 September 7;317(5843): 1393-7). Among the most conserved signal transduction systems in the heart are the MAPK cascades, which consists of sequentially acting protein kinases resulting in the activation of three principal terminal MAP kinases, p38 kinase, extracellular signal-regulated protein kinase (ERK), and c-jun N-terminal protein kinase (JNK) (Rose BA, et al. Physiol Rev 2010 October;90(4): 1507-46). MAPKs are serine/threonine kinases which regulate a number of critical cellular processes in the heart, such as cell growth, division, differentiation and survival through the regulation of cardiac gene expression (Rose BA, et al. Physiol Rev 2010 October;90(4): 1507-46). Due to the relative inability of adult cardiomyocytes to divide, the relevant function of MAPKs relates mainly to the stress response, hypertrophy (survival) and apoptosis, which are all fundamental components of the ventricular remodeling process (Petrich BG, et al. Trends Cardiovasc Med 2004 February; 14(2):50-5).

A number of studies suggest that activation of p38 MAPK aggravates LV matrix remodeling (Ma XL, et al. Circulation 1999 April 6;99(13): 1685-91), worsens cell survival following ischemia-reperfusion (Ren J, et al. J Mol Cell Cardiol 2005 April;38(4):617-23) and thereby exacerbates ventricular remodeling (Clark JE, et al. Pharmacol Ther 2007 November; 116(2): 192-206). Recently, a large number of studies strongly suggest that pharmacological inhibition of p38 MAPK can attenuate pathological cardiac remodeling and LV dysfunction (Kompa AR, et al. J Pharmacol Exp Ther 2008 June;325(3):741-50; See F, et al. J Am Coll Cardiol 2004 October 19;44(8): 1679-89). Nonetheless, a few well-designed studies using p38alpha -deficient (Taniike M, et al. Circulation 2008 January 29;117(4):545- 52) or p38alpha -dominant-negative (Braz JC, et al. J Clin Invest 2003 May;l 11(10): 1475- 86), mice suggest that reduced p38 signaling in the heart promotes myocyte growth and exacerbates dysfunction and hypertrophy. Furthermore, the rescue of p38 kinase activity by adenovirus-mediated gene transfer during postinfarction LV remodeling results in significant functional improvement with enhanced angiogenesis and a reduced rate of apoptosis (Tenhunen O, et al. FASEB J 2006 September;20(l 1): 1907-9). The reasons for these discrepant findings are not clear but it was recently demonstrated using a chemical genetic approach that p38alpha can have two distinct and diametrically opposed roles: aggravating injury during lethal ischemia whilst initiating protection during sublethal preconditioning ischemia (Kumphune S, et al. J Biol Chem 2010 January 29;285(5):2968-75; Sicard P et al. J Mol Cell Cardiol 2010 February 24). There are several reasons why under some circumstances the activation of a single isoform of p38 can lead to reduced, and under others increased, injury. These include the timing of activation, differential localisation and discrete binding partners. Perhaps most importantly these maybe be driven by different modes of p38 activation (Ota A, et al. Circ Res 2010 March 18).

A better understanding of the consequences of the different pathways leading to p38 MAPK activation in the heart is therefore needed.

Instead of the classical upstream kinase MAPKKK-MAPKK-MAPK (MTK1- MKK3/6-p38) canonical activation cascade, p38alpha is also known to auto-activate through at least 2 mechanisms which facilitate auto-phosphorylation. One is dependent on a Syk family kinase, ZAP-70, which can phosphorylate Tyr323 and induce auto-phosphorylation (Jirmanova L, et al. Blood 2011 September 22;118(12):3280-9). The other is mediated by the interaction with the non- enzymatic adaptor protein, transforming growth factor-beta- activated protein kinase- 1 (TAK1) binding protein- 1 (TAB1). These canonical and non- cannonical modes of activation are known to cause p38 to differentially localise within the cell and phosphorylate different substrates resulting in divergent phenotypic effects (Lu G, et al. J Biol Chem 2006 March 3;281(9):6087-95) (Figure 1).

The Growth arrest and DNA-damage-inducible 45 (Gadd45) protein is known to have an important role in promoting the classical canonical activation of p38 (Takekawa M, et al. Cell 1998 November 13;95(4):521-30). All Gadd45 family members bind a noncatalytic autoinhibitory region in the N-terminal portion of the kinase MEKK4 (also known as MTK1), and this binding relieves autoinhibition and activates MTK1 and therefore p38. MTK1 plays an important function in the regulation of cell death and is also involved in the pathogenesis of HF through both the p38 and JNK pathways (Mizote I, et al. J Mol Cell Cardiol 2010 February;48(2):302-9). Furthermore, Gadd45 has recently been shown to have a more novel role in inhibiting directly and indirectly the non-cannonical autophosphorylation activation of p38. In this way, Gadd45 can inhibit Syk family kinases and lead to the inactivation of p38 through the dephosphorylation of the Tyr323 site of p38alpha and p38beta (Salvador JM et al. Nat Immunol 2005 April;6(4):396-402). Thus Gadd45 may act as a nodal switch by enhancing one pathway (canonical) of p38 MAPK activation at the expense of another (non-canonical). Since these pathways have different downstream substrates and drive differential localisation of p38 (Lu G, et al. J Biol Chem 2006 March 3;281(9):6087-95), by amplifying one pathway and inhibiting the other, Gadd45 may be able to steer the physiological consequence of p38 activation without appreciable change in its level of activation. This in turn could explain the divergent findings seen with pharmacological inhibition. Thus, a better understanding of the role of Gadd45 in p38 regulation is crucial (Figure 1).

The Gadd45 family includes three isoforms, Gadd45alpha, Gadd45beta, and Gadd45gamma, encoded by separate genes, which are key players in cellular stress responses. Gadd45s are small (~18 kDa), evolutionarily conserved acidic proteins that are highly homologous to one another (55-57% overall identity at the amino acid level) and are primarily, but not exclusively, localised within the cell nucleus (Zhan Q, et al. Mol Cell Biol 1994 April;14(4):2361-71). Each of the Gadd45 isoforms is expressed in multiple murine tissues including heart, brain, spleen, lung, liver and skeletal muscle but their ratios vary (Zhang W, et al. Oncogene 1999 September 2;18(35):4899-907). Each of the Gadd45 proteins has been shown to elicit pleiotropic effects, inducing cell cycle arrest, DNA repair and ultimately promoting apoptosis in response to environmental and physiological stress including oxidative stress (Vairapandi M, et al. J Cell Physiol 2002 September;192(3):327- 38). The physiological effects of Gadd45 are mediated through interactions with proteins partner, including PCNA, cdkl , p21, and most importantly MEKK4 and p38 MAPK (Bulavin DV, et al. Mol Cell Biol 2003 June;23(l 1):3859-71.40). Thus, although complex, Gadd45 seems to promote cell death which may in part be through increased activation of p38 MAPK via the canonical pathway. Many studies have focused on the role of Gadd45 in cancer cells and support the idea that the repression of expression may be considered a survival mechanism, as cancer cells with low Gadd45 can evade the apoptotic pathway (Hollander MC, et al. Nat Genet 1999 October;23(2): 176-84). However, the exact mechanisms of how Gadd45s interact with protein partners during stress and its regulation are not well known and are subject to dispute.

Despite its relationship with p38 MAPK and intense interest in the cancer field, little is known about the role of Gadd45 in the heart. Tantalizingly a few studies have shown that acute stresses such as angiotensin II or transient ischemia can increase Gadd45beta and/or gamma mRNA levels (Liu L, et al. Anesthesiology 2009 November; 111(5): 1052-64) and could play a key role in ischemia/hypoxia-induced cardiomyocyte death. Sustained repression of Gadd45beta expression may be considered as a survival mechanism (Yoo J et al. J Biol Chem 2003 October 31;278(44):43001-7). Using silencing RNA in order to downregulate Gadd45beta in vivo and in vitro, the ischemia/hypoxia-induced apoptotic cardiomyocyte death was remarkably attenuated (Kim MY, et al. Cardiovasc Res 2010). p38 MAPK is also known to be activated by these stresses and it has been suggested that p38 MAPK is required for Gadd45beta-mediated apoptotic cell death in some cell types (Yoo J, et al. J Biol Chem 2003 October 31;278(44):43001-7). Thus, it is possible the cardiovascular effects of Gadd45 so far described are p38 MAPK- dependent. In addition, patients with chronic idiopathic or hypertrophic cardiomyopathy have a lower Gadd45beta transcript abundance than controls (Wang J, et al. Biochem Biophys Res Commun 2008 August 8;372(4):623-8). These results imply that low expression levels of Gadd45beta are associated with cardiac hypertrophy but it is unknown whether this association is causal, casual or adaptive. Based on this evidence the role of the Gadd45 in the heart is still unclear. SUMMARY OF THE INVENTION:

The present invention relates to methods for treating and diagnosing heart failure. In particular, the present invention is defined by the claims

DETAILED DESCRIPTION OF THE INVENTION:

Heart Failure (HF) is the leading cause of hospitalisation in people older than 65 and has major clinical and economical consequences. The diagnosis of heart failure itself is often difficult and when is made, it often too late and it remains difficult to assess stability of the patient. Furthermore, despite improved treatments for HF, their impact on mortality has been relatively modest. The inventors show that (1) Gadd45 gamma deletion reduced the development of heart failure after MI in mice (2) Gadd45 gamma is regulated by p38 MAPK and spleen tyrosine kinase in cardiomyocytes and (3) Gadd45 gamma expression level is associated with the severity and the consequences of myocardial infarction in patients. Therefore Gadd45 gamma represents a therapeutic target against cardiovascular diseases and an early biomarker of LV remodeling and HF.

The present invention relates to an inhibitor of Gadd45gamma activity or expression for use in a method for treating heart failure in a subject in need thereof.

The terms "subject," and "patient," used interchangeably herein, refer to a mammal, particularly a human who has been previously diagnosed with heart failure or who is at risk for having or developing heart failure. Typically, the subject suffers from a cardiovascular disease leading to heart failure. In some embodiments, the subject has experienced a myocardial infarction, in particular an acute myocardial infarction. The term "heart failure" (HF) as used herein embraces congestive heart failure and/or chronic heart failure. Functional classification of heart failure is generally done by the New York Heart Association Functional Classification (Criteria Committee, New York Heart Association. Diseases of the heart and blood vessels. Nomenclature and criteria for diagnosis, 6th ed. Boston: Little, Brown and co, 1964;114). This classification stages the severity of heart failure into 4 classes (I-IV). The classes (I-IV) are: Class I: no limitation is experienced in any activities; there are no symptoms from ordinary activities; Class II: slight, mild limitation of activity; the patient is comfortable at rest or with mild exertion;Class III: marked limitation of any activity; the patient is comfortable only at rest; Class IV: any physical activity brings on discomfort and symptoms occur at rest.

The term "treatment" means any treatment of a disease in a patient including: (i) preventing the disease, that is causing the clinical symptoms not to develop; (ii) inhibiting the disease, that is, arresting the development of clinical symptoms; and/or (iii) relieving the disease, that is, causing the regression of clinical symptoms. By way of example only, treating may include, preventing or reducing ventricular hypertrophy, preventing or reducing ventricular remodeling, improving ventricular function and/or alleviating symptoms, including, but not limited to exertional dyspnea, fatigue, chest pain, and combinations thereof. As used herein, the term "ameliorate", when used in reference to the severity of a pathologic condition, means that signs or symptoms associated with the condition are lessened, or improvement in the subject's condition. The signs or symptoms to be monitored will be characteristic of a particular pathologic condition and will be well known to skilled clinician, as will the methods for monitoring the signs and conditions. In one embodiment, the method of the invention may reduce death or hospitalization in subjects by treating the heart failure. Moreover, the method of the invention will achieve improved cardiac condition of post- myocardial infarction (MI) subjects. In particular the method of the invention is suitable for reducing the risk or progression of heart failure, increasing the left ventricle ejection fraction (LVEF), inhibiting left ventricle enlargement, reducing left ventricle end systolic volume, reducing left ventricle end diastolic volume, ameliorating left ventricle dysfunction, improving myocardial contractibility.

As used herein, the term "Gadd45gamma" has its general meaning in the art and refers to Growth Arrest and DNA-damage-inducible 45 protein isoform gamma (Zhang, W.; Bae, I.; Krishnaraju, K.; Azam, Naiyer et al. (1999). "CR6: A third member in the MyD118 and Gadd45 gene family which functions in negative growth control". Oncogene 18 (35): 4899- 907. doi: 10.1038/sj.onc.1202885. PMID 10490824. Beadling, C; Johnson, K.W.; and Smith, K.A. (1993). "Isolation of interleukin 2-induced immediate-early genes". Proceedings of the National Academy of Sciences 90 (7): 2719-23. doi: 10.1073/pnas.90.7.2719. PMC 46167. PMID 7681987). An exemplary amino acid sequence is SEQ ID NO: l .

SEQ ID NO: l : mtleevrgqd tvpestarmq gagkalhell lsaqrqgclt agvyesakvl nvdpdnvtfc vlaageedeg dialqihftl iqafccendi divrvgdvqr laaivgagee agapgdlhci lisnpnedaw kdpaleklsl fceesrsvnd wvpsitlpe

An "inhibitor of Gadd45gamma activity" has its general meaning in the art, and refers to a compound (natural or not) which has the capability of reducing or suppressing the activity of Gadd45gamma. For example the compound may block the interaction of Gadd45gamma with the Gadd45gamma binding partners such as MEK 4 (MTKl), or may bind to Gadd45gamma in manner that Gadd45gamma loss its capacity to interact with its binding partners. Typically, said inhibitor is a small organic molecule or a biological molecule (e.g. peptides, lipid, aptamer...). In a particular embodiment, the activity of Gadd45 gamma can be reduced using a

"dominant negative." To this end, constructs which encode, for example, defective Gadd45gamma polypeptide, can be used in gene therapy approaches to diminish the activity of Gadd45gamma on appropriate target cells. For example, nucleotide sequences that direct host cell expression of Gadd45 gamma in which all or a portion of the binding domain is altered or missing can be administered to the subject (either by in vivo or ex vivo gene therapy methods known in the art). Alternatively, targeted homologous recombination can be utilized to introduce such deletions or mutations into the subject's endogenous Gadd45gamma gene. The engineered cells will express non- functional Gadd45gamma polypeptides. In another embodiment the Inhibitor of Gadd45 gamma activity is an aptamer directed against Gadd45 gamma. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods. Then after raising aptamers directed against the Gadd45 gamma as above described, the skilled man in the art can easily select those blocking Gadd45 gamma activity. In still another embodiment, the Inhibitor of Gadd45gamma activity may be a small organic molecule.

An "inhibitor of Gadd45gamma expression" refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of the gene encoding for Gadd45 gamma.

Inhibitors of expression for use in the present invention may be based on anti-sense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense R A molecules and anti-sense DNA molecules, would act to directly block the translation of Gadd45gamma mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of Gadd45 gamma, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding Gadd45gamma can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732).

Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. Gadd45 gamma gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that Gadd45gamma gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known. All or part of the phosphodiester bonds of the siRNAs of the invention are advantageously protected. This protection is generally implemented via the chemical route using methods that are known by art. The phosphodiester bonds can be protected, for example, by a thiol or amine functional group or by a phenyl group. The 5'- and/or 3'- ends of the siRNAs of the invention are also advantageously protected, for example, using the technique described above for protecting the phosphodiester bonds. The siRNAs sequences advantageously comprises at least twelve contiguous dinucleotides or their derivatives.

As used herein, the term "siRNA derivatives" with respect to the present nucleic acid sequences refers to a nucleic acid having a percentage of identity of at least 90% with erythropoietin or fragment thereof, In particular of at least 95%, as an example of at least 98%, and more In particular of at least 98%.

As used herein, "percentage of identity" between two nucleic acid sequences, means the percentage of identical nucleic acid, between the two sequences to be compared, obtained with the best alignment of said sequences, this percentage being purely statistical and the differences between these two sequences being randomly spread over the nucleic acid acids sequences. As used herein, "best alignment" or "optimal alignment", means the alignment for which the determined percentage of identity (see below) is the highest. Sequences comparison between two nucleic acids sequences are usually realized by comparing these sequences that have been previously align according to the best alignment; this comparison is realized on segments of comparison in order to identify and compared the local regions of similarity. The best sequences alignment to perform comparison can be realized, beside by a manual way, by using the global homology algorithm developed by SMITH and WATERMAN (Ad. App. Math., vol.2, p:482, 1981), by using the local homology algorithm developped by NEDDLEMAN and WUNSCH (J. Mol. Biol, vol.48, p:443, 1970), by using the method of similarities developed by PEARSON and LIPMAN (Proc. Natl. Acd. Sci. USA, vol.85, p:2444, 1988), by using computer software using such algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, TFASTA in the Wisconsin Genetics software Package, Genetics Computer Group, 575 Science Dr., Madison, WI USA), by using the MUSCLE multiple alignment algorithms (Edgar, Robert C, Nucleic Acids Research, vol. 32, p: 1792, 2004 ). To get the best local alignment, one can In particular used BLAST software. The identity percentage between two sequences of nucleic acids is determined by comparing these two sequences optimally aligned, the nucleic acids sequences being able to comprise additions or deletions in respect to the reference sequence in order to get the optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical position between these two sequences, and dividing this number by the total number of compared positions, and by multiplying the result obtained by 100 to get the percentage of identity between these two sequences.

Ribozymes can also function as inhibitors of expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of Gadd45gamma mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable.

Both antisense oligonucleotides and ribozymes useful as inhibitors of expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any ^ ^ vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and In particular cells expressing Gadd45 gamma. In particular, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which nonessential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991). Preferred viruses for certain applications are the adenoviruses and adeno-associated

(AAV) viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. Actually 12 different AAV serotypes (AAVl to 12) are known, each with different tissue tropisms (Wu, Z Mol Ther 2006; 14:316-27). Recombinant AAV are derived from the dependent parvovirus AAV2 (Choi, VW J Virol 2005; 79:6801-07). The adeno-associated virus type 1 to 12 can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species (Wu, Z Mol Ther 2006; 14:316- 27). It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and microencapsulation.

In a preferred embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The inhibitor of Gadd45gamma activity or expression may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

In particular, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The inhibitor of Gadd45gamma activity or expression of the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifuGadd45 gamma agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

The inhibitor of Gadd45gamma activity or expression of the invention may be formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses can also be administered.

In addition to the compounds of the invention formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; liposomal formulations; time release capsules; and any other form currently used.

The present invention also relates to a method for screening a plurality of candidate compounds for use as a drugs for the treatment of heart failure comprising the steps consisting of (a) testing each of the candidate compounds for its ability to inhibit Gadd45gamma activity or expression and (b) and positively selecting the candidate compounds capable of inhibiting said Gadd45 gamma activity or expression.

Typically, the candidate compound is selected from the group consisting of small organic molecules, peptides, polypeptides or oligonucleotides. Other potential candidate compounds include antisense molecules, siR As, or ribozymes. Testing whether a candidate compound can inhibit Gadd45 gamma activity or expression can be determined using or routinely modifying assays known in the art. For example, the method may consist of a first step consisting of selecting candidate compounds that are able to bind to Gadd45 gamma. The method may then comprise a step consisting of contacting cells expressing Gadd45 gamma with the candidate compound, and measuring the Gadd45gamma mediated activity (e.g. p38 phosphorylation), and comparing the cellular response to a standard cellular response. Typically, the standard cellular response is measured in absence of the candidate compound. A decrease cellular response (e.g. a decrease in the p38 phosphorylation) over the standard indicates that the candidate compound is an inhibitor of Gadd45gamma activity. Typically the assay may consist in exposing cardiomyocytes (e.g. neonatal cadiomycoytes) to oxidative stress (e.g. H ₂ 0 ₂ ), bringing into contact said exposed cardiomyocytes with the candidate and determining the phosphorylation level of p38 and/or the ratio of BAX/BCL2 (e.g. Figure 2 for example, we use siR A to decrease Gadd45 gamma expression in neonatal expression (Figure 2 A). The use of siRNA to downregulate Gadd45 gamma is associated with a decreased in p38 phosphorylation and less BAX/BCL2 ratio in neonatal cardiomyocytes (Figure 2 B-D)). A decrease in the p38 phosphorylation and/or in the BAX/BCL2 ratio in comparison with conditions wherein the candidate compound is absent indicates that said candidate compound is an inhibitor of Gadd45gamma activity. The candidate compounds that have been positively selected may be subjected to further selection steps in view of further assaying its properties on heart failure. For example, the candidate compounds that have been positively selected may be subjected to further selection steps in view of further assaying its properties on animal models for heart failure.

The present invention also relates to a method for determining whether a subject is at risk of developing heart failure comprising the steps consisting of i) providing a blood sample from the subject, ii) determining the expression level of Gadd45gamma in leukocytes of said blood sample, iii) comparing the expression level determined at step ii) with a predetermined reference value and iv) concluding that the subject is at risk of developing heart failure when the expression level determined at step ii) is higher than the predetermined reference value.

Typically the subject has experienced a myocardial infarction, in particular an acute myocardial infarction. Methods for isolating leukocytes from a blood sample are well known in the art. For example, the leukocytes may be isolated from a PBMC sample. The term "PBMC" or "peripheral blood mononuclear cells" or "unfractionated PBMC", as used herein, refers to whole PBMC, i.e. to a population of white blood cells having a round nucleus, which has not been enriched for a given sub-population. Typically, the PBMC sample may have been subjected to a selection step to contain non-adherent PBMC (which contain T cells, B cells, natural killer (NK) cells, NK T cells and DC precursors). A PBMC sample according to the invention therefore contains leukocytes (B cells, T cells, NK cells, NKT cells). Typically, these cells can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates layers of blood, with the PBMC forming a cell ring under a layer of plasma. Additionally, PBMC can be extracted from whole blood using a hypotonic lysis buffer, which will preferentially lyse red blood cells. Such procedures are known to the expert in the art. Once isolated the leukocytes may be lysed and the expression level of Gadd45 gamma may be determined.

Determination of the expression level of a gene can be performed by a variety of techniques. Generally, the expression level as determined is a relative expression level.

More preferably, the determination comprises contacting the sample with selective reagents such as probes, primers or ligands, and thereby detecting the presence, or measuring the amount, of polypeptide or nucleic acids of interest originally in the sample. Contacting may be performed in any suitable device, such as a plate, microtiter dish, test tube, well, glass, column, and so forth In specific embodiments, the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array. The substrate may be a solid or semi-solid substrate such as any suitable support comprising glass, plastic, nylon, paper, metal, polymers and the like. The substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc. The contacting may be made under any condition suitable for a detectable complex, such as a nucleic acid hybrid or an antibody- antigen complex, to be formed between the reagent and the nucleic acids or polypeptides of the sample.

In a preferred embodiment, the expression level may be determined by determining the quantity of mRNA. Methods for determining the quantity of mR A are well known in the art. For example the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis) and/or amplification (e.g., RT-PCR). Preferably quantitative or semi-quantitative RT-PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous. Other methods of Amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin).

Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single- stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are "specific" to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).

The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.

Other methods for determining the expression level of said gene include the determination of the quantity of proteins encoded by said gene.

Such methods comprise contacting a biological sample with a binding partner capable of selectively interacting with a marker protein present in the sample. The binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.

The presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; Immunoelectrophoresis; immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith. The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex {e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with an antibody against the protein to be tested. A biological sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate (s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.

A further object of the invention relates to a method for preventing the development of heart failure in a subject comprising the steps consisting of i) determining whether the subject is at risk of developing heart failure by performing the above mentioned method, and ii) administering the subject with an inhibitor of Gadd45gamma activity or expression when the subject has been considered at risk of developing heart failure at step i).

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: Schematic representation of the possible interaction between Gadd45 and p38 MAPK during ischemia. Gadd45 may act as a nodal switch by enhancing canonical and inhibiting the non-cannonical p38MAPK activation.

Figure 2: Gadd45 γ influences p38 MAPK activity and induce apoptosis in stressed cardiomyocytes. Panel A: Downregulation of Gadd45 γ after 48h of siRNA exposition (n=3) Panel B: Representative western blot of the effect of Gadd45 γ silencing in p38 phosphorylation after an incremental concentration of H202 exposure for 15 min in culture neonatal cardiomyocytes. (n=3-4) Panel C Time course analysis of H202 (88μΜ) exposure on neonatal cardiomyocytes subject to a knockdown of Gadd45 γ. Panel D: Quantitative analysis of Bax/BCL2 ratio with or without siRNA Gadd45 γ. The use of siRNA to downregulate Gadd45 is associated with a decreased in p38 phosphorylation and less BAX/BCL2 ratio (n=3).

Figure 3: Relative expression of the three Gadd45 isoforms in murin heart. Panel

A: Western blot showing the affinity of each Gadd45 isoform antibodies using an increasing amount of recombinant proteins (RP). Gadd45 γ is more abundant than a and β in normal murine heart Panel B: Time course analysis of Gadd45 α, β and γ expression after 1 day, 1 week of permanent LAD occlusion. Gadd45 γ is downregulate after 1 day of permanent LAD ligation. However, after 1 week Gadd45 γ expression is increase and is higher than the baseline level (n=3 in each group). Panel C: Quantitative PCR analysis of Gadd45 isoform shown an increase in mR A levels of Gadd45 β and γ observed after 1 week of permanent LAD ligation. (n=6). Panel D- E: Echocardiography data of WT and Gadd45 γ KO animals after 1 month of permanent LAD ligation . Panel D: Left ventricle dimension during diastole and systole in WT and Gadd45 γ KO animals sham or after28 days post-MI (n = 8). Panel E Cardiac function (ejection fraction and fractional shortening) in WT and Gadd45 γ KO animals sham or after 28 days post-MI (n = 8).

Figure 4: Red picrosirius staining of WT and KO heart slice animals subject to 4 weeks of permanent LAD ligation. The fibotic area (purple) is smaller in the Gadd45 gamma KO compared to the WT (p<0.05)

Figure 5: Western blot analysis of the relation between p38 MAPK, SYK activation and Gadd45 γ expression. Panel A: p38 MAPK activity modulates Gadd45 γ expression in HEK293 cells. When p38 and MKK3 are co-expressed, p38 phosporylation is enhanced and it is associated with a decrease in Gadd45 a expression (n=5). Representative immunoblots are shown with quantitative analyses of repeat experiments (right panels) expressed as a ratio of total p38 protein. Panel B: SB203580 (2mg/Kg) infusion can inhibits p38 and at least partially restores Gadd45 protein levels after 1 day of LAD ligation (n=2). Panel C: SYK inhibition by 0.1 μΜ BAY 61-3606 decreases the expression of Gadd45 γ during oxidative stress.

Figure 6: Gadd45 isoforms transcripts levels in patients admitted for AMI (Panel A) according to troponin levels stratification (Panel A), Killip classification (Panel B). Relationship between Gadd45 gamma isoform transcripts expression and NT-proBNP (Panel C) and creatinine (Panel D).

EXAMPLE: Material & Methods

Animals: Gadd45 gamma Knock out mouse model and littermate WT (The Jackson Laboratory, USA) were used in this study. This work was conducted at the Institute of Metabolic and Cardiovascular Diseases (I2MC Toulouse France). All animal experiments were approved by the Animal Care and Use Committees of the University of Toulouse. We examined our hypotheses using our existing in vitro models of isolated neonatal, adult ventricular myocytes (Jacquet S, et al. Cardiovasc Res 2007 December l;76(3):465-72) and in vivo models of MI and remodeling (permanent ligation of the left coronary artery) (Clark JE, et al. Cardiovasc Res 2007 June l;74(3):466-70). Cell culture

In order to define the cellular mechanism of Gadd45 gamma in cardiomyocytes we used either neonatal cardiomyocytes or mouse adult ventricular myocytes (AVMs) primary culture. For neonatal cardiomyocytes, hearts of 2-3-day-old Sprague-Dawley rats were dissociated with collagenase type II, 0.1% (Biovalley). Myocyte enrichment was performed by centrifugation through a discontinuous Percoll gradient and resultant suspension of myocytes was plated onto gelatin-coated culture dishes.

AVMs were isolated, washed and resuspended in complete Ml 99 medium with penicillin (100 IU/ml), streptomycin (100 IU/ml), L-carnitine (2 mM), creatine (5 mM), and taurine (5 mM) as described previously (Jacquet S, et al. Cardiovasc Res 2007 December 1;76(3):465- 72) plated on laminin-coated 6-well plates prior to incubation in 5% C02/room air at 37 °C. After 1 h, the medium were aspirated, leaving only adherent cells and fresh prewarmed complete Ml 99 medium added with specified viral vectors.

We subjected these cells to oxidative stress (H ₂ 0 ₂ ). In order to study the effect of the absence of Gadd45 gamma we used siR A (Dharmacon) strategy to downregulate Gadd45 gamma in neonatal cardiomyocytes. Pharmacologic inhibitor in adult cardiomyocytes were used In order to inhibit p38 MAPK (SB203580, 10μΜ Tocris) and the spleen tyrosine kinase (SYK) (BAY 61 3606, 0.1 μΜ, Sigma).

Characterization of Gadd45 gamma regulation by p38 MAPK Using Transfected HEK293 Cells. HEK293 cells were transfected at 70% confluency in Opti-MEM I using Lipofectin (both from Invitrogen) with peptide 6 and the relevant plasmid DNAs. Wild-type p38alpha MAPK and MKK3 -expressing plasmids were obtained from Jiahuai Han (The Scripps Research Institute, La Jolla, CA).

In vivo heart failure model The basal cardiac phenotypes of Gadd45 gamma KO and littermate mice were characterised using echocardiography. To resolve the influence of Gadd45 on the late phase of post-MI remodeling we used our established murine model of permanent coronary ligation.

In brief, mice were anaesthetised by isofluorane inhalation, and ventilated by orotracheal intubation. A left thoracotomy was performed to expose the heart and, using a 8-0 nylon suture, the left anterior descending (LAD) coronary artery was tied by the edge of the left atrium. The thoracotomy site was closed whilst increasing positive end expiratory pressure and the mice recovered. Only LAD ligation was omitted in the sham procedure. Echocardiography

Animals were anesthetized with 1% isoflurane and examined with noninvasive echocardiography (echocardiograph Vivid 7 ultrasound; GE). Cardiac ventricular dimensions were measured on M-mode images at least five times for the number of animals indicated. Fibrosis

After 4 weeks, hearts of surviving mice were rapidly excise and prepared for histology. Hearts were fixed (10% paraformaldehyde) and sectioned (10 μιη) on paraffin. Fibrotic area was determined by red picrosirius staining and quantified using sigmascan software.

Western blot analysis.

Cardiomyocytes proteins were extracted; separated on 10 or 12% SDS-polyacrylamide gels; transferred to polyvinylidene difluoride membranes, which were blocked for 1 h with 4% nonfat milk in Tris-buffered saline (pH 7.4) containing 0.1% Triton X-100; and probed overnight at 4 °C with the appropriate primary antibody as follows: total p38 (catalog no. 9212; T-p38), diphospho-p38 (catalog no. 9211; polyclonal; p-p38), T-SYK (2712), p-SYK (2710) from Cell Signaling, Gadd45 gamma (catalog no. SAB4500177) from Sigma; Gadd45 alpha (Sc-792), Gadd45 beta (Sc- 133606), BAX, BCL-2 from Santacruz, p-BLNK (ab73204) from abeam. Recombinant Gadd45 alpha,beta and gamma were from santacruz. After washing and exposure for 2 h at room temperature to horseradish peroxidase-conjugated secondary antibody, antibody-antigen complexes were visualized by enhanced chemiluminescence as described previously.

Patients One hundred and twenty one consecutive patients aged >18 years and hospitalized <24 h after symptoms onset for acute myocardial infarction (MI) in the coronary care unit of Dijon University Hospital between 1st March and 31st July 2009 were included. Patients with malignancy were excluded from the study. MI was defined by an increase in serum troponin I (2 X upper limit of the hospital normal range) associated with symptoms of ischemia and/or characteristic electrocardiographic (ECG) signs. The present study complied with the Declaration of Helsinki and was approved by the ethics committee of University Hospital of Dijon. Each patient gave written consent before participation. Data collection

For each patient, data on demographics, risk factors (history of hypertension, diabetes, hyperlipidemia, obesity {defined as a body mass index (BMI) > 30}), a family history of CAD [i.e. parental history of premature (<55 years for men and <65 years for women) CAD in first degree relative] and prior MI were prospectively collected. Clinical data, including hemodynamic parameters on admission, left ventricular ejection fraction (LVEF) and location of infarction were recorded. LVEF was determined by echocardiography at 2 ± 1 days after admission.

Biological data

Blood samples were drawn at admission [time from symptom onset to blood sampling:

240(160-579) min] to assess biological variables.

C-reactive protein (CRP), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) concentrations were measured on a Dimension analyzer (Dade Behring, Newark, NE) using immunonephelometry assay. Low-density lipoprotein cholesterol (LDL-C) levels were calculated using the Friedewald formula. Plasma glucose concentrations [enzymatic method (glucose oxidase)] and creatinine levels were measured on a Vitros 950 analyzer (Ortho Clinical Diagnostics, Rochester, NY). Glycated hemoglobin Al C (HbAlc) was measured with ion exchange HPLC (Bio-Rad Laboratories, Richmond, CA). Creatine kinase (CK) peaks were assessed by sampling every 8 h during the first two days after admission (Dimension Vista Intelligent Lab System, Siemens). Plasma N-terminal pro B-type natriuretic peptide (NT-proBNP) was determined by ELISA with an Elecsys NT- proBNP sandwich immunoassay on Elecsys 2010 (Roche Diagnostics, Basel, Switzerland). Peak creatine kinase (CK) was assessed by sampling every 8 h during the first two days after admission (Dimension Vista Intelligent Lab System, Siemens). mRNA extraction and Quantitative RT-PCR

Blood samples were salted-out in a lysing solution (Tris HCL pH 7.5; 50 mM; MgC12

25 mM; NaCl 50 mM) to obtain a total leukocyte pellet. mRNA were obtained using a TRIZOL ^® (Invitrogen, Paisley, UK) extraction assay protocol on the leukocyte pellet, and reverse transcribed using Murine Moloney Leukemia Virus Reverse Transcriptase (Invitrogen, Paisley, UK).

The levels of the transcripts were quantified by the relative gene expression analysis method using Q-PCR technique, in duplicate at least two or three times. Briefly, for each amplification, aliquots of cDNA from 50 ng of total RNA were amplified by the Q-PCR technique on the Rotorgene 3000 (Corbett Research, Cambridge, UK), using the Quantifast SYBR Green PCR kit (Qiagen S.A., France) in a 25 μ L reaction volume. PCR amplification was run on a Light Cycler 480 (Roche Diagnostics, France). The GAPDH gene was selected as the reference (normalizing) gene after testing the homogenous expression of three genes (GAPDH, B2M and ACTB) using geNorm software. Each gene sequence was amplified in triplicate for 45 cycles under standardized conditions and the results were expressed as the mean of the three Cycle Threshold (Ct) values. The reaction mixture contained 200 ng of each primer and the PCR cycle parameters were: 94° for 15 s, 60° for 1 min, 72° for 40 s. The sequences of the primers selected by the Primer3 software (Whitehead Institute for Biomedical Research) were tested for their specificity by obtaining the melting curves at the end of each PCR.

The Gadd45 gamma specific primers were:

Forward: 5 ^* -CAGATCCATTTTACGCTGATCCA-3 (SEQ ID NO:2)

reverse: 5 '- ACT ATGTCGATGTCGTTCTC-3 (SEQ ID NO:3).

The PCR efficiency was established at 0.95 by the standard curve

RT-PCR was normalized by measuring the average Ct ratio in the investigated genes and in the control gene, glyceraldehyde-3 -phosphate dehydrogenase (GAPDH): forward 5 '-CAT CTC TGC TGC CCT CCC T-3 ^* (SEQ ID NO:4), reverse 5 ^* -ACG CCT GCT TCA CCACCT T-3 ^* (SEQ ID NO:5).

Statistical analysis Continuous variables are presented as medians (25-75th percentile) or means ± SD, as appropriate or as proportions. For continuous variables, a Kolmogorov-Smirnov analysis was performed to test normality. One Way ANOVA was performed to compare the groups (age, sex, killip, troponin levels). For the comparison between 2 groups (univariate predictor of gadd45 isoforms levels) Pearson's or Spearman's rank correlation was used for continuous variables.

Results Gadd45 gamma isoform expression is more abundant in the normal and stressed myocardium than alpha and beta:

In the first instance we determined the relative expression of the three Gadd45 isoforms in normal mouse myocardium (figure 3). The three antibodies for Gadd45 alpha, beta and gamma we tested have similar sensitivity and were able to detect up to 3 ng of recombinant proteins (RP) (Figure 3 A). The Gadd45 gamma isoform seem more abundant than alpha and beta in normal murine heart (Figure 3 A). The expression of Gadd45 alpha, beta, and gamma is increased by a number of stresses. In most instances the pattern of expression of each isoform is unique, consistent with each Gadd45 family member having a different role (Vairapandi M, et al. J Cell Physiol 2002 September;192(3):327-38).

In order to determine the relative expression of the three Gadd45 isoforms in a murine cardiomyopathy model we first used a model of acute MI (Left Anterior Descending Artery (LAD) ligation) in mice known to quickly develop dilated cardiomyopathy (Figure 3B and C). Our preliminary data suggest that Gadd45 gamma protein is degraded after 1 day of ischemia in both infarct and non infarct area (Figure 3B). However, the Gadd45 gamma mRNA and protein level increase above baseline after 1 week of MI in both infarct and non infarct area (Figure 3C). The overexpression is still marked after 1 month of MI (data not shown). Gadd45 alpha undergoes no fluctuations of mRNA and protein levels during ischemia (Figure 3 B and C). While Gadd45 beta mRNA and proteins levels are overexpressed after 1 week of ischemia.

Gadd45 gamma deletion reduces cardiac remodeling after chronic myocardial infarction: As the role of Gadd45 gamma is totally unknown in the development of HF, the main goal of these experiments is to determine how Gadd45 gamma influences cardiac remodeling after myocardial infarction. We studied the remodeling process using our in vivo model of MI in littermate or Gadd45gamma KO mice. Cardiac function was assessed by echocardiography 28 days after MI. Our results (n = 8 in each group) showed that the Gadd45gamma KO heart are significantly less dilated and had preserved cardiac function after MI compared to WT littermate group (Figure 3 panels D-E). The fibrotic area was determined in the WT and KO animals. The KO animals have less fibrotic area compared to the WT 4 weeks after MI (Fig 4 p<0.05) . These results demonstrate the importance of Gadd45 gamma in the development of post-infarction HF.

To summarise, Gadd45 gamma is the most abundant isoform in the heart and whose expression changes after different stresses (MI). Based on these results, we decided to focus our research on the study on Gadd45 gamma isoform. Gadd45 gamma KO animals present a better cardiac function and less fibrosis after MI compared to WT littermate group (Figure 3 panel D; Figure 4). These results demonstrate the importance of Gadd45 gamma in the development of post-infarction HF in mice. The next step is to understand the role of Gadd45 during stress in cells and how can we modulate that effect.

Gadd45 gamma is regulated in concert by p38 MAPK and spleen tyrosine kinase (SYK) in cells:

The effects of Gadd45 are related to its cellular levels and/or to its conformation promoting protein/protein interactions. A number of factors can modulate the activity of Gadd45 by adapting its expression level using transcriptional, post-transcriptional (Lai A, et al. Cell Cycle 2006 July;5(13): 1422-5). No specific inhibitors or regulators of Gadd45 are available.

The aim of this study is to determine the factors involved in regulating the expression of cardiac Gadd45 gamma and therefore give us the possibility to modulate cell fate in HF.

Gadd45 gamma has a particular expression profile after oxidative stress exposition in cardiomyocytes (Figure 2B-C) or in the heart following MI (Figure 3B). The Gadd45 gamma regulation seems to be fast, efficient and probably means different consequences for the cell. Lai et al. {Cell Cycle 2006 July;5(13): 1422-5) have reviewed the mechanisms that regulate Gadd45 abundance and a number of factors can modulate Gadd45 expression in both transcriptional and post translational levels. p38 MAPK can also regulate Gadd45 expression (Yin F, et al. Oncogene 2004 June 3;23(26):4614-23). Because this work was only performed at the Gadd45 alpha mR A level, we investigated Gadd45 gamma protein levels in HEK293 cells co-transfected with p38alpha and MKK3 (Figure 5A). Such co-transfection increases the activating dual phosphorylation (Thr 180/Tyr 182) of p38 MAPK and we observed a consequent decrease in Gadd45 gamma expression. This data suggests that Gadd45 gamma may be involved in a feedback loop controlling p38 MAPK activity and, together with the data summarised below, as a determinant of cell fate. We have preliminary data confirming an inverse relationship between murine cardiac p38 MAPK activity after MI and Gadd45 expression (Figure 5B). We performed permanent LAD ligation in outbred C57BL6 mice and examined p38 MAPK phosphorylation and Gadd45 gamma expression (n=2) in the presence and absence of SB203580. After 1 hour and 1 day of permanent occlusion p38 MAPK was highly phosphorylate. This situation was associated with a decrease in Gadd45 gamma protein levels compared to Sham. Moreover, SB203580 inhibits p38 MAPK and at least partially restores Gadd45 protein levels (Figure 5B) reinforcing the inverse relationship between p38 MAPK activity and Gadd45 gamma within the heart. This data, together with that summarised in the background, suggest that p38 MAPK may be a negative regulator of Gadd45 in the heart.

In contrast the use of SYK inhibitor, BAY 61-3606 (IC 50 = 7 nm) is able of reduce the phosphorylation of the downstream substrat BLNK and it is associated with a decreased in the expression of Gadd45 gamma in cells. It seems that this kinase represents an attractive target in the hope of limiting damage during stress (Figure 5 C)

To summarise, Gadd45 gamma is downregulate by p38MAPK and upregulate by SYK during oxdative stress.

Gadd45 gamma expression level in leukocyte is a potential biomarker of the development of HF after MI?:

Gadd45 is a key regulator of signals involved in the effects of cytokines (IL-12 and IL-18) and can promote cell infiltration (Shin GT, et al. Kidney int., 2008 73(11): 1251-65). It is highly probable that the expression of Gadd45 in leukocytes can promote cell infiltration in the heart after myocardial infarction and may be involved in acute cardiac remodeling. Therefore, the analysis Gadd45 expression can be considered as a biomarker of an early cardiac remodeling and thus the development of HF.

One hundred and twenty one patients were included in this study. Baseline characteristics of the study population are shown in Table 1. Risk factors n=121

Age (y) 64 ± 15

Female 37(30)

Hypertension 68(56)

IMC 27 ± 5

Diabetes 28(23)

Hypercholesterolemia 51 (42)

Smoking 40 (33)

Chronic treatments

ACE inhibitor 24(19)

Aspirin 34(28)

Beta-blocker 35(29)

Statin 42(34)

Oral antidiabetic 17(14)

Clinical data and delay

Anterior wall Ml 43(35)

LVEF (%) 55 ± 1 1

STEMI 58 (48)

Time to admission (min) 220 ± 120

Biological data on admission

Glucose (mmol/L) 7.8 ± 3.3

HbA1c (%) 5.7 ± 0.6

Creatinine clearance 84.7 ± 48.7

(ml/min)

Creatinine 1 17.0 ± 187.3

CRP (mg/L) 17.6 ± 34.9

HDL-cholesterol (mg/dL) 0.4 ± 0.1

LDL-cholesterol (mg/dL) 1.1 ± 0.3

Total-cholesterol (mg/dL) 1.8 ±0.4

Triglycerides (mg/dL) 1.4 ± 0.8

Leukocytes (x 10 ³ /mm ³ ) 10.4(8.6-12.9)

Table 1: Patients characteristics (n(%) means ± S.D)

The median age of the 121 patients was 64=1=15 years. Patients were predominantly male (84 (70%)). The results of RT-PCR were normalized by measuring the average ratio (triplicate) Ct between the target gene and the GAPDH gene control and in the words of deltaCT. We have determined that the beta isoform is the most expressed in leukocytes of patients admitted for MI followed by alpha and gamma (Data not shown, p <0.001 vs alpha or gamma). A statistical analysis allowed us to assess that neither age nor gender appears to influence the expression of Gadd45 isoforms (Data not shown). We next checked if the Gadd45 iso forms expression were correlated with the severity of the myocardial infarction and sign and biomarker of HF (Fig 6 A-D). Therefore, several parameters including Killip classification, ST elevation, risk factors, and biological data were analyzed. The Gadd45 alpha or beta expression during MI are not statistically associated or correlate with any of the parameters tested (data not shown). However, Gadd45 gamma isoform expression demonstrates an interesting profile during MI. First, Gadd45 gamma is statistically associated with high troponin levels and with sign of HF at admission (Killip) at admission (Fig 6A-B, p<0.05). Interestingly, Gadd45 gamma is positively correlate with high levels NT-proBNP (Fig 6 C, R:0.417 p<0.001) and creatinine (Fig 6D; R: 0.266 p<0.003) , two biomarker of cardiac and renal dysfunction. In conclusion to this part, it seems that Gadd45 gamma could be associated with the severity and the consequences of myocardial infarction in patients.

Conclusion:

These results demonstrate the importance of Gadd45 gamma/p38 MAPK complex in the development and persistence of HF, particularly in the balance of the cell death process. Gadd45 gamma KO mice have shown a better heart function after permanent coronary artery ligation compare to WT animals. Using cellular approach we characterize (1) the signalling pathway involving in the process of cell death dependant of Gadd45 gamma and p38 MAPK phosphorylation (2) the kinases (p38 MAPK and SYK) responsible of Gadd45 gamma expression level regulation.

Taking together these results highlight that Gadd45 gamma is clearly a therapeutic target against the development of HF. The possibility to use selective kinase inhibitor/activator or other chemical agent to reduce Gadd45 gamma apoptotic activity to limit the development of cardiomyopathy is particularly interesting. The development of more specific and selective therapeutic target against Gadd45 gamma to modulate cellular fate is therefore highly relevant. In addition, this study describe for the first time the importance of Gadd45 gamma as a biomarker of LV remodeling and HF after MI. Improving risk stratification of patients after MI is very challenging and predicting LV remodeling in clinical practice remains difficult. It is important to note that the "ideal biomarker" (Morrow DA, de Lemos JA circulation 2007) should be easily detected from a blood sample, strongly associated with the pathophysiologic process and allowing improved clinical decision making for management of patients. Therefore, monitoring the evolution of Gadd45 during MI could be interesting in terms of early biomarker of ventricular dysfunction and HF. More works is needed to do a better characterization of Gadd45 iso forms expression (ARNm, proteins) levels during MI, HF and cardiovascular diseases in general.

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.