Patent Application
20180258407
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 6, 2015, is named 116023_2_Sequence_Listing.txt and is 78,641 bytes in size.
The present invention relates to Abhydrolase containing domain 5 (ABHDS) and N-terminal fragments of HDAC4 (HDAC4-NT) and variants of the aforementioned peptides for the treatment and prevention of heart failure. The present invention further provides vectors for the cardiomyocyte-specific expression of said peptides and a test system comprising ABHDS for the identification of novel compounds which are useful for the treatment of heart failure.
Sustained catecholaminergic stress is known to promote heart failure (Cohn et al., 1984 “Plasma Norepinephrine as a Guide to Prognosis in Patients with Chronic Congestive Heart Failure” New England Journal of Medicine, 311: 819-823). In contrast to this, short-term catecholaminergic stimulation (e.g. by physical exercise) promotes cardiac health (Keteyian et al, 2010 “Clinical Role of Exercise Training in the Management of Patients with Chronic Heart Failure” Journal of Cardiopulmonary Rehabilitation and Prevention, 30:67-76). Thus, there appear to be different signaling pathways downstream of the β-adrenergic receptor mediating cardioprotective and pathophysiological effects of β-adrenergic receptor stimulation. One consequence of sustained catecholaminergic stress is myocardial remodeling which causes or exacerbates heart failure. Thus, therapeutic means for influencing said signaling pathways are desirable.
Full-length histone deacetylase 4 (HDAC4) is proteolytically processed by a previously unknown enzyme yielding an N-terminal fragment (HDAC4-NT) comprising 201 amino acids. In vitro, HDAC4-NT selectively represses myocyte enhancer factor 2 (MEF2) (Backs et al., 2011, “Selective repression of MEF2 Activity by PKA-dependent Proteolysis of HDAC4”, Journal of Cell Biology, 195: 403-415).
Abhydrolase containing domain 5 (ABHDS, also known as “comparative gene identification-58” (CGI-58)) is a protein which has been previously known to be involved in lipid metabolism (Grannemann et al., 2009 “Perilipin controls Lipolysis by regulating the Interactions of AB-hydrolase Containing 5 (Abhd5) and Adipose Triglyceride Lipase (Atgl)”, The Journal of Biological Chemistry 284: 34538-34544). Mutations of ABHDS cause the Chanarin-Dorfman syndrome, a rare genetic disease characterized by excessive accumulation of triacylglycerol in multiple tissues (Lass et al., 2006 “Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome” Cell Metabolism 3: 309-319). It has not been implicated in the regulation of cardiac remodeling.
Thus, the problem underlying the present invention can be viewed as the provision of novel means and methods for the treatment and prevention of heart failure.
In a first aspect, the present invention relates to abhydrolase containing domain 5 (ABHDS) or a variant thereof for use as a medicament.
In another aspect, the present invention relates to ABHDS or a variant thereof for use in the treatment or prevention of heart failure.
In yet another aspect, the present invention relates to a nucleic acid encoding ABHDS or a variant thereof for use as a medicament. In yet another aspect, the present invention relates to a nucleic acid encoding ABHDS or a variant thereof for use in the treatment or prevention of heart failure.
In yet another aspect, the present invention relates to a vector comprising a nucleic acid encoding ABHDS or a variant thereof.
In yet another aspect, the present invention relates to a vector comprising a nucleic acid encoding ABHDS or a variant thereof for use as a medicament.
In yet another aspect, the present invention relates to a vector comprising a nucleic acid encoding ABHDS or a variant thereof for use in the treatment or prevention of heart failure.
In yet another embodiment, the present invention relates to an elongated and/or multimerized variant of the N-terminal fragment of histone deacetylase 4 (HDAC4-NT).
In yet another embodiment, the present invention relates to HDAC4-NT or a variant thereof for use as a medicament.
In yet another embodiment, the present invention relates to HDAC4-NT or a variant thereof for use in the treatment or prevention of heart failure.
In yet another aspect, the present invention relates to a nucleic acid encoding an elongated and/or multimerized variant of HDAC-NT.
In yet another aspect, the present invention relates to a nucleic acid encoding HDAC4-NT or a variant thereof for use as a medicament.
In yet another aspect, the present invention relates to a nucleic acid encoding HDAC4-NT or a variant thereof for use in the treatment or prevention of heart failure.
In yet another aspect, the present invention relates to a vector comprising nucleic acid encoding HDAC4-NT or a variant thereof.
In yet another aspect, the present invention relates to a nucleic acid encoding HDAC4-NT or a variant thereof for use as a medicament.
In yet another aspect, the present invention relates to a vector comprising a nucleic acid encoding HDAC4-NT or a variant thereof for use in the treatment or prevention of heart failure.
In yet another aspect, the present invention relates to a method for identifying a compound which modulates the activity and/or localization of ABHDS or a variant thereof comprising the steps of
In yet another aspect, the present invention relates to the use of ABHDS for identifying a compound which suppresses myocardial remodeling.
Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.
In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.
Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, H. G. W. Leuenberger, B. Nagel, and H. Kölbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).
To practice the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, and recombinant DNA techniques are employed which are explained in the literature in the field (cf., e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). Furthermore, conventional methods of clinical cardiology are employed which are also explained in the literature in the field (cf., e.g., Practical Methods in Cardiovascular Research, S. Dhein et al. eds., Springer Verlag Berlin Heidelberg, 2005).
Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents, unless the content clearly dictates otherwise.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
It has been surprisingly found in the study underlying the present invention that inhibition of expression of ABHD5 prevents the proteolytic cleavage of full-length HDAC4, thus generating HDAC4-NT. Based on sequence analysis, it is likely that ABHD5 is a serine protease which cleaves HDAC4. Based on this finding, the ABHD5 can be used for generating HDAC4-NT in vivo, thus protecting the myocardium from heart failure.
Therefore, the present invention relates to abhydrolase containing domain 5 (ABHD5) or a variant thereof for use as a medicament.
In another embodiment, the present invention relates to ABHD5 or a variant thereof for use in the treatment or prevention of heart failure.
The term “ABHD5” relates to a polypeptide having an amino acid sequence as defined by one of the sequences SEQ ID NO: 1 to 5 or a variant thereof. Preferably, ABDHS has an amino acid as defined by SEQ ID NO: 1 (human ABDHS) or a variant thereof.
“Variants” are preferably N-terminally and/or C-terminally truncated variants, amino acid substitution or deletion variants, or prolonged variants of the sequences of SEQ ID NO: 1 to 5. Variants comprise furthermore an amino acid sequence comprising modified amino acid(s), unnatural amino acid(s) or peptidomimetic(s) or further compounds which can mimic a peptide backbone/structure. Preferably, variants are selected from C-terminally truncated variants of SEQ ID NO.: 1 to 5; amino acid substitution or deletion variants; variants comprising modified amino acid(s), unnatural amino acid(s) or peptidomimetic(s) or further compounds which can mimic a peptide backbone/structure.
Deletion variants are, preferably, characterized by C-terminal deletion of up to 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30 amino acids. Independently of the presence or absence of C-terminal deletions further preferred deletion variants are characterized by N-terminal deletion of up to 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30 amino acids.
The term “ABDHS variants” preferably includes proteins which have at least 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity, preferably sequence identity over the entire length of the protein according to SEQ ID NO: 1 to 5 or proteins based on SEQ ID NO: 1 to 5 carrying above outlined N- and/or C-terminal deletions using the best sequence alignment and/or over the region of the best sequence alignment, wherein the best sequence alignment is obtainable with art known tools, e.g. Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5, with the amino acid sequence set forth in SEQ ID NOs: 1, 2, 3, 4, or 5. It is preferred that when a given ABDHS variant is aligned with a ABDHS according to SEQ ID NO:1, 2, 3, 4 or 5 that alignment will be over the entire length of the two proteins and, thus, that the alignment score will be determined on this basis.
In especially preferred deletion variants, the first 29 or, more preferably, the first 31 amino at the N-terminus are deleted. Human ABHDS comprises tryptophan residues at positions 19, 23 and 27. Rat and mouse ABHDS comprise tryptophan residues at positions 21, 25 and 29. Said tryptophan residues are responsible for the binding of ABHDS to lipid droplets. Deletion of these tryptophan residues creates a variant of ABHDS which has less affinity to lipid droplets. Thus, such deletion variants move more easily to the nucleus and the cytosol where they mediate cleavage of full-length HDAC4 yielding HDAC4-NT.
Deletions in further preferred deletion variants are designed to remove only the first or only the first and second tryptophan residues. Thus, these deletion variants have a lower affinity to lipid droplets as compared to full-length ABHDS but higher affinity as compared to deletion variants missing the first 27 or 30 amino acids.
Preferred substitution variants are generated by substituting up to 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30 amino acids against natural amino acids, unnatural amino acids or peptidomimetics. Preferably, the amino acids of the wild type protein or a deletion variant thereof are substituted for natural amino acids. .more preferably, said substitutions are conservative substitutions.
A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases, where two or more amino acid sequences differ from each other by conservative substitutions, the number of substitutions may be adjusted upwards to correct for the conservative nature of the substitution. Thus, in the case of conservative amino acid substitutions up to 40, up to 50 or even up to 70 amino acids may be substituted. Examples of groups of amino acids that have side chains with similar chemical properties include:
Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine . Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet G.H. et al, 1992 “Exhaustive matching of the entire protein sequence database.” Science 1992, 256:1443-1445. A “moderately conservative” replacement is any change having a non-negative value in the PAM250 log-likelihood matrix.
In especially preferred substitution variants preferably 1, more preferably 2 and most preferably 3 of the tryptophan residues mentioned above (positions 19, 23 and 27 in human ABHDS; positions 22, 26 and 30 in mouse or rat ABHDS) are substituted by a different amino acid. Preferably, said substitution is not a conservative substitution and more preferably, the amino acid which takes the place of tryptophan is alanine.
In one preferred embodiment, positions 19 and 23 of human ABHADS or positions 22 and 26 of rat ABHDS are substituted, preferably with alanine.
In another preferred embodiment, positions 23 and 27 of human ABHADS or positions 26 and 30 of rat ABHDS are substituted, preferably with alanine.
A “variant” as defined above is, preferably, a functional variant. A functional variant is a variant of the wild type ADHDS as described above which retains its capability to mediate cleavage of full-length HDAC4 in vitro and, preferably, in vivo. Preferably, the ADHDS variant has at least 50% of the capability of ADHDS according to SEQ ID NO: 1 to 5, preferably of SEQ ID NO: 1 to mediate cleavage of full-length HDAC4 in vitro and, preferably, in vivo, more preferably at least 60%, 70%, 80%, 90%, 95% or 100% of this activity.
An assay for testing whether a variant of ABDHS is still functional can be based on a cell line which does not express ABDHS due to a knock out. As described in the examples, inhibition of ABHDS expression inhibits cleavage of HDAC4. If the cells are then transfected with an expression vector encoding the variant of ABHDS to be tested, a functional variant restores the cell's capability of generating HDAC4-NT from full-length HDAC4, while a non-functional variant does not. Alternatively, the variant of ABDHS may be administered to the cells as a peptide.
An even more preferred assay can be conducted in vitro: Since ABDHS is a putative protease, functional variants of ABDHS are able to cleave full-length HDAC4 if the required ions and cofactors are present.
The term “heart failure” as used in the present application refers to any conditions characterized by the inability of the heart to pump a sufficient amount of blood to meet the body's oxygen demand.
Heart failure may affect the right ventricle, the left ventricle or both ventricles. The present invention relates to the treatment or prevention of all of the aforementioned types of heart failure and all degrees of heart failure as set forth below.
The typical symptom experienced by a patient suffering from left-ventricular heart failure is shortness of breath (dyspnea). In mild forms of heart failure, the patient is not limited in ordinary physical activity but experiences dyspnea during periods of increased physical exercise. In more severe cases of heart failure, dyspnea is experienced during ordinary or even light physical activity. In the most severe cases, the patient even experiences dyspnea at rest. The typical symptom of right ventricular heart failure is the congestion of systemic capillaries leading to the accumulation of fluids in various parts of the body. Fluid accumulates in the feet and legs (in people standing up) or in the sacral area (in people lying down). In severe cases fluid accumulates in the abdominal cavity and/or the liver.
Heart failure may be divided into inherited and acquired forms of the disease. Inherited forms of the disease include hypertrophic cardiomyopathy, dilated cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, isolated ventricular non-compaction and mitochondrial myopathy. Acquired heart failure is typically caused by coronary artery disease, arterial hypertension, loss of viable myocardium due to myocardial infarction, cigarette smoking, obesity, diabetes, metabolic syndrome, kidney disease, environmental stress, depressive or mood disorders, lipid storage disease, cancer, chronic inflammation, postpartal cardiomyopathy, stress induced cardiomyopathy, transient cardiac dysfunction or valvular heart disease. It is particularly preferred that acquired cardiomyopathies are treated, in particular postpartal cardiomyopathy, stress induced cardiomyopathy, transient cardiac dysfunction.
The means and methods of the present invention are suited for the treatment of all the aforementioned types of heart failure.
Heart failure is caused or accompanied by myocardial remodeling. In myocardial remodeling, terminally differentiated cardiomyocytes increase in size in order to increase the contractility of the myocardium. However, this leads to increasing stiffness of the heart and concomitantly to difficulties in filling the ventricles during diastole. Moreover, the ventricles enlarge contributing to a more spherical form of the heart. This spherical shape of the heart decreases the stroke volume. Since myocardial remodeling tends to decrease myocardial output rather than increasing it, the process of myocardial remodeling causes and exacerbates heart failure.
As a consequence of myocardial remodeling, the risk of cardiac dysrhythmia increases. The term “cardiac dysrhythmia” refers to all types abnormal electric activity of the myocardium. Preferably, cardiac dysrhythmia is tachycardia (pathologically increased heart beat) or bradycardia (pathologically decreased heart beat). Cardiac dysrhythmia may originate from the atria or the ventricles. Particularly relevant types of dysrhythmia are atrial fibrillation and ventricular tachycardia.
Therefore, the means and methods of the present invention which suppress or reverse the process of myocardial remodeling during heart failure are well suited for the treatment or prevention of myocardial remodeling, thus increasing cardiac output and decreasing the risk of complications of cardiac remodeling, particularly cardiac dysrhythmia as defined above.
The success of the treatment according to the present invention may be verified by echocardiography or magnetic resonance imaging. Moreover, decreasing levels of natriuretic peptides, particularly nT-proBNP after onset of treatment indicate that the treatment is successful.
The patient receiving the pharmaceutical compounds and pharmaceutical compositions disclosed in the present application is preferably a primate, rodent, pig, sheep, cow or goat. The rodent is, preferably, a rat and more preferably a mouse. The primate is, preferably, a human, chimpanzee or macaque. Most preferably, the patient is a human.
Preferably, the patient suffers from heart failure.
As used herein, “treat”, “treating” or “treatment” of a heart failure means refers to at least one of the following: (a) reducing the severity of heart failure, (b) limiting or preventing the symptoms typical for heart failure, (c) reversing myocardial remodeling, (d) preventing further progress of myocardial remodeling, and (e) decreasing the likelihood of the negative consequences of myocardial remodeling described above. Preferably, a patient to be treated already suffers from heart failure and/or myocardial remodeling.
As used herein, “prevent”, “preventing” or “prevention” refer to the inhibition of the onset of heart failure and/or myocardial remodeling in a patient not yet suffering from the aforementioned disorders. Thus, a patient receiving preventive treatment is healthy with respect to the disorder to be prevented by said treatment. In a preferred embodiment of the present invention, the patient suffers from heart failure but does not yet show signs and symptoms of myocardial remodeling.
It is understood by the person skilled in the art that “treatment” or prevention” may not be successful in every patient receiving the pharmaceutical compounds or composition of the present invention. However, the terms “treatment” and “prevention” require that a significant proportion of patient benefits from said treatment.
In patients suffering from primary heart failure as defined above, treatment according to the present invention preferably cures heart failure because it removes or alleviates the underlying cause.
In patients suffering from secondary heart failure as defined above, the treatment according to the present invention preferably decreases the speed of progression of heart failure or even stops progression of heart failure completely.
In yet another aspect, the present invention relates to a nucleic acid encoding ABHDS or a variant thereof for use as a medicament.
In yet another aspect, the present invention relates to a nucleic acid encoding ABHDS or a variant thereof for use in the treatment or prevention of heart failure.
The term “nucleic acid” refers to a polymeric macromolecule made from nucleotide monomers. Nucleotide monomers are composed of a nucleobase, a five-carbon sugar (such as but not limited to ribose or 2′-deoxyribose), and one to three phosphate groups. Typically, a polynucleotide is formed through phosphodiester bonds between the individual nucleotide monomers. In the context of the present invention preferred nucleic acid molecules include but are not limited to ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Moreover, the term “polynucleotide” also includes artificial analogs of DNA or RNA, such as peptide nucleic acid (PNA).
The nucleic acid of the present invention encodes ABHDS or a variant thereof. Since the genetic code is degenerated, i.e. each amino acid is encoded by more than one nucleic acid, each peptide or its variant may be encoded by a multiplicity of different nucleic acid sequences.
Preferably, the nucleic acid encoding ABHDS is selected from the group consisting of SEQ ID NOs: 6, 7, 8, 9 and 10. More preferably, the nucleic acid encoding ABHDS has a sequence as defined by SEQ ID NO: 6.
For an effective treatment of heart failure, the increase of the amount of functional ABHDS is advantageous. The administration of ABHDS may pose practical difficulties. Therefore, the use of expression systems which mediate expression of ABHDS by the cardiomyocytes of the patient are a solution for the problem underlying the present invention.
Thus, in yet another embodiment, the present invention relates to a vector comprising a nucleic acid encoding ABHDS or a variant thereof.
In yet another embodiment, the present invention relates to a vector comprising a nucleic acid encoding ABHDS or a variant thereof for use as a medicament.
In yet another embodiment, the present invention relates to a vector comprising a nucleic acid encoding ABHDS or a variant thereof for use in the treatment or prevention of heart failure.
As used herein, the term “vector” refers to at least one nucleic acid or to a mixture of at least one nucleic acid and at least one protein which is capable of introducing the nucleic acid comprised therein into a cell. At least one nucleic acid comprised by the vector consists of or comprises at least one nucleic acid encoding ABHDS or a variant thereof. In addition to the nucleic acid consisting of or comprising the nucleic acid encoding ABHDS or the variant thereof, additional nucleic acids and/or polypeptides may be introduced into the cell. The addition of additional nucleic acids and/or polypeptides is especially preferred if said additional nucleic acids and/or polypeptides are required to introduce the nucleic acid encoding ABHDS or the variant thereof into the cell.
In the context of the present invention it is preferred that ABHDS or the variant thereof is expressed within the cell upon introduction of the vector or vectors. Examples of suitable vectors include but are not limited to plasmids, cosmids, phages, viruses or artificial chromosomes.
In preferred embodiments, the vector is selected from the group consisting of plasmids, cosmids, phages, viruses, and artificial chromosomes. More preferably, a vector suitable for practicing the present invention is a phage vector, preferably lambda phage and filamentous phage vectors, or a viral vector.
Preferred viral vectors are based on naturally occurring vectors, which are modified to be replication incompetent also referred to as non-replicating. Non-replicating viruses require the provision of proteins in trans for replication. Typically those proteins are stably or transiently expressed in a viral producer cell line, thereby allowing replication of the virus. The viral vectors are, thus, preferably infectious and non-replicating. The skilled person is aware of how to render various viruses replication incompetent.
In a preferred embodiment of the present invention the vector is selected from the group consisting of adenovirus vectors, adeno-associated virus (AAV) vectors (e.g., AAV type 6, type 1, type 5, type 9 and type 2), alphavirus vectors (e.g., Venezuelan equine encephalitis virus (VEE), sindbis virus (SIN), semliki forest virus (SFV), and VEE-SIN chimeras), herpes virus vectors (e.g. vectors derived from cytomegaloviruses, like rhesus cytomegalovirus (RhCMV) (14)), arena virus vectors (e.g. lymphocytic choriomeningitis virus (LCMV) vectors (15)), measles virus vectors, pox virus vectors (e.g., vaccinia virus, modified vaccinia virus Ankara (MVA), NYVAC (derived from the Copenhagen strain of vaccinia), and avipox vectors: canarypox (ALVAC) and fowlpox (FPV) vectors), vesicular stomatitis virus vectors, retrovirus, lentivirus, viral like particles, and bacterial spores.
The most preferred vector is an adenovirus, more preferably adeno associated virus (AAV) type 1, type 6 or type 9. For use in humans, AAV types 1 or 6 are preferred, for use in mice AAV type 9.
An especially preferred vector given in SEQ ID NO: 18. Also preferred is a vector as defined by SEQ ID NO: 19. Said sequences comprise HDAC4-NT or luciferase instead of a ABHDS. However, the exchange of these inserts for ABHDS is easily accomplished.
To direct expression of ABHDS or the variant thereof, the nucleic acid encoding it is operationally linked to an internal promoter and/or enhancer that is recognized by the transcriptional machinery of the cell. Suitable promoters may be derived from the genome of mammalian cells (e. g., MHCII promoter, EFlalpha promoter) or from mammalian viruses (e.g., the cytomegalovirus promoter, the spleen focus-forming virus SFFV promoter). Especially preferred are promoters which enable the expression of the above-mentioned gene in cardiomyocytes.
One preferred promoter is defined by SEQ ID NO: 22. In an especially preferred embodiment, the promoter is a human troponin promoter, more preferably the human troponin T promoter, as this promoter is especially well suited for cardiomyocyte-specific expression of transgenes. Preferably, the human troponin T promoter has a nucleic acid sequence as defined by SEQ ID NO: 21.
In another preferred embodiment, the vector encodes a recognition site for micro-RNA 122 between the coding sequence of the HDAC4, ABHDS or their variants and the terminator sequence so that said recognition site becomes part of the transcript which also encodes HDAC4, ABHDS or a variant thereof produced from this vector. Since micro-RNA 122 is found in many types of cells but not in cardiomyocytes, said recognition site causes breakdown of the mRNA in those types of cells where micro-RNA 122 is present, thus preventing translation of the transcript. Preferably the recognition site for micro-RNA 122 has a nucleic acid sequence as defined by SEQ ID NO: 20.
Since the two elements described above are advantageous for the cardiomyocyte-specific expression of many other peptides besides HDAC4-NT and ABHDS, the present invention relates in a further embodiment to the use of a recognition site for micro-RNA 122 for the cardiomyocyte-specific expression of transgenes. Preferably, said recognition site has a nucleic acid sequence as defined by SEQ ID NO: 20.
In yet another embodiment, the present invention relates to the use of a combination of the human troponin promoter and the recognition site for micro-RNA 122 for the cardiomyocyte-specific expression of transgenes. Preferably, said recognition site has a nucleic acid sequence as defined by SEQ ID NO: 20.
In yet another embodiment, the present invention relates to a vector encoding a recognition site for micro-RNA 122 between the promoter and the terminator sequence so that said recognition site becomes part of the transcript which also encode HDAC4-NT or a variant thereof produced from this vector. Preferably, said recognition site has a nucleic acid sequence as defined by SEQ ID NO: 20. Preferably, the vector additionally comprises a human troponin promoter. More preferably, the human troponin promoter controls transcription of the nucleic sequence which comprises the aforementioned recognition site for micro-RNA 122.
In a particularly preferred embodiment of the present invention, the vector comprises a human troponin T promoter as defined above and a nucleic acid sequence encoding a recognition site for micro-RNA 122 as defined above.
As used herein, “operatively linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest.
Specific initiation signals may also be required for efficient translation of coding sequences. These signals include the ATG initiation codon and adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may additionally need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be in-frame (or in-phase) with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
HDAC4-NT not just represses the transcription factor MEF2 in vitro, thus inhibiting cardiomyocyte hypertrophy (Backs et al., 2011, cited supra). The study underlying the present invention surprisingly showed that overexpression of HDAC4 by an adenoviral expression system in vivo (i) prevents myocardial remodeling, and (ii) does not cause intolerable side effects. Given the fact, that similar approaches with other peptides proved non feasible in vivo due to side effects (Czubryt et al. 2003, Proc Natl Acad Sci USA. 100:1711-6), this finding was unexpected and highlights the advantages of HDAC4-NT as a therapeutic means.
Therefore, in yet another embodiment, the present invention relates to an elongated and or multimerized variant of the N-terminal fragment of histone deacetylase 4 (HDAC4-NT).
In yet another embodiment, the present invention relates to HDAC4-NT or a variant thereof for use as a medicament.
In yet another embodiment, the present invention relates to HDAC4-NT or a variant thereof for use in the treatment or prevention of heart failure.
All definitions given above for the terms “heart failure”, “patient”, “treatment” and “prevention” with respect to ABHDS also apply to embodiments of the invention relating to HDAC4-NT and the variants thereof.
The term “HDAC4-NT” relates to a polypeptide having an amino acid sequence as defined by one of the sequences SEQ ID NO: 13 (human HDAC4-NT) or 14 (mouse HDAC4-NT) or a variant thereof. Preferably, HDAC4-NT has an amino acid sequence as defined by SEQ ID NO: 13 or a variant thereof.
“Variants” of HDAC4-NT are preferably N-terminally and/or C-terminally truncated variants, amino acid substitution or deletion variants, multimerized or elongated variants of the sequences defined by SEQ ID NO: 13 or 14. Variants comprise furthermore an amino acid sequence comprising modified amino acid(s), unnatural amino acid(s) or peptidomimetic(s) or further compounds which can mimic a peptide backbone/structure. Preferably, variants are selected from C-terminally truncated variants of SEQ ID NO: 13 or 14; amino acid substitution or deletion variants; variants comprising modified amino acid(s), unnatural amino acid(s) or peptidomimetic(s) or further compounds which can mimic a peptide backbone/structure.
Deletion variants are, preferably, characterized by C-terminal deletion of up to 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30 amino acids. Independently of the presence or absence of C-terminal deletions further preferred deletion variants are characterized by N-terminal deletion of up to 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30 amino acids. A preferred deletion variant comprises amino acid positions 2 to 201 of SEQ ID NO: 13 or 14, more preferably of SEQ ID NO: 13. This variant is also referred to as “HDAC4 2-201”.
The term “HDAC4-NT variants” preferably includes proteins which have at least 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity, preferably sequence identity over the entire length of the protein according to SEQ ID NO: 13 or 14 or proteins based on SEQ ID NO: 13 or 14 carrying above outlined N- and/or C-terminal deletions using the best sequence alignment and/or over the region of the best sequence alignment, wherein the best sequence alignment is obtainable with art known tools, e.g. Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5, with the amino acid sequence set forth in SEQ ID NOs: 13 or 14. It is preferred that when a given HDAC4-NT variant is aligned with a HDAC4-NT according to SEQ ID NO: 13 or 14 that alignment will be over the entire length of the two proteins and, thus, that the alignment score will be determined on this basis.
Preferably, a deletion variant retains amino acid positions 169 to 185 of SEQ ID NO: 13 as these positions are important for binding of HDAC-NT to MEF2.
Preferred substitution variants are generated by substituting up to 1, 2, 3, 4, 5, 10, 15, 20, 25 or 30 amino acids against natural amino acids, unnatural amino acids or peptidomimetics. Preferably, the amino acids of the wild type protein or a deletion variant thereof are substituted for natural amino acids, more preferably, said substitutions are conservative substitutions.
A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases, where two or more amino acid sequences differ from each other by conservative substitutions, the number of substitutions may be adjusted upwards to correct for the conservative nature of the substitution. Thus, in the case of conservative amino acid substitutions up to 40, up to 50 or even up to 70 amino acids may be substituted. Examples of groups of amino acids that have side chains with similar chemical properties include
Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet G.H. et al, 1992 “Exhaustive matching of the entire protein sequence database.” Science 1992, 256:1443-1445. A “moderately conservative” replacement is any change having a non-negative value in the PAM250 log-likelihood matrix.
Further preferred variants of HDAC4-NT are elongated variants which comprise additional amino acids at the C-terminus. More preferred are variants which comprise amino acid positions 2 to 202, 1 to 220, 1 to 216, 1 to 212 or 1 to 208 of full-length HDAC4 as defined by SEQ ID NO: 11 (human) or 12 (mouse). Even more preferably, a variant of HDAC4-NT comprises amino acid positions 2 to 202, 1 to 208 or 1 to 220 of SEQ ID NO: 11 or 12. Most preferably, an elongated variant of HDAC4-NT comprises amino acid positions 2 to 202, 1 to 220 or 1 to 208 of SEQ ID NO: 11. It is to be understood that said elongated variants may be modified by amino acid substitutions as set forth above. The variant comprising amino acid positions 2 to 202 is also referred to as HDAC4 2-202.
In a further preferred embodiment, the variant of HDAC4-NT is a variant generated by multimerization of HDAC4-NT. A multimerized variant of HDAC4-NT is a polypeptide which comprises the amino acid sequence of HDAC4-NT or any of the variants described above not just once but in at least 2, at least 3, at least 4 or at least 5 repeats. Thus, a single polypeptide comprises more than one binding site for MEF2. It is to be understood that a multimerized variant of HDAC4-NT or any of the above-described deletion, elongation or substitution variants thereof may be generated by repeating the same amino acid sequence (homogeneous multimer) or by combining more than one of the aforementioned variants (heterogeneous multimer), thus generating a polypeptide whose repeated sequence motifs are similar but not identical.
In an especially preferred embodiment of the present invention, amino acid positions 1 to 201, 2 to 201, 2 to 202, 1 to 208 or 1 to 220 are multimerized. In one preferred embodiment, the multimerization is homogeneous, i.e. only a sequence comprising amino acid positions 2 to 201, 2 to 202, 1 to 201, 1 to 208 or 1 to 220 is repeated. In another preferred embodiment, the multimerization is heterogeneous, i.e. the multimer comprises a combination of at least two different sequences.
Any “variant” defined above is, preferably, a functional variant. A functional variant is a variant of the wild type HDAC4-NT as described above which retains its capability to repress myocyte enhancer factor 2 (MEF2). Preferably, the HDAC4-NT variant has at least 50% of the
MEF2 repressing ability of HDAC4-NT comprising amino acid positions 1 to 220 of SEQ ID NO: 11 or 12. More preferably at least 60%, 70%, 80%, 90%, 95% or 100%.
An assay for testing whether a variant of HDAC4-NT is still functional disclosed by Backs et al., 2011 cited supra. Briefly, a reporter gene such as luciferase is coupled to a MEF2-regulated promoter. If this construct is expressed in a cell, the signal generated by the reporter gene is decreased in the presence of repressors of MEF2. Thus, variants of HDAC4-NT can be expressed in the cell and their ability to repress MEF2 can be determined by measuring the signal of the reporter gene relative to the signal in a control experiment with the presence of HDAC4-NT or a variant thereof.
It is to be understood that the most preferred variant of HDAC4 is not always the variant which has the highest activity as determined by the repression of MEF2 in the assay described above. As excessive repression of MEF2 may have deleterious effects, HDAC4 variants with decreased activity may be preferred as these have a wider therapeutic index, thus decreasing the risk of undesired side effects.
For this reason, HDAC4 2-201 and 2-202 are equally preferred. HDAC4 2-202 is especially preferred in those cases, where side effects have to be avoided, while HDAC4 2-201,
In yet another aspect, the present invention relates to a nucleic acid encoding HDAC4-NT or a variant thereof for use as a medicament.
In yet another aspect, the present invention relates to a nucleic acid encoding HDAC4-NT or a variant thereof for use in the treatment or prevention of heart failure.
The term “nucleic acid” refers to a polymeric macromolecule made from nucleotide monomers. Nucleotide monomers are composed of a nucleobase, a five-carbon sugar (such as but not limited to ribose or 2′-deoxyribose), and one to three phosphate groups. Typically, a polynucleotide is formed through phosphodiester bonds between the individual nucleotide monomers. In the context of the present invention preferred nucleic acid molecules include but are not limited to ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Moreover, the term “polynucleotide” also includes artificial analogs of DNA or RNA, such as peptide nucleic acid (PNA).
The nucleic acid of the present invention encodes HDAC4-NT or a variant thereof. Since the genetic code is degenerated, i.e. each amino acid is encoded by more than one nucleic acid, each peptide or its variant may be encoded by a multiplicity of different nucleic acid sequences. Preferably, a nucleic acid encoding HDAC4-NT is defined by SEQ ID NO: 17.
For an effective treatment of heart failure, the increase of the amount of HDAC4-NT or a variant thereof is advantageous. The administration of HDAC4-NT as peptide may pose practical difficulties. Therefore, the use of expression systems which mediate expression of HDAC4-NT or a variant thereof by the cardiomyocytes of the patient are a solution for the problem underlying the present invention.
Thus, in yet another embodiment, the present invention relates to a vector comprising a nucleic acid encoding HDAC4-NT or a variant thereof.
In yet another embodiment, the present invention relates to a vector comprising a nucleic acid encoding HDAC4-NT or a variant thereof for use as a medicament.
In yet another embodiment, the present invention relates to a vector comprising a nucleic acid encoding HDAC4-NT or a variant thereof for use in the treatment or prevention of heart failure.
As used herein, the term “vector” refers to at least one nucleic acid or to a mixture of at least one nucleic acid and at least one protein which is capable of introducing the nucleic acid comprised therein into a cell. At least one nucleic acid comprised by the vector consists of or comprises at least one nucleic acid encoding HDAC4-NT or a variant thereof. In addition to the nucleic acid consisting of or comprising the nucleic acid encoding HDAC4-NT or the variant thereof, additional nucleic acids and/or polypeptides may be introduced into the cell. The addition of additional nucleic acids and/or polypeptides is especially preferred if said additional nucleic acids and/or polypeptides are required to introduce the nucleic acid encoding HDAC4-NT or the variant thereof into the cell.
In the context of the present invention it is preferred that HDAC4-NT or the variant thereof is expressed within the cell upon introduction of the vector or vectors. Examples of suitable vectors include but are not limited to plasmids, cosmids, phages, viruses or artificial chromosomes.
In preferred embodiments, the vector is selected from the group consisting of plasmids, cosmids, phages, viruses, and artificial chromosomes. More preferably, a vector suitable for practicing the present invention is a phage vector, preferably lambda phage and filamentous phage vectors, or a viral vector.
Preferred viral vectors are based on naturally occurring vectors, which are modified to be replication incompetent also referred to as non-replicating. Non-replicating viruses require the provision of proteins in trans for replication. Typically those proteins are stably or transiently expressed in a viral producer cell line, thereby allowing replication of the virus. The viral vectors are, thus, preferably infectious and non-replicating. The skilled person is aware of how to render various viruses replication incompetent.
In a preferred embodiment of the present invention the vector is selected from the group consisting of adenovirus vectors, adeno-associated virus (AAV) vectors (e.g., AAV type 1, type 2, type 5, type 6 and type 9), alphavirus vectors (e.g., Venezuelan equine encephalitis virus (VEE), sindbis virus (SIN), semliki forest virus (SFV), and VEE-SIN chimeras), herpes virus vectors (e.g. vectors derived from cytomegaloviruses, like rhesus cytomegalovirus (RhCMV) (14)), arena virus vectors (e.g. lymphocytic choriomeningitis virus (LCMV) vectors (15)), measles virus vectors, pox virus vectors (e.g., vaccinia virus, modified vaccinia virus Ankara (MVA), NYVAC (derived from the Copenhagen strain of vaccinia), and avipox vectors: canarypox (ALVAC) and fowlpox (FPV) vectors), vesicular stomatitis virus vectors, retrovirus, lentivirus, viral like particles, and bacterial spores.
An especially preferred vector with HDAC4-NT as insert is given in SEQ ID NO: 18. Also preferred is a vector as defined by SEQ ID NO: 19. Said sequence comprises luciferase instead of a peptide of the present invention. However, the exchange of the luciferase for HDAC4-NT is easily accomplished.
The most preferred vector is an adenovirus, more preferably adeno associated virus (AAV) type 1, type 6 or type 9. For use in humans, AAV types 1 or 6 are preferred, for use in mice AAV type 9.
To direct expression of HDAC4-NT or the variant thereof, the nucleic acid encoding it is operationally linked to an internal promoter and/or enhancer that is recognized by the transcriptional machinery of the cell. Suitable promoters may be derived from the genome of mammalian cells (e.g., MHCII promoter, EFlalpha promoter) or from mammalian viruses (e.g., the cytomegalovirus promoter, the spleen focus-forming virus SFFV promoter). Especially preferred are promoters which enable the expression of the above-mentioned gene in cardiomyocytes.
One preferred promoter is defined by SEQ ID NO: 22. In an especially preferred embodiment, the promoter is a human troponin promoter as this promoter is especially well suited for cardiomyocyte-specific expression of transgenes. Preferably, the human troponin promoter has a nucleic acid sequence as defined by SEQ ID NO: 21.
In another preferred embodiment, the vector encodes a recognition site for micro-RNA 122 between the promoter and the terminator sequence so that said recognition site becomes part of the transcript produced from this vector. Since micro-RNA 122 is found in many types of cells but not in cardiomyocytes, said recognition site causes breakdown of the mRNA in those types of cells where micro-RNA 122 is present, thus preventing translation of the transcript. Preferably the recognition site for micro-RNA 122 has a nucleic acid sequence as defined by SEQ ID NO: 20.
In a particularly preferred embodiment of the present invention, the vector comprises a human troponin T promoter as defined above and a nucleic acid sequence encoding a recognition site for micro-RNA 122 as defined above.
As used herein, “operatively linked” means incorporated into a genetic construct so that expression control sequences effectively control expression of a coding sequence of interest.
Specific initiation signals may also be required for efficient translation of coding sequences. These signals include the ATG initiation codon and adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may additionally need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be in-frame (or in-phase) with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
In a preferred embodiment of the present invention, the peptides, nucleic acids or vectors of the present invention are part of a pharmaceutical composition. These embodiments relate to all parts of the invention, i.e. to ABHDS and the variants thereof as well as HDAC4-NT and the variants thereof.
The term “composition” refers to the combination of a vector, nucleic acid or peptide of the present invention and at least one further compound selected from the group consisting of pharmaceutically acceptable carriers and pharmaceutical excipients.
“Pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
The term “carrier”, as used herein, refers to a pharmacologically inactive substance such as but not limited to a diluent, excipient, or vehicle with which the therapeutically active ingredient is administered. Such pharmaceutical carriers can be liquid or solid. Liquid carrier include but are not limited to sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously or intranasally by a nebulizer.
Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
In yet another embodiment, the present invention relates to a method for identifying a compound which modulates the activity and/or localization of ABHDS or a variant thereof comprising the steps of
It is preferred to use the variant of ABHDS whose activity is to be determined in the test system. However, if it is expected that two variants of ABHDS will show the same response to the candidate compound, it is also possible to use only one of these variants and to extrapolate to the effect of said compound on the other candidate compound. Similarly, if wild-type ABHDS and a variant thereof can be expected to show the same response, it may be possible—and thus also preferred—to use a test system comprising wild type ABHDS to test the modulation of the variant and vice versa.
A test system is a composition of reagents comprising ABHDS or a variant thereof, wherein said ABHDS generates a signal which allows the differentiation between a test compound which modulates the activity of ABHDS and a test compound which does not.
If localization of ABHDS is used as an indicator of its activity, the test system is, preferably, a cell expressing ABHDS or a variant thereof, more preferably a cardiomyocyte.
If the enzymatic activity of ABHDS is to be analyzed, the test system is, preferably a cell as described above. However, due to their simplicity, cell free systems are more preferred.
Preferably, the modulation of the activity of ABHDS is determined by comparing a test system to which the test compound is added and an otherwise identical test system without the test compound.
Preferably, the activity determined with the test system is the ability of ABHDS to mediate cleavage of HDAC4 or a variant of HDAC4 which does not bind to MEF2 into a fragment which binds to MEF2. Preferably, said fragment is HDAC4-NT as described further above in the present application.
Preferably, the modulation of the activity of ABHDS is an increase of its activity. Said increase may be caused by increased affinity of ABHDS for HDAC4 or by an increased turnover number. The cleavage of HDAC4 may be determined as explained in the examples section.
Moreover, the activity of ABHDS or a variant thereof may be influenced by its binding affinity to lipid droplets as set forth above. Thus, the localization of ABHDS may be used as an indicator of its activity. The less ABHD is bound to lipid droplets, the higher its activity.
We further claim potential tests were the link to HDAC4 or the proteolytic activity of abhd5 are directly or indirectly tested. This could be achieved by the subcellular localization (bound to LD, vs homogenous expression in the cytosol/nucleus) of ABHD4 may be analyzed by expressing GFP- or RFP or other fluophor-tagged ABHDS in cell-based assays (high-content screening).
Protein-protein interaction assays (e.g. alpha screen, FRET assays, mammalian two hybrid screen) to test the binding between ABHDS and perilipin or between ABHDS and HDAC4.
The test compound may be any peptide or small molecule. A peptide as referred to in this embodiment comprises at least 2, more preferably at least 3 even more preferably at least 4 and most preferably at least 5 amino acids linked by peptide bonds. Preferably, said amino acids are amino acids found in nature, more preferably proteinogenic amino acids. However, it is also preferred to use peptides comprising at least one amino acid having a residue not found in nature.
A small molecule is, preferably, an organic molecule having a molecular weight of not more than 2000 Da, more preferably not more than 1600 Da, even more preferably not more than 1200 Da and, most preferably, not more than 800 Da.
Since the proteolytic cleavage of HDAC4 mediated by ABHDS generates an N-terminal fragment (HDAC4-NT) which is useful for the treatment or prevention of heart failure, the identification of modulators which increase the activity of ABHDS, preferably in cell based systems and more preferably in vivo, is of great clinical importance. Hence, the test system of the invention is a valuable tool for the discovery of novel pharmaceutical compounds useful for the treatment or prevention of heart failure.
In yet another embodiment, the present invention relates to the use of ABHD5 for identifying a compound which suppresses myocardial remodeling.
The following examples are merely intended to illustrate the invention. They shall not limit the scope of the claims in any way.
Materials and Methods
Transthoracic echocardiography. Echocardiography was performed using a Sonos 5500 with a S12 transducer (12 MHz). The echocardiographer was blinded with respect to the treatment group. Mice were shaved and left ventricular parasternal short-axis views were obtained in M-mode imaging at the papillary muscle level. Three consecutive beats were used for measurements of left ventricular end-diastolic internal diameter (LVEDD) and left ventricular end-systolic internal diameter (LVESD). Fractional shortening (FS) was calculated as FS %=[(LVEDD−LVESD)/LVEDD]×100%.
Generation of an adeno associated virus (HDAC4-AAV). HDAC4 aa2-201 was cloned into a double-stranded AAV-vector downstream of a CMV-enhanced short (260 bp) myosin light chain promoter (CMVenh/MLC260). AAV9 vectors were produced with the three plasmid transfection method.
RNA analysis. Total RNA was isolated from ventricular tissue using TRIzol (Invitrogen, Germany). Total RNA was digested with DNase, and cDNA synthesis from 500 ng of RNA was carried out using a SuperScript first-strand synthesis system for RT-PCR (Invitrogen). Quantitative real-time PCR (qPCR) was performed with Universal ProbeLibrary (Roche) by using TaqMan Universal PCR Mastermix (Applied Biosystems) and detection on a 7500 Fast Cycler (Applied Biosystems) as described previously [1].
Transverse aortic constriction. TAC to a 27 gauge stenosis was performed in 9-10 week-old male black six mice (charles river), mice as described previously [2]. AAV vectors were intravenously injected into the tail vein of male adult mice as 150-200 μL bolus using a sterile syringe and 29-gauge needle. Animals were euthanized by cervical dislocation. Organs were dissected and rapidly frozen in liquid nitrogen.
Western blotting. Proteins from heart tissue and cultured cardiomyocytes were isolated, and Western blot analysis was performed according to protocols described previously [1]. Primary antibodies used were anti-flag (santa cruz), anit-myc (santa cruz), anti-gfp (abcam). Primary antibody incubation was followed by corresponding horseradish peroxidase (HRP)-conjugated secondary anti-mouse and anti-rabbit antibodies and ECL detection. Relative protein levels were detected by densitometry using the Image J program.
Histology. Hematoxylin and eosin (H&E) and Masson's trichrome stainings were performed as previously described [3]. Cardiomyocyte size was assessed on H&E-stained sections by using Image J software (http://rsb.info.nih.gov/ij/). More than 200 randomly chosen cardiomyocytes from each group were analyzed to measure cross-sectional cardiomyocyte area. To quantify cardiac fibrosis, 20 trichrome-stained sections (magnification 20×) from the left ventricle were randomly selected, and morphometric analysis by using Image J was performed. Photographs were acquired with an Olympus SZH zoom stereo dissection scope with an Optronics DEI-750 CCD digital camera. All data were analyzed by a single observer blinded to the mouse genotypes.
Results:
Overexpression of N-terminal HDAC4 via an adeno associated virus (AAV) in mice is cardioprotective. HDAC4-NT is able to inhibit the transcription factor myocyte enhancer factor 2 (MEF2). [4] MEF2 is thought to be involved in pathological cardiac remodeling. [5] We therefore hypothesized that HDAC4-NT could have beneficial and cardioprotective effects in vivo. By cloning HDAC4-NT into a cardiotrophic AAV substrain (AAV9) under the control of a cardiomyocyte specific promoter (CMVenh-MLC260), we were able to transduce cardiomyocytes in vivo via a single tail vein injection. Mice were injected 4 weeks before they were exposed to transthoracic aortic constriction (TAC)-surgery as a model for cardiac stress and the development of heart failure. By doing so, animals that were treated with AAV-HDAC4-NT showed reduced cardiac hypertrophy, improved cardiac function and normalized gene regulation from genes that are known to play an important role in pathological cardiac remodeling (
We further aimed to investigate the endogenous role of HDAC4-NT. In wildtype animals, HDAC4-NT production is increased after physiological exercise. We generated conditional HDAC4-knockout animals, lacking HDAC4 in cardiomyocytes only and exposed these animals to running exercise. HDAC4-KO animals showed a reduced exercise tolerance with a reduced left ventricular function after running exercise. Reduced exercise performance was rescued when HDAC4-KO were treated with AAV-HDAC4-NT, indicating that HDAC4-NT is crucial for sustained cardiac function after cardiac stress (
Abhd5 is a critical HDAC4 protease. To get insides about the upstream regulation of HDAC4 cleavage, we performed a siRNA-screen with a set of potential serine-proteases. By doing so, we identified a protein called 1-acylglycerol-3-phosphate O-acyltransferase (abhd5) playing a crucial role in PKA induced HDAC4 cleavage. HDAC4 cleavage was not longer present when abhd5 was knocked down by different siRNAs even when PKA was co-expressed with HDAC4 (
Abhd5 overexpression leads to MEF2 inhibition and is cleavage dependent. By using a MEF2-luciferase assay, we found that abhd5 is able to completely normalize MEF2 activity induced by fetal calf serum (FCS), which is commonly used to induce cardiomyocyte hypertrophy. EGFP control virus did not show any beneficial effects in this system (
Abhd5 inhibits cardiomyocyte hypertrophy in vitro. We next hypothesized, that abhd5 induced HDAC4-NT is able to inhibit cardiomyocyte hypertrophy in vitro. Indeed, abhd5 was able to inhibit FCS induced cardiomyocyte hypertrophy. This effect was independent from a control virus with EGFP. (
Discussion:
We found that HDAC4-NT is cardioprotective in mice and thus could serve as a potential pharmacological tool for the treatment of heart failure in human. This was surprising because earlier approaches to protect the heart by overexpression of a class II HDAC failed [6]. These earlier approaches used transgenic expression of full length HDACs and specific mutations of phosphosites to render HDACs signal-resistant and to force them to localize to the nucleus. However, class II HDAC mutant overexpression showed dramatic harmful effects by inducing mitochondrial dysfunction and apoptosis. In this work we provide two novel ways to circumvent harmful effects. First, we use the AAV system which allowed us to closely titrate the dosage of HDAC expression. The AAV system was already used in clinical trials (SERCA gene therapy). Second, by using an partial HDAC4 peptide we specifically seem to prevent the disease process of the heart and not essential functions such as mitochondrial function or cell survival. As a proof of concept, by expressing HDAC4 1-201 via AAVs, we show here for the first time, that this concept is a new promising therapeutic approach. Given the previous experience with the overexpression of putatively protective proteins in the same animal model, this was not expected.
Upstream Regulation Could Serve as an Additional Tool:
With the identification of a critical PKA dependent protease (abhd5) we have identified the upstream signaling molecule that induced cardioprotective HDAC4 proteolysis. Abhd5 was not described as a protease and the involvement in potential cardioprotective pathways is new. Under normal conditions, abhd5 is bound to lipid droplets (LDs) and is involved in the regulation of lipolysis.
This application is a Continuation of U.S. application Ser. No. 14/768,050, filed on Aug. 14, 2015. U.S. application Ser. No. 14/768,050 claims the benefit of International Application No. PCT/EP2014/053042, filed on Feb. 17, 2014, and U.S. Provisional Application No. 61/765,440, filed on Feb. 15, 2013, the disclosures of which are herein incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61765440 | Feb 2013 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 14768050 | Aug 2015 | US |
| Child | 15881935 | US |
